一.技术领域

癌症是目前严重威胁人们健康的一种疾病，据报道，全球每年癌症新发病人约1000万， 我国每年新发癌症患者近100万，死亡约150万。目前对癌症治疗并没有非常有效的方法， 如果早期发现可以使用手术，但大部分是在后期发现，只能通过放疗或化疗来进行治疗。放 疗法一般使用阿霉素等药物，具有很大的副作用，主要有骨髓抑制而造成的各种血相降低， 胃肠道反应，脱发，肝功能损伤和神经系统损伤等。而放疗的副作用更大，给病人带来巨大 的痛苦。并且放化疗的有效率也不高，在30％左右。

鉴于此，急需找到一种副作用小并且有效率高的方法来对癌症进行治疗，随着现代生物 技术的不断发展，这种方法成为了可能：由于放化疗副作用大的一个十分重要的原因是其毒 性缺乏选择性，其在杀伤癌症细胞的同时也大量地杀伤了正常细胞，而生物导向技术有望解 决这个问题，选用一些导向分子，如特异表达受体的配体或者受体的特异性抗体等来作为导 向，连接上毒素，即是生物靶向药物，可以靶向的到达肿瘤部位，选择性地杀伤肿瘤细胞， 达到治疗肿瘤的同时毒性很小的目的。

二.背景技术

关于生物靶向性药物，目前的导向分子有人促黄体生成激素释放因子(LHRH)，表皮生长 因子(EGF)和白细胞介素2等，其中LHRH仅有10个氨基酸组成，分子量小，融合后不会影 响毒素蛋白的空间构象，关于毒素部分，可选的有假单胞菌外毒素A(PE)，白喉毒素，蓖麻 毒素等，目前较常见的是假单胞菌外毒素A(PE)，PE是一个由613个氨基酸组成的蛋白分子， 去掉N末端1-252位氨基酸后就成为了PE40分子，由于分子量的减少，PE40成为一个常用 的毒素。目前有报道的生物靶向性药物有白细胞介素2-白喉毒素，表皮生长因子-白喉毒素和 LHRH-PE40，特异抗体连接细胞毒素等，但是该类毒素的临床用量较大，导致副作用较大， 所以截止目前为止，生物靶向性药物只有白细胞介素2-白喉毒素在美国上市，而其他种类的 药物还处于临床或临床前阶段，本专利针对生物靶向性药物效价低、用量大的特点，研究了 一系列的生物导向融合蛋白，其具有生物活性高，用量小的特点。

三.发明内容

本专利主要是研究LHRH-PE40系列融合蛋白的基因工程改造，从而提高其杀伤癌细胞 的生物活性，我们主要从以下2方面进行改造：1.对融合蛋白的LHRH部分进行改造，增 强其与癌细胞结合能力；2.对融合蛋白的细胞毒素PE40部分进行改造，增强其癌细胞杀伤 能力；3同时对融合蛋白的LHRH和PE40两方面进行改造，增强其癌细胞杀伤能力，从而 达到减少用药量，降低体内毒副反应，延缓抗体产生时间，降低生产成本，增强治疗效果的 目的，具有广阔的临床效益和社会效益。

LHRH作为融合蛋白导向部分氨基酸序列为：

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly

LHRH突变体氨基酸序列分别为：

Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly

Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly

Glu-His-Trp-Ser-Tyr-Leu-Leu-Arg-Pro-Gly

假单胞杆菌外毒素A PE40作为融合蛋白毒素部分氨基酸序列为

253-279

Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg- His-Arg-Gln-Pro-Arg

280-300

Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala

301-330

Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly- Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg

331-364

Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn

365-399

Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly- Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu

400-613

Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg- Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe- Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile- Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro- Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro- Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile- Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu- Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg- Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro- Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys

(一).首先对融合蛋白假单胞杆菌外毒素A部分进行改造：

1.本专利对假单胞杆菌外毒素A PE40(氨基酸253-613)进行缺失替换突变，缺失替换其C 末端5个氨基酸即609-613位氨基酸REDLK置换为XYEL，其中X代表赖氨酸或精氨酸， Y代表天冬氨酸，谷氨酰胺或谷氨酸，E代表谷氨酸，L代表亮氨酸。

