本发明以及可用于实现其具体实施方式的材料和方法在下面 的
实施例中将进一步说明,但不用于限制本发明的范围。
实施例
实施例1:材料和方法
1.1-痘病毒
CPV、EV和VV通过感染铺满的
单层Bsc-I细胞而在体外进行 增殖。
1.2-骆驼痘病毒CrmB的克隆和VaV CrmB的产生
利用寡核苷酸CMLV 264 Eco (5’-GCGGAATTCATGAAGTCCGTATTATACTCG)和CMLV 264 Xho (5’-GCGCTCGAGTAAAAAGTGGGTGGGTTTGG)并且纯化的CMLV DNA作为
模版通过PCR来扩增骆驼痘病毒的菌株CMS的ORF 264 (相应于CrmB基因)。PCR产物被克隆入EcoRI/XhoI消化的 pBacl(Novagen)以产生质粒pRA1。通过DNA测序确定扩增基因中 缺少突变。根据制造商的说明,利用“QuikChange Multi Site-Directed Mutagenesis Kit(Strategene)”,通过质粒pRA1的多位点定点突变 来获得相应于VaV(菌株Bangladesh 1975;ORF 188)的DNA。 引入的突变示于图6中。在若干连续的几轮定点突变后,获得质粒 pRA105。这种质粒包含编码融合至由原始pBac1质粒提供的C-末 端His标签的VaV(BSH 1975菌株)CrmB的序列。通过直接测序 确认了存在所有的突变并且不存在不希望的其它突变。
1.3-表达来自EV或CPV的CrmD的重组杆状病毒 (baculovirus)和牛痘病毒的构建
为了在杆状病毒系统中的表达,将编码全长或截短型CrmD(表 3)的DNA用特异性寡核苷酸(表4)进行PCR-扩增,并克隆入 pBAC-1中。通过在转染有重组pBAC-1质粒和如所述(Alcami et al., 1998)的线性杆状病毒DNA的SF21昆虫细胞中的同源重组而产 生重组杆状病毒。
vTNFR也从VV表达系统进行表达。感兴趣的基因被克隆入 pMJ601,用于从强大VV启动子表达该基因(Davison & Moss,1990)。 通过同源重组将感兴趣的基因插入VV基因组的胸苷激酶座位,在 溴脱
氧尿苷的存在下选择该重组VV并且通过
颜色选择进行鉴定 (□-半乳糖苷酶的表达并用X-gal
染色)。VTNFR从VV Western Reserve(一种不编码TNF结合活性的病毒株)被表达 (Alcami et al.,1999)。
1.4-表达融合于CPV 35kDa蛋白的VaV CrmB、VaV CrmB CRD(1-4)、VaV CrmB CTD、EV E12、EV E184、CPV V218和 VaV CrmB CRD(1-4)的重组杆状病毒的产生
融合于C-末端His标签的全长VaV CrmB基因被亚克隆入 EcoRI/SphI消化的pFastBac1(Invitrogen)以产生pRA107。利用 pRA107作为模版,利用寡核苷酸 VaV 188 Eco(5’-GCGGAATTCATGAAGTCCGTATTATACTTG)和VaV 188 CRDs1-4 Xho (5’- GCGCTCGAGACACGATGTGTCGTTAACTGG),通过PCR扩增包括四个CRD 并相应于残基1(M)到192(C)的VaV CrmB的N-末端结构域。 该扩增的片段用由EcoRI/XhoI消化的pBac1(Novagen)提供的C- 末端his标签符合读框地被克隆,以生成pRA99。用EcoRI/SphI消 化pRA99并且携带融合于His标签的VaV CrmB CRD1-4的片段象 先前一样被克隆入pFastBac1以产生pRA106。利用pRA107作为模 版,将VaV CrmB的CTD(残基T194到L348)用寡核苷酸VaV188 Cter-PfIMI(5’-CGCCCACCCAATGGAACTAGGACGACCAC-TACCGG)和 H347R 3进行PVR扩增。该片段用PfmII和XhoI消化并且克隆入 用同样的酶消化的pRA107中。这产生了pRA108,一种编码由VaV CrmB的29N-末端残基(其包括预测的
信号肽)和随后的CrmB的 CTD以及另外的His标签构成的融合蛋白的质粒。在所有情况下, 通过对全部插入物进行测序而确认了在pRA106、pRA107和 pRA108中不存在不希望的突变。
利用寡核苷酸E1(5’-GCGGGA-TCCATGATAAACATAAACATAAACACAATAC) 和E2(5’-GCGGCGGCCGCAT-TAATAGTTCTAGTAGCGCAAG)并且纯化的 EV(菌株naval.Cam)DNA作为模版通过PCR来扩增EV基因E12。 将该PCR产物克隆入Bam HI/NotI消化的pBac1(Novagen)生成 质粒pMS51中。E12基因被亚克隆入Bam HI/Xho I消化的pRA106 以产生质粒pAH18,其包含融合于pFastbac(Invitrogen)主链中编 码His-标签的C-末端序列的E12基因。利用寡核苷酸 5’V218 EcoRI(5’-CGCGAATTCATGATGATATACGGATTAATAGC)和 3’V218 Sall(5’-GCGGTCGACACCATCGACACCACTCATC)并且 纯化的病毒DNA作为模版,PCR-扩增CPV Brighton Red菌株ORF V218。PCR产物被克隆入EcoRI/Xho I消化的pRA106以产生质粒 pAH17,其包含融合于pFastbac(Invitrogen)主链中编码His-标签 的C-末端序列的CPV V218基因。利用寡核苷酸5’35KBR-S23 (5’-CGCCTCGAGTCATTCTCATCCTCATCCTC)和3’35KBR (-stop)(5’-CGCCTCGAGGACACACGCTATAAGTTTTGC)并且纯化 的病毒DNA作为模版,PCR-扩增相应于CPV(菌株Brighton Red) 35kDa蛋白的残基S23-V246的片段。PCR产物被克隆入Xho I消 化的pRA106以产生质粒pRA109。该质粒携带编码 VaV CrmB CRD(1-4)的序列,其符合读框地融合于pFastBac主链 (Invitrogen)中编码CPV 35kDa vCKBP(不具有其信号肽,而具有 C-末端His标签)的序列。
如制造商所述,利用Bac-to-Bac表达系统(Invitrogen)来获得 重组杆状病毒。简单地说,质粒pRA106、pRA107、pRA108、pRA109、 pAH17和pAH18被转化入感受态DH10Bac细菌(一个转座事件), 产生相应的重组杆粒。这些产物被纯化并转染入High5昆虫细胞, 并且转染后三天,从细胞培养物的上清液
收获重组杆状病毒 vBac106、vBac107、vBac1068、vBac109、vBacAH17和vBacAH18。 在一个单独的步骤中进一步扩增这些病毒以产生用于蛋白质生产 的高效价重组病毒原种。
用寡核苷酸5’E184(5’-GCGGAATTCATGTATAAAAAAGTAATAACGTTT)和 3’E184 XhoI(5’-CGCCTCGAGAAAATCATATTTTGAATAATATGTA)并且 纯化的EV DNA作为模版,PCR-扩增来自EV的E184基因。PCR 产物被克隆入用EcoRI和XhoI消化的pRA106质粒,产生pAH11。 确认正确的DNA序列。如上所述,质粒pAH11被用于产生重组杆 状病毒vBacAH11,表达融合于组氨酸标签的EV E184蛋白以促进 该蛋白的纯化。
1.5-蛋白质表达和纯化
为了表达该重组蛋白,将High5细胞以高感染复数 (multiplicity)(10pfu/细胞)加以感染,并且在感染后三天(d.p.i.) 收集来自感染培养物的上清液。通过蛋白质印迹法,和/或在全长和 N-末端VaV CrmB构建体的情况下的溶液中TNF结合分析来确认这 些上清液中蛋白质的存在(Alcami et al.,1999)。利用YM-3膜 (Millipore)在搅拌
超滤池(Amicon)上将该样品浓缩至约2.5ml, 并且在PD-10脱盐柱(Amersham-Pharamcia Biosciences)上将缓冲液变 换为结合缓冲液(50mM
磷酸盐、300mM NaCl、10mM咪唑,pH7.4)。 按照制造商的说明,利用Vivapure金属鳌合柱(Vivascience)亲和 -纯化His-标记的重组蛋白。在考
马斯-蓝染色的SDS-聚丙烯酰胺凝 胶上检查纯化蛋白的纯度和数量。为了TNF保护和趋化性抑制实 验,针对PBS
透析重组蛋白。
1.6-通过
表面等离子共振(SPR)进行生物分子相互作用分析
利用BIAcore X生物
传感器(Biacore,Uppsala,Sweden)来确定 细胞因子结合特异性和亲和力常数。对于配体筛选实验,在每个情 况下,纯化的重组蛋白被胺连接至CM5芯片达到约5000RU (5000pg/mm2)的水平。随后以5μl/min的流速注射在HBS-EP缓 冲液(10mM Hepes,150mM NaCl,3mM EDTA,0.005%(v/v)