2.本专利对假单胞杆菌外毒素A PE40进行缺失突变，缺失其N末端氨基酸253-279及氨基 酸365-380部分，取其氨基酸280-364和氨基酸381-613部分按原顺序首尾相连得到蛋白即 为PE35。

3.本专利对PE35C末端5个氨基酸REDLK置换为XYEL，其中X代表赖氨酸或精氨酸， Y代表天冬氨酸，谷氨酰胺或谷氨酸，E代表谷氨酸，L代表亮氨酸。

4.本专利取假单胞杆菌外毒素A缺失氨基酸365-380部分，取其氨基酸253-364和氨基酸 381-613部分按原顺序首尾相连得到蛋白即为PE38。

5.本专利对PE38C末端5个氨基酸REDLK置换为XYEL，其中X代表赖氨酸或精氨酸， Y代表天冬氨酸，谷氨酰胺或谷氨酸，E代表谷氨酸，L代表亮氨酸。

6.本专利取假单胞杆菌外毒素A缺失氨基酸253-279部分，取其氨基酸280-613部分得到蛋 白即为PE37。

7.本专利对PE37C末端5个氨基酸REDLK置换为XYEL，其中X代表赖氨酸或精氨酸， Y代表天冬氨酸，谷氨酰胺或谷氨酸，E代表谷氨酸，L代表亮氨酸。

(二).另外对融合蛋白的LHRH部分进行改造：

1.本专利将天然LHRH的N末端第一位的焦谷氨酸改为谷氨酸，然后将其与突变后的PE40 进行融合连接，或者将突变了的LHRH与PE40进行融合连接，形成高亲和力融合蛋白。

2.本专利将一、中融合蛋白LHRH部分的N末端第6位突变为Ala或Trp或Leu。

(三).将LHRH或突变了的LHRH部分的N末端与毒素部分的C末端进行连接，并在LHRH 或突变了的LHRH的C末端连接上XYEL或REDLK。

(四).将末尾含有XYEL或REDLK序列的融合蛋白末端序列XYEL或REDLK重复串联2-5 次。

总结：对融合蛋白LHRH部分的突变使其对肿瘤细胞的亲和力增强；对融合蛋白毒素部 分的改变使得融合蛋白的毒力增强；二个部分的同时改变使得融合蛋白的杀伤肿瘤细胞的活 性更是大大增强。

四.具体实施方式

实例一

首先从cDNA文库中用PCR方法得到LHRH与PE40的基因片段。然后再用PCR与基 因重组方法把融合蛋白PE40部分C末端5个氨基酸突变为KDEL，并获得重组基因载体 pLHRH-PE40(KDEL)，对插入载体的基因片段LHRH-PE40(KDEL)进行序列测定。验证其基 因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重 组表达载体导入表达宿主菌中，经培养表达，疏水层析和离子交换层析得到目的蛋白 LHRH-PE40(KDEL)，用MTT细胞测活法测定LHRH-PE40(KDEL)的杀伤肿瘤细胞的活力。

此序列为LHRH-PE40(KDEL)(序列1)

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val- Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr -Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp- Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr- His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp- Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln- Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg- Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val- Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly- Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro- Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile- Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu

实例二

从pLHRH-PE40(KDEL)载体用PCR方法把融合蛋白PE40部分C末端4个氨基酸突变 为RQEL，得到目的基因片段LHRH-PE40(RQEL)进行序列测定。验证其基因序列的正确性 后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入 表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH-PE40(RQEL)，用MTT细胞测活法测 定LHRH-PE40(RQEL)的杀伤肿瘤细胞的活力。

此序列为LHRH-PE40(RQEL)(序列2)

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val- Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr -Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp- Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr- His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp- Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln- Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg- Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val- Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly- Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro- Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile- Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Gln-Glu-Leu