表面活性剂P20;pH7.4)中的100nM的商业上可获得的细胞因子 (Peprotech,R&D Systems),并且监测结合和解离。在每次注射后 利用10mM甘氨酸-HCl pH2.0再生该表面。对于动力学分析,将重 组蛋白以低
密度(Rmax<200RU)固定。随后不同浓度的相应分析 物以30μl/min的流速注射超过2分钟并且使其解离另外5分钟。 利用BIAevaluation 3.2软件分析所有的Biacore感应图 (sensorgram)。通过减去基准流动细胞反应而去除体积折射率变 化,并且从全部分析物感应图减去空白注射的平均反应以去除系统 假象(artifact)。动力学数据被全面拟合至1∶1的langmuir模型。
1.7-TNF所致的细胞毒性的抑制
如前所述在小鼠L929细胞上进行TNF诱导的细胞毒性实验 (Loparev et al.,1998,Saraiva & Alcami,2001,Schreiber et al.,1997,Smith et al.,1996)。 在补充有放线菌素D(4□g/ml)(Sigma)的完全DMEM中,将TNF (20ng/ml)(R&D Systems)用纯化的重组蛋白在37℃预孵育2h。随 后将该混合物加入到96孔板中前一天接种的2×104个细胞上,并 且按照制造商的指导,利用“CellTiter 96 Aqueous One Solution cell proliferation assay”(Promega),在16-18 小时后评估细胞死亡。
1.8-趋化性实验
如前所述,利用具有3□m孔径的
过滤器的24孔Transwell板 (Costar)来评估MOLT-4细胞的迁移(Zaballos et al.,1999)。简 单地说,将人类CCL25(100nM)(R&D Systems)单独地或在数量 增加的纯化重组蛋白存在的条件下在37℃孵育30分钟,并放置在 下室内。然后,在包含0.1%FCS的100□l完全RPMI中将5×105 个MOLT-4细胞被加入到上孔,并且在37℃,5%CO2,95%加湿 的孵育箱中孵育该板。在4小时后,通过流式细胞仪确定MOLT-4 细胞向下室的迁移。
实施例2:结果
2.1-由EV和CPV编码的vTNFR CrmD结合趋化因子
为了寻找结合趋化因子的新型病毒分泌蛋白质,我们用 1251-CXCL8进行了交联实验,并且鉴定了由痘病毒EV编码的新 型分泌vCKBP(图3a)。这种活性在VV样品中缺少,并且具有比 由VV和其它痘病毒编码的35kDa vCKBP更大的分子大小,vCKPB 是一种可以交联CXCL8但是由于其对CXCL8的低亲和力而不阻断 其生物活性的蛋白质 (Alcami et al.,1998,Graham et al.,1997,Lalani et al.,1998)。出乎意料的是, 我们发现由EV编码的vTNFR CrmD(已知结合TNF)具有另外的 结合CXCL8的性质(图3b)。利用在VV表达系统中表达的截短型 的CrmD蛋白,我们已经证实,CrmD的3个N-末端CRD对于阻 断TNF活性是必要的(图4a、4b和4c),而CTD对于TNF结合 不是必需的,但是CTD为CrmD赋予了结合趋化因子的能力(图 3b)。CrmD的不同结构域看起来涉及TNF和趋化因子结合,因为 在TNF存在的情况下,CXCL8交联至CrmD不能被阻断(图3c), 而在CXCL8存在的情况下,TNF结合至CrmD不被抑制(未示出)。 利用表面等离子共振(SPR,BIAcore X)技术,我们检测了纯化的 EV CrmD与所有商业上可获得的人源和鼠源趋化因子的可能相互 作用,并且已经确定CrmD以高亲和力与许多趋化因子结合(图5)。
2.2-由CPV和VaV编码的vTNFR CrmB结合趋化因子
为了检测编码延伸的CTD的其它vTNFR是否结合趋化因子, 我们在杆状病毒系统中表达了来自CPV的CrmB,并且证实它在交 联实验中结合CXCL8(图3d)。CrmB也由人类病原体VaV(天花 的病因)编码。我们通过来自相关的骆驼痘病毒的CrmB基因的大 量定点突变产生了VaV的CrmB基因(图6),并且在杆状病毒系 统中表达了该蛋白。我们首先检测了纯化的VaV CrmB蛋白结合 TNF(图7)并抑制TNF生物活性(图8a)。我们还通过SPR检测 了CrmB与所有可获得的人类趋化因子的结合,并且我们证明了 CrmB以高亲和力与大量趋化因子结合(图9)。缺少CTD的截短 型VaV CrmB不结合趋化因子(图7)。此外,VaV CrmB的CTD 的表达和纯化已经证明了CTD编码CrmB的趋化因子结合活性(图 7)。这通过表明TNF和趋化因子结合至CrmB中不同的位点(未示 出)的SPA分析得以确认。
2.3-在体外由VaV编码的vTNFR CrmB阻断由CCL25诱导的 MoIt4细胞的迁移
vTNFR CrmB和CrmD对一些趋化因子的高亲和力表明这些病 毒蛋白质可以用作鳌合趋化因子的假目标受体,并且阻止趋化因子 结合到白细胞上的其特异性受体和诱导触发细胞迁移的信号。我们 证实,VaV CrmB抑制表达相关受体的MoIt4细胞响应于趋化因子 CCL25的迁移(图8b)。
2.4.-与vTNFR的CTD(CTD同源物)相关的蛋白质结合趋 化因子
如上所述,由痘病毒编码的三种蛋白质具有与vTNFR(CrmB 和CrmD)的CTD类似的氨基酸序列(图1和图2)。称为CTD1、 CTD2、CTD3的这些蛋白质具有表明它们是被分泌的N-末端信号 肽。这些蛋白质、EV E12(CTD1)、EV184(CTD2)和CPV B21R (CTD3)的两种在杆状病毒系统中的表达已表明两种蛋白质均被 分泌入培养基中。检测纯化的EV E12蛋白对于全部小鼠趋化因子 的结合,并且发现其以高亲和力结合多种趋化因子(表2)。另外, 我们已通过SPR确定:纯化的蛋白CPV B21R和V E184结合来自 小鼠的CCL21,CCL24,CCL25,CCL27,CCL28,CXCL10,CXCL11,CXCL12β,CXCL13和 CXCL14,以及人类CCL26。已经证实VV VVR B7R(CTD2)被 转运到内质网腔并且被留在细胞内而不是被分泌(Price et al.,2000)。 一些CTD同源物可以起作用以阻断趋化因子(如CCL27)的活性, 已知其也在细胞内被表达(Gortz et al.,2002)。在鼠皮层内模式下缺 乏B7R基因的VV突变体被减弱(Price et al.,2000)。
2.5.-来自EV的vTNFR CrmD和来自VaV的CrmB结合其它 TNF配体超家族成员
TNF是具有结构类似的免疫调节剂的大家族(称为TNF配体 (TNFL)超家族)的成员(Locksley et al.,2001,Wallach,2001)。 通过SPR检测EV CrmD和VaV CrmB结合所有商业上可获得的 TNF配体超家族成员,并发现其结合APRIL(TNFL 13)(CrmD Kd 110 pM;CrmB Kd 2nM)和LIGHT(TNFL14)(CrmD Kd 140nM;CrmB Kd 2nM)。 这表明CrmD和CrmB(和可能是其它vTNFR)可以抑制多种TNF 配体超家族成员的生物活性。
2.6-vTNFR的CTD可以影响CRD以高亲和力结合TNF的能 力
如上所证实的,包括N-末端CRD(1,2)的截短的vTNFR CrmD 丧失了对于TNF的亲和力,并且不阻断TNF生物活性,而CRD(1-3) 结合TNF并且抑制其活性(图4a、4b、4c)。令人惊奇的是,当 CRD(1,2)融合至CTD被表达时,它恢复TNF抑制活性,表明CTD 可以提高vTNFR的N-末端CRD的TNF结合活性(图4d)。
2.7-病毒编码的蛋白质形成有独立结合免疫配体的结构域
这里所示的数据表明,vTNFR CrmB和CrmD由两个独立的结 构域构成(图1和图2)。N-末端CRD具有结合TNF的能力,而 CTD以独立的方式结合趋化因子。这通过如下发现得以证实:当独 立表达时,来自CrmB的CRD(1,4)保留TNF结合活性,而来自 CrmB的CTD具有趋化因子结合活性。病毒蛋白质的这些区域代表 结合免疫调节剂的结构模
块或结构域的这个概念通过以下发现被 着重强调:(1)纯化的VaV CrmB的CTD结合趋化因子(图7); (2)由CPV编码的CrmB和CrmD的CTD可以被交换并且仍提供 趋化因子结合活性(未示出);(3)与vTNFR CTD相关的三种不同 的蛋白质(EV E12、EV E184和CPV B21R)编码趋化因子结合活 性(表2);以及(4)CPV 35kDa vCKBP与VaV CrmB的CRD(1.4)的融 合在不影响TNF抑制活性的情况下为该蛋白提供了结合趋化因子 的能力(图10)。因此,这些病毒结构域可以被交换或者结合以产 生阻断多种细胞因子的活性的免疫调节蛋白。我们提出vTNFR的 CTD限定结合免疫系统涉及的免疫蛋白(如趋化因子)或其他蛋白 质的新型蛋白结构/结构域,并为vTNFR赋予结合除了TNF之外的 其他免疫调节蛋白的能力。
表1.由牛痘病毒和疱疹病毒编码的趋化因子结合蛋白 蛋白 病毒 结合特异性 参考文献 vCKBP1 vCKBP2 vCKBP3 vCKBP4 M-T7 35kDa, M-T1, vCC1 M3 gG 痘病毒:黏液瘤病毒 痘病毒:痘苗病毒、 牛痘病毒、缺肢畸形 病毒、黏液瘤病毒、 天花病毒、口疮病毒 γ疱疹病毒:小鼠γ 疱疹病毒68α疱疹 病毒:马疱疹病毒, 牛疱疹病毒 宽CC 趋化因子 宽 (Lalani et al.,1997) (Alcami et al., 1998,Graham et al., 1997,Seet et al., 2003,Smith et al., 1997, Smith & Alcami, 2000)(Parry et al., 2000,van Berkel et al.,2000)(Bryant et al.,2003)
表2.通过SPR获得的EV E12蛋白对于不同趋化因子的亲和力常 数(KD)。纯化的重组蛋白被低水平硫醇连接,并且利用BIAcoreX 以高流速注射不同浓度的所示细胞因子。每种情况下的KD利用 BIAevaluation软件来确定。 趋化因子 KD(nM) mCCL21 0.5 mCCL25 0.7 mCCL27 2 mCXCL11 1 mCXCL13 1.5 mCXCL14 1.5
表3:制备的为了表达vTNFR的重组质粒。涉及的插入物是利用所 示的寡核苷酸对通过PCR从病毒DNA进行扩增(寡核苷酸序列见表 4)。EV是菌株Hampstead,CPV是菌株Brighton Red。 pBacI 插入物 寡核苷酸 Ta RS 质粒 EV crmD 5’CrmD-7 55 BamHI pMS1 3’CrmD-9 XhoI EV crmD-CRD 1,2 5’CrmD-7 50 BamHI pMS42 3’CrmD-29 NotI EV crmD-CRD 1,2,3 5’CrmD-7 50 BamHI pMS46 3’PT-3 NotI EV crmD-CRD 1,2,3,4 5’CrmD-7 50 BamHI pMS48 3’PT-4 NotI EV crmD-CTD 5’PT-1 50 NotI pPT6 3’PT-2 XhoI EV crmD-CRD 1,2-CTD EV crmD Ct subcloned into NotI/XhoI of pMS42 pPT1 EV crmD-CRD 1,2,3-CTD EV crmD Ct subcloned into NotI/XhoI of pMS46 pPT2 CPV crmB-CTD 5’PT-5 50 NotI pPT5 3’PT-6 XhoI EV crmD-CRD 1,2,3,4-CPV crmB CTD CPV crmB Ct subcloned into NotI/XhoI of pMS48 pPT3 CPV crmB-CRD 1,2,3,4-EV crmD CTD 5’SF-1 50 EcoRI 3’PT-7 NotI pPT4 PMJ-601 插入物 寡核苷酸 Ta RS RpMJ 601 EV crmD 5’CrmD-7 50 BamHI pMS11 3’CrmD-15 KpnI EV crmD-CRD 1,2 5’CrmD-7 50 BamHI pMS21 3’CrmD-23 HindIII
表4:在杆状病毒和VV系统中用于表达EV CrmD和CPV CrmB 的寡核苷酸
寡核苷酸序列(5’>3’)
5’CrmD-7 CGCGTTTAAACGGATCCATGATGAAGATGACACCATCATA
3’CrmD-9 CGCCTCGAGATCTCTTTCACAATCATTTGGTGG
3’CrmD-15 CGCGGTACCTCAATCTCTTTCACAATCATTTGG
3’CrmD-22 CGCGGTACCTTAATCTATGCTGTTAAAGGACAGATCAC
3’CrmD-23 GCGAAGCTTTTACCATGGGTAGTATCCGGATGCACAGACAC
3’CrmD-24 GCGAAGCTTTTACCATGGACAAGAGGTCTTGTTAACAGGATAC
3’CrmD-29 GCGGCGGCCGCGTAGTATCCGGATGCACAGACAC
5’PT-1 GCGGCGGCCGCCAATTCGAGTATAGGAAGCAGCAGTAC
3’PT-2 GCGCTCGAGATCTCTTTCACAATCATTTGGTGG
3’PT-3 GCGGCGGCCGCATCTATGCTGTTAAAGGACAGATCAC
3’PT-4 GCGGCGGCCGCACAAGAGGTCTTGTTAACAGGATAC
5’PT-5 GCGGCGGCCGCCACTCGGACGACCACTACCGGTCTC
3’PT-6 GCGCTCGAGTAAAAAGTGGGTGGGATACTGGGAA
3’PT-7 GCGGCGGCCGCACACGATGTGTCGTTGACGGGATAC
5’SF-1 GCGGGTACCGAATTCACCATGGAGTCATATATATTGCTATTGC
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序列表序列表
<110>安东尼奥·阿尔卡米
<120>
病毒TNF受体和相关蛋白质的趋化因子结合活性<130>1
<160>28
<170>PatentIn version 3.3
<210>1
<211>963
<212>DNA
<213>牛痘病毒菌株Brighton Red
<220>
<221>基因
<222>(1)..(963)
<223>基因V221
<400>1
atgatgaata tgacaccatc atacatcttg ttggtatata tgttcgtagt cgtaagtgga 60
gatgttcctt atgaacacat taatgggaaa tgtaacggta ccgactataa tagtaataat 120
ctatgttgta aacaatgcga tcctggaatg tatatgactc attcctgtaa taccacttct 180
aatacaaaat gtgacaagtg cccagatggc acctttacat ccattcctaa tcatattccc 240
acgtgtctaa gttgtcgagg caaatgtagc agtaatcatg tagagactaa atcgtgtagt 300
aacacacagg acagagtatg tgtctgtgca tccggatact actgcgaatt tgaaggatca 360
aacggttgca ggctatgtgt accacaaaca aagtgtgatt ctggttacgg tgtatatggc 420
tactcatcta aaggagatgt aatatgtaaa aagtgtccgg gtaatataga taaatgtgat 480
ctgtccttta acagcataga tgtagaaatt aatatgtatc ctgttaacaa gacctcttgt 540
aattcgagta taggaagtag cagtaccata tcaacttccg agttaacaat tactctaaaa 600
catgaggatt gtactactgt ctttattgga gattactatt cagtcgttga taaactagca 660
acttcaggtt tctttacaaa cgataaagta catcaagacc tcacaacgca gtgcaagatt 720
aatctagaaa tcaaatgtaa ttctggagga gaatctagac aactaacacc cacgacgaag 780
gtatacttta tgcctcattc agaaacggta actgtggtag gagactgtct ctctaatctc 840
gatgtctata tagtatatgc caatacggac gcgatatatt ccgacatgga tgtcgtcgct 900
tatcatacta gttatatact aaatgttgat catattccac caaatgattg tgaaagagat 960
tga 963
<210>2
<211>320
<212>PRT
<213>牛痘病毒菌株Brighton Red
<220>
<221>MISC_FEATURE
<222>(1)..(320)
<223>细胞因子应答修饰物D(CrmD)
<400>2
Met Met Asn Met Thr Pro Ser Tyr Ile Leu Leu Val Tyr Met Phe Val
1 5 10 15
Val Val Ser Gly Asp Val Pro Tyr Glu His Ile Asn Gly Lys Cys Asn
20 25 30