实例三

从pLHRH-PE40(RQEL)载体用PCR方法把融合蛋白PE40部分C末端4个氨基酸突变 为KEEL，得到目的基因片段LHRH-PE40(KEEL)进行序列测定。验证其基因序列的正确性 后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入 表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH-PE40(KEEL)，用MTT细胞测活法测 定LHRH-PE40(KEEL)的杀伤肿瘤细胞的活力。

此序列为LHRH-PE40(KEEL)(序列3)

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val- Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr -Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp- Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr- His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp- Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln- Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg- Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val- Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly- Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro- Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile- Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Glu-Glu-Leu

实例四

从pLHRH-PE40(KEEL)载体用PCR方法把融合蛋白PE40部分C末端4个氨基酸突变 为REEL，得到目的基因片段LHRH-PE40(REEL)进行序列测定。验证其基因序列的正确性后， 扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达 宿主菌中，经培养表达，纯化得到目的蛋白LHRH-PE40(REEL)，用MTT细胞测活法测定 LHRH-PE40(REEL)的杀伤肿瘤细胞的活力。

此序列为LHRH-PE40(REEL)(序列4)

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val- Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr -Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp- Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr- His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp- Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln- Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg- Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val- Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly- Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro- Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile- Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Glu-Leu

实例五

用PCR方法把融合蛋白PE40部分缺失氨基酸365-380部分，取其氨基酸254-364和氨 基酸381-613部分按原顺序首尾相连得到的DNA序列即为蛋白PE38目的基因，然后对目的基 因片段LHRH-PE38进行序列测定。验证其基因序列的正确性后，扩增保存，并把目的基因 片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的 蛋白LHRH-PE38，用MTT细胞测活法测定LHRH-PE38的杀伤肿瘤细胞的活力。

此序列为LHRH-PE38(序列5)

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu- Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr- Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly -Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val- Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu- Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu- Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro- Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr- Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val- Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro- Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu- Asp-Leu-Lys

实例六

用PCR方法把融合蛋白PE40部分缺失氨基酸253-279，365-380部分，取其氨基酸 280-364和氨基酸381-613部分按原顺序首尾位相连得到的DNA序列即为蛋白PE35目的基 因，对目的基因片段LHRH-PE35进行序列测定。验证其基因序列的正确性后，扩增保存，并 把目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯 化得到目的蛋白LHRH-PE35，用MTT细胞测活法测定LHRH-PE35的杀伤肿瘤细胞的活力。

此序列为LHRH-PE35(序列6)

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val- Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg- Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln- Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn -Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr- Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn- Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly- Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln- Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala- Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro- Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu- Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu- Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile -Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala- Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys

实例七

从pLHRH-PE35载体用PCR方法把融合蛋白PE35部分C末端4个氨基酸突变为KDEL， 得到目的基因片段LHRH-PE35(KDEL)进行序列测定。验证其基因序列的正确性后，扩增保 存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌 中，经培养表达，纯化得到目的蛋白LHRH-PE35(KDEL)，用MTT细胞测活法测定 LHRH-PE35(KDEL)的杀伤肿瘤细胞的活力。

此序列为LHRH-PE35(KDEL)(序列7)

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val- Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg- Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln- Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn -Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr- Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn- Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly- Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln- Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala- Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro- Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu- Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu- Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile -Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala- Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu

实例八

从pLHRH-PE40载体上，用PCR方法把融合蛋白LHRH部分N末端第6位氨基酸突变 为Trp，得到目的基因片段LHRH(Trp6)-PE40进行序列测定。验证其基因序列的正确性后， 扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达 宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Trp6)-PE40，用MTT细胞测活法测定 LHRH(Trp6)-PE40的杀伤肿瘤细胞的活力。

此序列为LHRH(Trp6)-PE40(序列8)

Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val- Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr -Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp- Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr- His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp- Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln- Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg- Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val- Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly- Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro- Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile- Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys

实例九

从LHRH(Trp6)-PE40载体用PCR方法把融合蛋白PE40部分C末端5个氨基酸突变为 KDEL，得到目的基因片段LHRH(Trp6)-PE40(KDEL)进行序列测定。验证其基因序列的正 确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体 导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Trp6)-PE40(KDEL)，用MTT 细胞测活法测定LHRH(Trp6)-PE40(KDEL)的杀伤肿瘤细胞的活力。

此序列为LHRH(Trp6)-PE40(KDEL)(序列9)

Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val- Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr -Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp- Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr- His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp- Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln- Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg- Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val- Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly- Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro- Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile- Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu

实例十

从pLHRH-PE38载体用PCR方法把融合蛋白LHRH部分N末端第6位氨基酸突变为 Trp，得到目的基因片段LHRH(Trp6)-PE38进行序列测定。验证其基因序列的正确性后，扩 增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿 主菌中，经培养表达，纯化得到目的蛋白LHRH(Trp6)-PE38，用MTT细胞测活法测定 LHRH(Trp6)-PE38的杀伤肿瘤细胞的活力。

此序列为LHRH(Trp6)-PE38(序列10)

Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu- Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr- Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly -Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val- Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu- Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu- Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro- Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr- Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val- Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro- Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu- Asp-Leu-Lys

实例十一

从LHRH(Trp6)-PE38载体用PCR方法把融合蛋白PE38部分C末端5个氨基酸突变为 KDEL，得到目的基因片段LHRH(Trp6)-PE38(KDEL)进行序列测定。验证其基因序列的正 确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体 导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Trp6)-PE38(KDEL)，用MTT 细胞测活法测定LHRH(Trp6)-PE38(KDEL)的杀伤肿瘤细胞的活力。

此序列为LHRH(Trp6)-PE38(KDEL)(序列11)

Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu- Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr- Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly -Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val- Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu- Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu- Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro- Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr- Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val- Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro- Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp- Glu-Leu

实例十二

从pLHRH-PE35载体用PCR方法把融合蛋白LHRH部分N末端第6位氨基酸突变为 Ala，得到目的基因片段LHRH(Ala6)-PE35进行序列测定。验证其基因序列的正确性后，扩 增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体导入表达宿 主菌中，经培养表达，纯化得到目的蛋白LHRH(Ala6)-PE35，用MTT细胞测活法测定 LHRH(Ala6)-PE35的杀伤肿瘤细胞的活力。

此序列为LHRH(Ala6)-PE35(序列12)

Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val- Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg- Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln- Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn -Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr- Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn- Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly- Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln- Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala- Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro- Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu- Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu- Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile -Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala- Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys

实例十三

从LHRH(Ala6)-PE35载体用PCR方法把融合蛋白PE35部分C末端5个氨基酸突变为 KDEL，得到目的基因片段LHRH(Ala6)-PE35(KDEL)进行序列测定。验证其基因序列的正 确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体 导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Ala6)-PE35(KDEL)，用MTT 细胞测活法测定LHRH(Ala6)-PE35(KDEL)的杀伤肿瘤细胞的活力。

此序列为LHRH(Ala6)-PE35(KDEL)(序列13)

Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val- Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg- Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln- Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn -Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr- Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn- Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly- Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln- Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala- Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro- Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu- Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-pro-Glu-Glu-Glu- Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile -Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala- Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu

实例十四

从LHRH(Ala6)-PE35载体用PCR方法把融合蛋白PE35部分C末端5个氨基酸突变为 RQEL，得到目的基因片段LHRH(Ala6)-PE35(RQEL)进行序列测定。验证其基因序列的正 确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体 导入表达宿主菌中，经培养表达，纯化得到目的蛋白LHRH(Ala6)-PE35(RQEL)，用MTT 细胞测活法测定LHRH(Ala6)-PE35(RQEL)的杀伤肿瘤细胞的活力。

此序列为LHRH(Ala6)-PE35(RQEL)(序列14)

Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val- Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg- Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln- Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn -Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr- Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn- Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly- Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln- Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala- Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro- Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu- Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu- Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile -Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala- Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Gln-Glu-Leu

实例十五

从cDNA文库中用PCR方法得到LHRH与PE40的基因片段后，用PCR与基因重组方 法把PE40的C末端和LHRH的N末端相连，并在LHRH的C末端加上KDEL，获得重组 基因载体pPE40-LHRH-KDEL，对插入载体的基因片段PE40-LHRH-KDEL进行序列测定。 验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中， 再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE40-LHRH-KDEL， 用MTT细胞测活法测定PE40-LHRH-KDEL的杀伤肿瘤细胞的活力。