Gly Thr Asp Tyr Asn Ser Asn Asn Leu Cys Cys Lys Gln Cys Asp Pro
35 40 45
Gly Met Tyr Met Thr His Ser Cys Asn Thr Thr Ser Asn Thr Lys Cys
50 55 60
Asp Lys Cys Pro Asp Gly Thr Phe Thr Ser Ile Pro Asn His Ile Pro
65 70 75 80
Thr Cys Leu Ser Cys Arg Gly Lys Cys Ser Ser Asn His Val Glu Thr
85 90 95
Lys Ser Cys Ser Asn Thr Gln Asp Arg Val Cys Val Cys Ala Ser Gly
100 105 110
Tyr Tyr Cys Glu Phe Glu Gly Ser Asn Gly Cys Arg Leu Cys Val Pro
115 120 125
Gln Thr Lys Cys Asp Ser Gly Tyr Gly Val Tyr Gly Tyr Ser Ser Lys
130 135 140
Gly Asp Val Ile Cys Lys Lys Cys Pro Gly Asn Ile Asp Lys Cys Asp
145 150 155 160
Leu Ser Phe Asn Ser Ile Asp Val Glu Ile Asn Met Tyr Pro Val Asn
165 170 175
Lys Thr Ser Cys Asn Ser Ser Ile Gly Ser Ser Ser Thr Ile Ser Thr
180 185 190
Ser Glu Leu Thr Ile Thr Leu Lys His Glu Asp Cys Thr Thr Val Phe
195 200 205
Ile Gly Asp Tyr Tyr Ser Val Val Asp Lys Leu Ala Thr Ser Gly Phe
210 215 220
Phe Thr Asn Asp Lys Val His Gln Asp Leu Thr Thr Gln Cys Lys Ile
225 230 235 240
Asn Leu Glu Ile Lys Cys Asn Ser Gly Gly Glu Ser Arg Gln Leu Thr
245 250 255
Pro Thr Thr Lys Val Tyr Phe Met Pro His Ser Glu Thr Val Thr Val
260 265 270
Val Gly Asp Cys Leu Ser Asn Leu Asp Val Tyr Ile Val Tyr Ala Asn
275 280 285
Thr Asp Ala Ile Tyr Ser Asp Met Asp Val Val Ala Tyr His Thr Ser
290 295 300
Tyr Ile Leu Asn Val Asp His Ile Pro Pro Asn Asp Cys Glu Arg Asp
305 310 315 320
<210>3
<211>960
<212>DNA
<213>鼠痘病毒菌株Naval
<220>
<221>基因
<222>(1)..(960)
<223>基因E3
<400>3
atgatgaaga tgacaccatc atacatcttg ttggtatata tgttcgtagt cgtaagtgga 60
gatgttccgt atacacccat taatgggaaa tgtaacggta cagactataa cagtaataat 120
ctatgttgta aacaatgcaa tcctggaatg tatatgactc attcctgtaa taccacttct 180
aatacaaaat gtgacaagtg cccagatgac acctttacat ccattcctaa tcatagtccc 240
gcgtgtctaa gttgtcgagg caaatgtagc agtaatcaag tagagactaa atcgtgtagt 300
aacacacagg acagagtatg tgtctgtgca tccggatact actgcgaatt tgaaggatca 360
aacggttgca ggctatgtgt accacaaaca aagtgtggtt ctggttacgg tgtatatggc 420
tactcatcta aaggagatgt aatatgtaaa aagtgtccgg gtaatataga taaatgtgat 480
ctgtccttta acagcataga tgtagaaatt aatatgtatc ctgttaacaa gacctcttgt 540
aattcgagta taggaagcag cagtaccata tcaacttccg agttaacaat tactctaaca 600
catgaggatt gtactcctgt ctttattgga gattactatt cagtcgttga taaactagca 660
acttcaggtt tctttacaaa cgataaagta catcaagacc tcacaacgca gtgcaagatt 720
aatctagaaa tcaaatgtaa ttctggaaga gaatctagac aactaacacc cacgacgaag 780
gtatacctta tgcctcattc agaaacggta actgtggtag gagactgtct ctctaatctc 840
gatgtctata tagtatatgc caatacggac gcgatatatt ccgacatgga cgtcgtcgcg 900
tatcatacta gttatatact aaatgttgat catattccac caaatgattg tgaaagagat 960
<210>4
<211>320
<212>PRT
<213>鼠痘病毒菌株Naval
<220>
<221>MISC_FEATURE
<222>(1)..(320)
<223>细胞因子应答修饰物D(CrmD)
<400>4
Met Met Lys Met Thr Pro Ser Tyr Ile Leu Leu Val Tyr Met Phe Val
1 5 10 15
Val Val Ser Gly Asp Val Pro Tyr Thr Pro Ile Asn Gly Lys Cys Asn
20 25 30
Gly Thr Asp Tyr Asn Ser Asn Asn Leu Cys Cys Lys Gln Cys Asn Pro
35 40 45
Gly Met Tyr Met Thr His Ser Cys Asn Thr Thr Ser Asn Thr Lys Cys
50 55 60
Asp Lys Cys Pro Asp Asp Thr Phe Thr Ser Ile Pro Asn His Ser Pro
65 70 75 80
Ala Cys Leu Ser Cys Arg Gly Lys Cys Ser Ser Asn Gln Val Glu Thr
85 90 95
Lys Ser Cys Ser Asn Thr Gln Asp Arg Val Cys Val Cys Ala Ser Gly
100 105 110
Tyr Tyr Cys Glu Phe Glu Gly Ser Asn Gly Cys Arg Leu Cys Val Pro
115 120 125
Gln Thr Lys Cys Gly Ser Gly Tyr Gly Val Tyr Gly Tyr Ser Ser Lys
130 135 140
Gly Asp Val Ile Cys Lys Lys Cys Pro Gly Asn Ile Asp Lys Cys Asp
145 150 155 160
Leu Ser Phe Asn Ser Ile Asp Val Glu Ile Asn Met Tyr Pro Val Asn
165 170 175
Lys Thr Ser Cys Asn Ser Ser Ile Gly Ser Ser Ser Thr Ile Ser Thr
180 185 190
Ser Glu Leu Thr Ile Thr Leu Thr His Glu Asp Cys Thr Pro Val Phe
195 200 205
Ile Gly Asp Tyr Tyr Ser Val Val Asp Lys Leu Ala Thr Ser Gly Phe
210 215 220
Phe Thr Asn Asp Lys Val His Gln Asp Leu Thr Thr Gln Cys Lys Ile
225 230 235 240
Asn Leu Glu Ile Lys Cys Asn Ser Gly Arg Glu Ser Arg Gln Leu Thr