此序列为PE40-LHRH-KDEL(序列15)

Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg- His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala- Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser- Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu- Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala- Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp- Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly- Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg- Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser- Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile- Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile- Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser- Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu- Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro- Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp- Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro -Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Lys-Asp- Glu-Leu

实例十六

从pPE40-LHRH-KDEL载体上，用PCR方法把融合蛋白PE40部分缺失氨基酸365-380 部分，取其氨基酸254-364和氨基酸381-613部分及LHRH-KDEL部分按原顺序首尾相连得 到得到目的基因片段PE38-LHRH-KDEL进行序列测定。验证其基因序列的正确性后，扩增 保存，并把目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养 表达，纯化得到目的蛋白PE38-LHRH-KDEL，用MTT细胞测活法测定PE38-LHRH-KDEL 的杀伤肿瘤细胞的活力。

此序列为PE38-LHRH-KDEL(序列16)

Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg- His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala- Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser- Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu- Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala- Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu- Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg- Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe- Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile- Leu-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro- Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro- Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile- Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu- Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg- Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro- Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-Lys-Asp-Glu-Leu

实例十七

从pPE40-LHRH-KDEL载体用PCR方法把融合蛋白PE40部分缺失氨基酸254-279， 365-380部分，取其氨基酸280-364和氨基酸381-613部分及LHRH-KDEL按原顺序首尾位 相连得到PE35-LHRH-KDEL进行序列测定。验证其基因序列的正确性后，扩增保存，并把 目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化 得到目的蛋白PE35-LHRH-KDEL，用MTT细胞测活法测定PE35-LHRH-KDEL的杀伤肿瘤 细胞的活力。

此序列为PE35-LHRH-KDEL(序列17)

Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala -Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly- Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala- Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro -Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp- Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala- His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala- Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe- Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly- Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg- Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu- Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly- Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly -Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser- Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Lys- Asp-Glu-Leu

实例十八

从pPE35-LHRH-KDEL载体上，用PCR方法把融合蛋白C末端4个氨基酸突变为 REDLK，得到目的基因片段PE35-LHRH-REDLK进行序列测定。验证其基因序列的正确性 后，扩增保存，并把目的基因片段，插入表达载体中，再把重组表达载体导入表达宿主菌中， 经培养表达，纯化得到目的蛋白PE35-LHRH-REDLK，用MTT细胞测活法测定 PE35-LHRH-REDLK的杀伤肿瘤细胞的活力。

此序列为PE35-LHRH-REDLK(序列18)

Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala -Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly- Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala- Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro -Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp- Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala- His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala- Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe- Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly- Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg- Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu- Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly- Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly -Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser- Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Arg- Glu-Asp-Leu-Lys

实例十九

从pPE40-LHRH-KDEL载体用PCR方法把融合蛋白LHRH部分N末端第6位氨基酸突 变为Trp，得到目的基因片段PE40-LHRH(Trp6)-KDEL进行序列测定。验证其基因序列的正 确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体 导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE40-LHRH(Trp6)-KDEL，用MTT细 胞测活法测定PE40-LHRH(Trp6)-KDEL的杀伤肿瘤细胞的活力。

此序列为PE40-LHRH(Trp6)-KDEL(序列19)

Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg- His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala- Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser- Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu- Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala- Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp- Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly- Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg- Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser- Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile- Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile- Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser- Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu- Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro- Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp- Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro -Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Lys-Asp- Glu-Leu

实例二十

从pPE35-LHRH-KDEL载体上，用PCR方法把融合蛋白LHRH部分N末端第6位氨基 酸突变为Ala，得到目的基因片段PE35-LHRH(Ala6)-KDEL进行序列测定。验证其基因序列 的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达 载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE35-LHRH(Ala6)-KDEL，用MTT 细胞测活法测定PE35-LHRH(Ala6)-KDEL的杀伤肿瘤细胞的活力。

此序列为PE35-LHRH(Ala6)-KDEL(序列20)

Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala- Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly- Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala- Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala -Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly- Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His- Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln- Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile -Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile- Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser- Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu- Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro- Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp- Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro -Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Lys-Asp- Glu-Leu

实例二十一

从pPE40-LHRH(Trp6)-KDEL载体上，用PCR方法把融合蛋白C末端4个氨基酸突变为 REDLK，得到目的基因片段PE40-LHRH(Trp6)-REDLK进行序列测定。验证其基因序列的正 确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体 导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE40-LHRH(Trp6)-REDLK用MTT细胞 测活法测定PE40-LHRH(Trp6)-REDLK的杀伤肿瘤细胞的活力。

此序列为PE40-LHRH(Trp6)-REDLK(序列21)

Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg- His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala- Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser- Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu- Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala- Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp- Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly- Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg- Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser- Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile- Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile- Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser- Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu- Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro- Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp- Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro -Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Arg-Glu- Asp-Leu-Lys

实例二十二

从pPE35-LHRH(Ala6)-KDEL载体上，用PCR方法把融合蛋白C末端4个氨基酸突变为 REDLK，得到目的基因片段PE35-LHRH(Ala6)-REDLK进行序列测定。验证其基因序列的正 确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再把重组表达载体 导入表达宿主菌中，经培养表达，纯化得到目的蛋白PE35-LHRH(Ala6)-REDLK，用MTT细 胞测活法测定PE35-LHRH(Ala6)-REDLK的杀伤肿瘤细胞的活力。

此序列为PE35-LHRH(Ala6)-REDLK(序列22)

Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala -Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly- Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala- Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro -Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp- Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala- His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala- Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe- Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly- Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg- Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu- Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly- Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly -Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser- Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Arg- Glu-Asp-Leu-Lys

实例二十三

从pLHRH-PE40载体上，用PCR方法把融合蛋白PE40部分C末端5个氨基酸突变为 KDELKDELKDEL，得到目的基因片段LHRH-PE40-KDELKDELKDEL进行序列测定。验证 其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再 把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白 LHRH-PE40-KDELKDELKDEL，用MTT细胞测活法测定LHRH-PE40-KDELKDELKDEL的 杀伤肿瘤细胞的活力。

此序列为LHRH-PE40-KDELKDELKDEL(序列23)

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val- Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr -Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp- Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr- His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp- Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln- Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg- Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val- Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly- Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro- Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile- Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu-Lys-Asp-Glu- Leu-Lys-Asp-Glu-Leu

实例二十四

从pLHRH-PE40-KDELKDELKDEL载体上，用PCR方法把融合蛋白LHRH部分N末 端第6位氨基酸突变为Trp，得到目的基因片段LHRH(Trp6)-PE40-KDELKDELKDEL进行序 列测定。验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表 达载体中，再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白 LHRH(Trp6)-PE40-KDELKDELKDEL，用MTT细胞测活法测定 LHRH(Trp6)-PE40-KDELKDELKDEL的杀伤肿瘤细胞的活力。

此序列为LHRH(Trp6)-PE40-KDELKDELKDEL(序列24)

Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Se-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val- Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr -Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp- Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr- His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp- Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln- Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg- Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val- Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly- Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro- Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile- Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu-Lys-Asp-Glu-Leu- Lys-Asp-Glu-Leu

实例二十五

从pLHRH-PE35载体上，用PCR方法把融合蛋白PE35部分C末端5个氨基酸突变为 KDELKDELKDEL，得到目的基因片段LHRH-PE35-KDELKDELKDEL进行序列测定。验证 其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中，再 把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白 LHRH-PE35-KDELKDELKDEL，用MTT细胞测活法测定LHRH-PE35-KDELKDELKDEL的 杀伤肿瘤细胞的活力。

此序列为LHRH-PE35-KDELKDELKDEL(序列25)

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val- Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg- Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln- Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn -Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr- Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn- Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly- Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln- Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala- Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro- Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu- Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu- Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile -Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala- Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu-Lys-Asp-Glu- Leu-Lys-Asp-Glu-Leu