245 250 255
Pro Thr Thr Lys Val Tyr Leu Met Pro His Ser Glu Thr Val Thr Val
260 265 270
Val Gly Asp Cys Leu Ser Asn Leu Asp Val Tyr Ile Val Tyr Ala Asn
275 280 285
Thr Asp Ala Ile Tyr Ser Asp Met Asp Val Val Ala Tyr His Thr Ser
290 295 300
Tyr Ile Leu Asn Val Asp His Ile Pro Pro Asn Asp Cys Glu Arg Asp
305 310 315 320
<210>5
<211>609
<212>DNA
<213>鼠痘病毒菌株Naval
<220>
<221>基因
<222>(1)..(609)
<223>基因E12
<400>5
atgataaaca taaacataaa cacaatacta atattcgcat cattatttgt tgcatcgttt 60
gcaaatgatt atcctccacc cggtttcttc gaaaacaaat acattacaga tacatttaat 120
tacatatcta tagattttga actatatcca gttaacgtat catcttgtaa tcgactaagt 180
acaaaacaat catccgatat tatcacgact tctgaattaa caattactgt taatagtaca 240
gactgcgatc cagtctttgt aacagaatat tattctgtaa aggataaaac tgctgtagcc 300
ggacttttca cagatactac aaaaaaacaa aatacatcca agatgtgtac gctgaatgta 360
gaagtaaaat gtaacgctga aacggaacct gtattaatcg gtaattttac acgtgttcct 420
gaaacagcat caacccacgc tgaaaatttc actttaatag gcaactgtct atcagatctc 480
catctctata ttgcgtacgt caataccgat gagggatttg aagaggatac tgctactatt 540
catataggaa acatgatcga tattagcggt atacctccaa atacttgcgc tactagaact 600
attaattag 609
<210>6
<211>202
<212>PRT
<213>鼠痘病毒菌株Naval
<220>
<221>MISC_FEATURE
<222>(1)..(202)
<223>CTD-1
<400>6
Met Ile Asn Ile Asn Ile Asn Thr Ile Leu Ile Phe Ala Ser Leu Phe
1 5 10 15
Val Ala Ser Phe Ala Asn Asp Tyr Pro Pro Pro Gly Phe Phe Glu Asn
20 25 30
Lys Tyr Ile Thr Asp Thr Phe Asn Tyr Ile Ser Ile Asp Phe Glu Leu
35 40 45
Tyr Pro Val Asn Val Ser Ser Cys Asn Arg Leu Ser Thr Lys Gln Ser
50 55 60
Ser Asp Ile Ile Thr Thr Ser Glu Leu Thr Ile Thr Val Asn Ser Thr
65 70 75 80
Asp Cys Asp Pro Val Phe Val Thr Glu Tyr Tyr Ser Val Lys Asp Lys
85 90 95
Thr Ala Val Ala Gly Leu Phe Thr Asp Thr Thr Lys Lys Gln Asn Thr
100 105 110
Ser Lys Met Cys Thr Leu Asn Val Glu Val Lys Cys Asn Ala Glu Thr
115 120 125
Glu Pro Val Leu Ile Gly Asn Phe Thr Arg Val Pro Glu Thr Ala Ser
130 135 140
Thr His Ala Glu Asn Phe Thr Leu Ile Gly Asn Cys Leu Ser Asp Leu
145 150 155
160
His Leu Tyr Ile Ala Tyr Val Asn Thr Asp Glu Gly Phe Glu Glu Asp
165 170
175
Thr Ala Thr Ile His Ile Gly Asn Met Ile Asp Ile Ser Gly Ile Pro
180 185 190
Pro Asn Thr Cys Ala Thr Arg Thr Ile Asn
195 200
<210>7
<211>609
<212>DNA
<213>牛痘病毒菌株Brighton Red
<220>
<221>基因
<222>(1)..(609)
<223>基因V014
<400>7
atgataaaca taaacataaa cacaatacta atattcgcat cattatttgt tgcatcgttt 60
gcaaatgatt atcctccacc cggtttcttc gaagacaaat acattacaaa tacatttaac 120
tacatatcta tagattttga actatatcca gttaacgtat catcttgtaa tcgactaagt 180
acaaaacaat catcagatgt tatatcgact tctgaattga caattactgt taatagtaca 240
gattgtgatc cagtctttgt aacagaatat tactctgtaa aggataaaac tgctatagcc 300
ggacttttca cagatactac aaaaaaacaa aatacatcca agatgtgtac gctgaatata 360
gaagtaaaat gtaacgctga aacggaacct gtattaatcg gtaattttac acgcgttcct 420
gaaaaagcat caacacacgc tgaaaatttc actttaatag gcaactgtct atcagatctc 480
catctctata ttgcgtatgt caataccgat gaggaatttg aagaggatac tgctactgtt 540
catataggaa acaaactcga tattaacggt atacctccaa atatgtgcgc taccagaacc 600
attaattag 609
<210>8
<211>202
<212>PRT
<213>牛痘病毒菌株Brighton Red
<220>
<221>MISC_FEATURE
<222>(1)..(202)
<223>CTD-1
<400>8
Met Ile Asn Ile Asn Ile Asn Thr Ile Leu Ile Phe Ala Ser Leu Phe
1 5 10 15
Val Ala Ser Phe Ala Asn Asp Tyr Pro Pro Pro Gly Phe Phe Glu Asp
20 25 30
Lys Tyr Ile Thr Asn Thr Phe Asn Tyr Ile Ser Ile Asp Phe Glu Leu
35 40 45
Tyr Pro Val Asn Val Ser Ser Cys Asn Arg Leu Ser Thr Lys Gln Ser
50 55 60
Ser Asp Val Ile Ser Thr Ser Glu Leu Thr Ile Thr Val Asn Ser Thr
65 70 75
80
Asp Cys Asp Pro Val Phe Val Thr Glu Tyr Tyr Ser Val Lys Asp Lys
85 90 95
Thr Ala Ile Ala Gly Leu Phe Thr Asp Thr Thr Lys Lys Gln Asn Thr
100 105 110
Ser Lys Met Cys Thr Leu Asn Ile Glu Val Lys Cys Asn Ala Glu Thr
115 120 125
Glu Pro Val Leu Ile Gly Asn Phe Thr Arg Val Pro Glu Lys Ala Ser
130 135 140
Thr His Ala Glu Asn Phe Thr Leu Ile Gly Asn Cys Leu Ser Asp Leu
145 150 155 160
His Leu Tyr Ile Ala Tyr Val Asn Thr Asp Glu Glu Phe Glu Glu Asp