实例二十六

从pLHRH-PE35-KDELKDELKDEL载体用PCR方法把融合蛋白LHRH部分N末端第6 个氨基酸突变为Ala，得到目的基因片段LHRH(Ala6)-PE35-KDELKDELKDEL进行序列测定。 验证其基因序列的正确性后，扩增保存，从该载体上切取下目的基因片段，插入表达载体中， 再把重组表达载体导入表达宿主菌中，经培养表达，纯化得到目的蛋白 LHRH(Ala6)-PE35-KDELKDELKDEL，用MTT细胞测活法测定 LHRH(Ala6)-PE35-KDELKDELKDEL的杀伤肿瘤细胞的活力。

此序列为LHRH(Ala6)-PE35-KDELKDELKDEL(序列26)

Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val- Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg- Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln- Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn -Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr- Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn- Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly- Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln- Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala- Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro- Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu- Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu- Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile -Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala- Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu-Lys-Asp-Glu- Leu-Lys-Asp-Glu-Leu

实例二十七

用MTT比色法测定细胞增殖中活细胞的数目，以此确定融合蛋白的杀伤各种肿瘤细胞 的活力，所选的肿瘤细胞包括SPC(人肺腺癌细胞)、HL60(人白血病细胞)、MGC(人胃癌细胞)、 PC-3(人前列腺癌细胞)和MCF-7(人乳腺癌细胞)。取96孔细胞培养板3块，每孔接种细胞数 为1×104、每块培养板每种细胞重复接种20个孔，37℃、5％CO2条件下培养，然后分别加入 等比稀释融合蛋白样品，接着培养24，48及72h后，每孔加5g/L MTT 20μl再培养4h， 然后去上清液加入二甲亚砜200μl，将培养板放在微量振荡器上振荡10min，待MTT被细胞 还原形成甲颗粒完全溶解后，立即用酶标仪、波长570nm测定其吸光度，然后通过标准计算 公式计算出目的蛋白活性。上述实验重复3次，计算其平均IC50值。

结果：见表1，以LHRH-PE40为对照，实例1-7中融合蛋白突变体是单独对PE40部分 进行的突变，活性大约提高了5-20倍；实例8中的融合蛋白突变体是单独对LHRH部分进 行的突变，活性提高了10倍；实例9-14中的融合蛋白突变体是结合了LHRH和PE40部分 二者的突变体，活性提高了50-1000倍；实例15-22中的融合蛋白突变体活性提高了20-500 倍；实例22-26中的融合蛋白突变体活性提高了100-800倍。

表1.实例中的融合蛋白细胞活性   序号   细胞IC50   融合蛋白   SPC   (ug/mL)   HL60   (ug/mL)   MGC   (ug/mL)   PC-3   (ug/mL)   MCF-7   (ug/mL)   0   LHRH-PE40   28.2   19.8   52.1   2.75   1.48   1   LHRH-PE40(KDEL)   6.8   15.4   20.1   0.58   0.54   2   LHRH-PE40(RQEL)   10.7   19.1   16.7   1.3   0.48   3   LHRH-PE40(KEEL)   8.2   22.4   21.2   0.86   0.62   4   LHRH-PE40(REEL)   7.6   15.7   18.3   1.2   0.37   5   LHRH-PE38   3.2   2.4   4.3   0.25   0.22   6   LHRH-PE35   2.1   1.3   6.8   0.18   0.25   7   LHRH-PE35(KDEL)   1.5   1.4   2.6   0.14   0.12   8   LHRH(Trp6)-PE40   3.1   2.1   5.3   0.28   0.16   9   LHRH(Trp6)-PE40(KDEL)   1.1   1.8   1.2   0.061   0.032   10   LHRH(Trp6)-PE38   0.24   0.28   0.66   0.019   0.022   11   LHRH(Trp6)-PE38(KDEL)   0.062   0.055   0.17   0.0052   0.0038   12   LHRH(Ala6)-PE35   0.18   0.22   0.74   0.057   0.041   13   LHRH(Ala6)-PE35(KDEL)   0.022   0.025   0.48   0.0013   0.0021   14   LHRH(Ala6)-PE35(RQEL)   0.046   0.032   0.28   0.0037   0.0018   15   PE40-LHRH-KDEL   2.6   1.4   3.9   0.17   0.048   16   PE38-LHRH-KDEL   2.2   0.65   2.8   0.095   0.041   17   PE35-LHRH-KDEL   3.41   0.66   2.2   0.11   0.026   18   PE35-LHRH-REDLK   2.7   0.42   1.9   0.074   0.023   19   PE40-LHRH(Trp6)-KDEL   0.12   0.077   0.22   0.012   0.0052   20   PE35-LHRH(Ala6)-KDEL   0.074   0.041   0.16   0.0071   0.0022   21   PE40-LHRH(Trp6)-REDLK   0.33   0.35   0.69   0.26   0.21   22   PE35-LHRH(Ala6)-REDLK   0.062   0.081   0.14   0.044   0.022   23   LHRH-PE40(KDEL)3   0.41   0.16   0.32   0.35   0.16   24   LHRH(Trp6)-PE40(KDEL)3   0.085   0.055   0.062   0.077   0.043   25   LHRH(Ala6)-PE35(KDEL)3   0.022   0.031   0.064   0.0018   0.0011

五.序列表

(1)序列1

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val- Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr -Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr -Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His- Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu- Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp- Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser- Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu- Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly- Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr- Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser- Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-(Lys-Asp-Glu-Leu)n

说明：该序列的N末端第6位Gly可替换成为Ala、Trp或Leu；该序列的C末端第3位Asp 可替换成Gln或Glu；该序列C末端第4位Lys可替换成Arg。n小于等于5大于等于1。并 且该序列C末端的氨基酸-(Lys-Asp-Glu-Leu)n可替换成-(Arg-Glu-Asp-Leu-Lys)n，n小于等 于5大于等于1。

(2)序列2

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala- Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val- Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg- Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg- Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu- Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr- Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly -Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val- Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu- Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu- Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro- Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr- Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val- Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro- Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-(Lys-Asp- Glu-Leu)n

说明：该序列的N末端第6位Gly可替换成为Ala、Trp或Leu；该序列的C末端第3位Asp 可替换成Gln或Glu；该序列C末端第4位Lys可替换成Arg。n小于等于5大于等于1。并 且该序列C末端的氨基酸-(Lys-Asp-Glu-Leu)n可替换成-(Arg-Glu-Asp-Leu-Lys)n，n小于等 于5大于等于1。

(3)序列3

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val- Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg- Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln- Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn -Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr- Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn- Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly- Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln- Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala- Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro- Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu- Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu- Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile -Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala- Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-(Lys-Asp-Glu-Leu)n

说明：该序列的N末端第6位Gly可替换成为Ala、Trp或Leu；该序列的C末端第3位Asp 可替换成Gln或Glu；该序列C末端第4位Lys可替换成Arg。n小于等于5大于等于1。并 且该序列C末端的氨基酸-(Lys-Asp-Glu-Leu)n可替换成-(Arg-Glu-Asp-Leu-Lys)n，n小于等 于5大于等于1。

(4)序列4

Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val- Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg- Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln- Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn -Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys -Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe- Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu- Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu- Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp- Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp- Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly- Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly- His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr- Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn- Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp- Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-(Lys-Asp-Glu-Leu)n

说明：该序列的N末端第6位Gly可替换成为Ala、Trp或Leu；该序列的C末端第3位Asp 可替换成Gln或Glu；该序列C末端第4位Lys可替换成Arg；n小于等于5大于等于1。并 且该序列C末端的氨基酸-(Lys-Asp-Glu-Leu)n可替换成-(Arg-Glu-Asp-Leu-Lys)n，n小于等 于5大于等于1。

(5)序列5

Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala -Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly- Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala- Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro -Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp- Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala- His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala- Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe- Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly- Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg- Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu- Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly- Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly -Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser- Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-(Lys -Asp-Glu-Leu)n

说明：该序列的C末端第9位Gly可替换成为Ala、Trp或Leu；该序列的C末端第3位Asp 可替换成Gln或Glu；该序列C末端第4位Lys可替换成Arg；n小于等于5大于等于1。并 且该序列C末端的氨基酸-(Lys-Asp-Glu-Leu)n可替换成-(Arg-Glu-Asp-Leu-Lys)n，n小于等 于5大于等于1。