165 170 175
Thr Ala Thr Val His Ile Gly Asn Lys Leu Asp Ile Asn Gly Ile Pro
180 185 190
Pro Asn Met Cys Ala Thr Arg Thr Ile Asn
195 200
<210>9
<211>1047
<212>DNA
<213>天花主要病毒菌株Bangladesh
<220>
<221>基因
<222>(1)..(1047)
<223>基因G2R
<400>9
atgaagtccg tattatactt gtatatattg tttctctcat gtataataaa cggaagagat 60
gcagcaccgt atacaccacc caatggaaag tgtaaagaca ccgaatacaa acgccataat 120
ctgtgttgtt tatcgtgtcc tccgggaaca tacgcttcca gattatgtga tagcaagact 180
aacacacaat gtacaccgtg tggttcgggt acctttacat ctcgcaataa tcatttaccc 240
gcttgtctaa gttgtaacgg aagatgcaat agtaatcagg tagagacgcg atcgtgtaac 300
acgactcaca atagaatctg tgaatgctct cccggatatt attgtcttct taaaggatca 360
tccggatgca aggcatgtgt ttcccaaaca aaatgtggaa taggatacgg agtatccgga 420
cacacgtctg ttggagacgt catctgttct ccgtgtggtt tcggaacata ttctcacacc 480
gtctcttccg cagataaatg cgaacccgta cccaacaata catttaacta tatcgatgtg 540
gaaattacac tgtatccagt taacgacaca tcgtgtactc ggacgaccac taccggtctc 600
agcgaatcca tcttaacgtc ggaactaact attactatga atcatacaga ttgcaatccc 660
gtatttcgtg aggaatactt ctctgtcctt aataaggtag caacttcagg attttttaca 720
ggagaaaata gatatcaaaa tatttcaaag gtgtgtactt taaattttga gattaaatgt 780
aataacaaag gttcttcctt caaacagcta acgaaagcaa agaatgatga cggtatgatg 840
tcgcattcgg agacggtaac tctagcgggt gactgtctat ctagcgtcga catctatata 900
ctatatagta ataccaatgc tcaagactac gaaactgata caatctctta tcgtgtgggt 960
aatgttctcg atgatgatag ccatatgccc ggtagttgca atatacataa accgatcact 1020
aattccaaac ccacccgctt tttatag 1047
<210>10
<211>348
<212>PRT
<213>天花主要病毒菌株Bangladesh
<220>
<221>MISC_FEATURE
<222>(1)..(348)
<223>细胞因子应答修饰物B(CrmB)
<400>10
Met Lys Ser Val Leu Tyr Leu Tyr Ile Leu Phe Leu Ser Cys Ile Ile
1 5 10 15
Asn Gly Arg Asp Ala Ala Pro Tyr Thr Pro Pro Asn Gly Lys Cys Lys
20 25 30
Asp Thr Glu Tyr Lys Arg His Asn Leu Cys Cys Leu Ser Cys Pro Pro
35 40 45
Gly Thr Tyr Ala Ser Arg Leu Cys Asp Ser Lys Thr Asn Thr Gln Cys
50 55 60
Thr Pro Cys Gly Ser Gly Thr Phe Thr Ser Arg Asn Asn His Leu Pro
65 70 75 80
Ala Cys Leu Ser Cys Asn Gly Arg Cys Asn Ser Asn Gln Val Glu Thr
85 90 95
Arg Ser Cys Asn Thr Thr His Asn Arg Ile Cys Glu Cys Ser Pro Gly
100 105 110
Tyr Tyr Cys Leu Leu Lys Gly Ser Ser Gly Cys Lys Ala Cys Val Ser
115 120 125
Gln Thr Lys Cys Gly Ile Gly Tyr Gly Val Ser Gly His Thr Ser Val
130 135 140
Gly Asp Val Ile Cys Ser Pro Cys Gly Phe Gly Thr Tyr Ser His Thr
145 150 155 160
Val Ser Ser Ala Asp Lys Cys Glu Pro Val Pro Asn Asn Thr Phe Asn
165 170 175
Tyr Ile Asp Val Glu Ile Thr Leu Tyr Pro Val Asn Asp Thr Ser Cys
180 185 190
Thr Arg Thr Thr Thr Thr Gly Leu Ser Glu Ser Ile Leu Thr Ser Glu
195 200 205
Leu Thr Ile Thr Met Asn His Thr Asp Cys Asn Pro Val Phe Arg Glu
210 215 220
Glu Tyr Phe Ser Val Leu Asn Lys Val Ala Thr Ser Gly Phe Phe Thr
225 230 235 240
Gly Glu Asn Arg Tyr Gln Asn Ile Ser Lys Val Cys Thr Leu Asn Phe
245 250 255
Glu Ile Lys Cys Asn Asn Lys Gly Ser Ser Phe Lys Gln Leu Thr Lys
260 265 270
Ala Lys Asn Asp Asp Gly Met Met Ser His Ser Glu Thr Val Thr Leu
275 280 285
Ala Gly Asp Cys Leu Ser Ser Val Asp Ile Tyr Ile Leu Tyr Ser Asn
290 295 300
Thr Asn Ala Gln Asp Tyr Glu Thr Asp Thr Ile Ser Tyr Arg Val Gly
305 310 315 320
Asn Val Leu Asp Asp Asp Ser His Met Pro Gly Ser Cys Asn Ile His
325 330 335
Lys Pro Ile Thr Asn Ser Lys Pro Thr Arg Phe Leu
340 345
<210>11
<211>1068
<212>DNA
<213>牛痘病毒菌株Brighton Red
<220>
<221>基因
<222>(1)..(1068)
<223>基因V005
<400>11
atgaagtcat atatattgct attgctgctt tcatgtataa tcataataaa cagcgatata 60
acaccgcatg aaccatccaa cggaaagtgt aaagacaacg aatacaaacg ccatcatcta 120
tgttgtttat cgtgtcctcc gggaacatac gcttccagat tatgcgatag caagactaac 180
acaaacacac aatgtacgcc gtgtgcgtcg gacaccttta cgtctcgcaa taatcattta 240
cccgcttgtc taagttgtaa cggaagatgc gatagtaatc aggtagagac gcgatcgtgt 300
aacacgactc acaatagaat ctgtgattgt gctcccggat attattgttt tctcaaagga 360
tcatccggat gcaaggcatg tgtttcccaa acaaagtgtg gaataggata cggagtatcc 420
ggacacacgc ctaccggaga cgtcgtctgt tctccgtgtg gtctcggaac atattctcac 480
accgtctctt ccgtagataa atgcgaaccc gtacccagta atacctttaa ctatatcgat 540
gtggaaatta atctgtatcc cgtcaacgac acatcgtgta ctcggacgac cactaccggt 600
ctcagtgaat ccatctcaac ttcggaacta acgattacta tgaatcataa agactgcgat 660
cccgtctttc gtaatggata cttctccgtt cttaatgagg tagcaacttc agggttcttt 720
acaggacaaa atagatatca gaatatttca aaggtatgca ctctgaattt cgagattaaa 780
tgtaataaca aagattctta ttcttcctcc aaacagttaa cgaaaacaaa gaatgatgac 840
gactccatca tgccgcattc ggaatcggta actctagtgg gcgactgtct atccagcgtc 900
gacatctata tactatatag taataccaat actcaagact acgaaactga tacaatctct 960
tatcatgtgg gtaatgttct cgatgtcgat agccatatgc ccggtaggtg cgatacacat 1020
aaactgatta ctaattccaa ttcccagtat cccacccact ttttatag 1068
<210>12
<211>355
<212>PRT
<213>牛痘病毒菌株Brighton Red
<220>
<221>MISC_FEATURE
<222>(1)..(355)
<223>细胞因子应答修饰物B(CrmB)
<400>12
Met Lys Ser Tyr Ile Leu Leu Leu Leu Leu Ser Cys Ile Ile Ile Ile
1 5 10 15
Asn Ser Asp Ile Thr Pro His Glu Pro Ser Asn Gly Lys Cys Lys Asp
20 25 30
Asn Glu Tyr Lys Arg His His Leu Cys Cys Leu Ser Cys Pro Pro Gly
35 40 45
Thr Tyr Ala Ser Arg Leu Cys Asp Ser Lys Thr Asn Thr Asn Thr Gln
50 55 60
Cys Thr Pro Cys Ala Ser Asp Thr Phe Thr Ser Arg Asn Asn His Leu
65 70 75 80
Pro Ala Cys Leu Ser Cys Asn Gly Arg Cys Asp Ser Asn Gln Val Glu
85 90 95
Thr Arg Ser Cys Asn Thr Thr His Asn Arg Ile Cys Asp Cys Ala Pro
100 105 110
Gly Tyr Tyr Cys Phe Leu Lys Gly Ser Ser Gly Cys Lys Ala Cys Val
115 120 125
Ser Gln Thr Lys Cys Gly Ile Gly Tyr Gly Val Ser Gly His Thr Pro
130 135 140
Thr Gly Asp Val Val Cys Ser Pro Cys Gly Leu Gly Thr Tyr Ser His
145 150 155 160
Thr Val Ser Ser Val Asp Lys Cys Glu Pro Val Pro Ser Asn Thr Phe
165 170 175
Asn Tyr Ile Asp Val Glu Ile Asn Leu Tyr Pro Val Asn Asp Thr Ser
180 185 190
Cys Thr Arg Thr Thr Thr Thr Gly Leu Ser Glu Ser Ile Ser Thr Ser
195 200 205
Glu Leu Thr Ile Thr Met Asn His Lys Asp Cys Asp Pro Val Phe Arg
210 215 220
Asn Gly Tyr Phe Ser Val Leu Asn Glu Val Ala Thr Ser Gly Phe Phe
225 230 235 240
Thr Gly Gln Asn Arg Tyr Gln Asn Ile Ser Lys Val Cys Thr Leu Asn
245 250 255
Phe Glu Ile Lys Cys Asn Asn Lys Asp Ser Tyr Ser Ser Ser Lys Gln
260 265 270
Leu Thr Lys Thr Lys Asn Asp Asp Asp Ser Ile Met Pro His Ser Glu
275 280 285
Ser Val Thr Leu Val Gly Asp Cys Leu Ser Ser Val Asp Ile Tyr Ile
290 295 300
Leu Tyr Ser Asn Thr Asn Thr Gln Asp Tyr Glu Thr Asp Thr Ile Ser
305 310 315 320
Tyr His Val Gly Asn Val Leu Asp Val Asp Ser His Met Pro Gly Arg
325 330 335
Cys Asp Thr His Lys Leu Ile Thr Asn Ser Asn Ser Gln Tyr Pro Thr
340 345 350
His Phe Leu
355
<210>13
<211>582
<212>DNA
<213>牛痘病毒菌株Brighton Red
<220>
<221>基因
<222>(1)..(582)
<223>基因V218
<400>13
atgatgatat acggattaat agcctgtctt atattcgtga cttcatccac cgctagtccc 60
ctttacattc ccgttattcc acccattacg gaagataaat cgtttaatag tgtagaggta 120
ttagtttcct tgtttagaga tgagcaaaaa gactatactg taacttcgca gttcaataac 180
tacactatcg ataccaaaga ctggactatc aacgtactat ccacacctga tggtctggag 240
ataccattga ccaatataac ttattggtca cggtttccaa ctataggtca tgcattgttc 300
aaatcagagt ccgaggatat cttccaaaag aacatgagta ttctaggtgt ctctattgaa 360
tgtaagaagc catcgacatc atttactttt ttgaccgtgc gtaaaatatc tcgagtattt 420
aatagatttc cagatatggc ttactatcga ggagactgtc tagaagtcgt ttatgtaaca 480
atgacttata aaaatactaa aactggagag actgattaca catacctctc taatgtgggg 540
attcctgaat actatcggtt gatgagtggt gtcgatggtt ga 582
<210>14
<211>193
<212>PRT
<213>牛痘病毒菌株Brighton Red
<220>
<221>MISC_FEATURE
<222>(1)..(193)
<223>CTD-3
<400>14
Met Met Ile Tyr Gly Leu Ile Ala Cys Leu Ile Phe Val Thr Ser Ser
1 5 10 15
Thr Ala Ser Pro Leu Tyr Ile Pro Val Ile Pro Pro Ile Thr Glu Asp
20 25 30
Lys Ser Phe Asn Ser Val Glu Val Leu Val Ser Leu Phe Arg Asp Glu
35 40 45
Gln Lys Asp Tyr Thr Val Thr Ser Gln Phe Asn Asn Tyr Thr Ile Asp
50 55 60
Thr Lys Asp Trp Thr Ile Asn Val Leu Ser Thr Pro Asp Gly Leu Glu
65 70 75 80
Ile Pro Leu Thr Asn Ile Thr Tyr Trp Ser Arg Phe Pro Thr Ile Gly
85 90 95
His Ala Leu Phe Lys Ser Glu Ser Glu Asp Ile Phe Gln Lys Asn Met
100 105 110
Ser Ile Leu Gly Val Ser Ile Glu Cys Lys Lys Pro Ser Thr Ser Phe
115 120 125
Thr Phe Leu Thr Val Arg Lys Ile Ser Arg Val Phe Asn Arg Phe Pro
130 135 140
Asp Met Ala Tyr Tyr Arg Gly Asp Cys Leu Glu Val Val Tyr Val Thr
145 150 155 160
Met Thr Tyr Lys Asn Thr Lys Thr Gly Glu Thr Asp Tyr Thr Tyr Leu
165 170 175
Ser Asn Val Gly Ile Pro Glu Tyr Tyr Arg Leu Met Ser Gly Val Asp
180 185 190
Gly
<210>15
<211>573
<212>DNA
<213>牛痘病毒菌株Western Reserve
<220>
<221>基因
<222>(1)..(573)
<223>基因VACWR206
<400>15
atgatgatat acggattaat agcgtgtctt atattcgtga cttcatccat cgctagtcca 60
ctttatattc ccgttattcc acccatttcg gaagataaat cgttcaatag tgtagaggta 120
ttagtttcct tgtttagaga tgaccaaaaa gactatacgg taacttctca gttcaataac 180
tacactatcg ataccaaaga ctggactatc ggcgtactat ccacacctga tggtttggat 240
ataccattga ctaatataac ttattggtca cggtttacta taggtcgtgc attgttcaaa 300
tcagagtctg aggatatttt ccaaaagaaa atgagtattc taggtgtttc tatagaatgt 360
aagaagtcgt cgacattact tacttttttg accgtgcgta aaatgactcg agtatttaat 420
aaatttccag atatggctta ttatcgagga gactgtttaa aagccgttta tgtaacaatg 480
acttataaaa atactaaaac tggagagact gattacacgt acctctctaa tggggggttg 540
cctgcatact atcgtaatgg ggtcgatggt tga 573
<210>16
<211>190
<212>PRT
<213>牛痘病毒菌株Western Reserve
<220>
<221>MISC_FEATURE
<222>(1)..(190)
<223>CTD-3
<400>16
Met Met Ile Tyr Gly Leu Ile Ala Cys Leu Ile Phe Val Thr Ser Ser
1 5 10 15
Ile Ala Ser Pro Leu Tyr Ile Pro Val Ile Pro Pro Ile Ser Glu Asp
20 25 30
Lys Ser Phe Asn Ser Val Glu Val Leu Val Ser Leu Phe Arg Asp Asp
35 40 45
Gln Lys Asp Tyr Thr Val Thr Ser Gln Phe Asn Asn Tyr Thr Ile Asp
50 55 60
Thr Lys Asp Trp Thr Ile Gly Val Leu Ser Thr Pro Asp Gly Leu Asp
65 70 75 80
Ile Pro Leu Thr Asn Ile Thr Tyr Trp Ser Arg Phe Thr Ile Gly Arg
85 90 95
Ala Leu Phe Lys Ser Glu Ser Glu Asp Ile Phe Gln Lys Lys Met Ser
100 105 110
Ile Leu Gly Val Ser Ile Glu Cys Lys Lys Ser Ser Thr Leu Leu Thr
115 120 125
Phe Leu Thr Val Arg Lys Met Thr Arg Val Phe Asn Lys Phe Pro Asp
130 135 140
Met Ala Tyr Tyr Arg Gly Asp Cys Leu Lys Ala Val Tyr Val Thr Met
145 150 155 160
Thr Tyr Lys Asn Thr Lys Thr Gly Glu Thr Asp Tyr Thr Tyr Leu Ser
165 170 175
Asn Gly Gly Leu Pro Ala Tyr Tyr Arg Asn Gly Val Asp Gly
180 185 190
<210>17
<211>549
<212>DNA
<213>牛痘病毒菌株Western Reserve
<220>
<221>基因
<222>(1)..(549)
<223>基因VACWR189
<400>17
atgtataaaa aactaataac gtttttattt gtaataggtg cattagcatc ctattcgaat 60
aatgagtaca ctccgtttaa taaactgagt gtaaaactct atatagatgg agtagataat 120
atagaaaatt catatactga tgataataat gaattggtgt taaattttaa agagtacaca 180
atttctatta ttacagagtc atgcgacgtc ggatttgatt ccatagatat agatgttata 240
aacgactata aaattattga tatgtatacc attgactcgt ctactattca acgcagaggt 300
cacacgtgta gaatatctac caaattatca tgccattatg ataagtaccc ttatattcac 360
aaatatgatg gtgatgagcg acaatattct attactgcag agggaaaatg ctataaagga 420
ataaaatatg aaataagtat gatcaacgat gatactctat tgagaaaaca tactcttaaa 480
attggatcta cttatatatt tgatcgtcat ggacatagta atacatatta ttcaaaatat 540
gatttttaa 549
<210>18
<211>182
<212>PRT
<213>牛痘病毒菌株Western Reserve
<220>
<221>MISC_FEATURE
<222>(1)..(182)
<223>CTD-2
<400>18
Met Tyr Lys Lys Leu Ile Thr Phe Leu Phe Val Ile Gly Ala Leu Ala
1 5 10 15
Ser Tyr Ser Asn Asn Glu Tyr Thr Pro Phe Asn Lys Leu Ser Val Lys
20 25 30
Leu Tyr Ile Asp Gly Val Asp Asn Ile Glu Asn Ser Tyr Thr Asp Asp
35 40 45
Asn Asn Glu Leu Val Leu Asn Phe Lys Glu Tyr Thr Ile Ser Ile Ile
50 55 60
Thr Glu Ser Cys Asp Val Gly Phe Asp Ser Ile Asp Ile Asp Val Ile
65 70 75 80
Asn Asp Tyr Lys Ile Ile Asp Met Tyr Thr Ile Asp Ser Ser Thr Ile
85 90 95
Gln Arg Arg Gly His Thr Cys Arg Ile Ser Thr Lys Leu Ser Cys His
100 105 110
Tyr Asp Lys Tyr Pro Tyr Ile His Lys Tyr Asp Gly Asp Glu Arg Gln
115 120 125
Tyr Ser Ile Thr Ala Glu Gly Lys Cys Tyr Lys Gly Ile Lys Tyr Glu
130 135 140
Ile Ser Met Ile Asn Asp Asp Thr Leu Leu Arg Lys His Thr Leu Lys
145 150 155 160
Ile Gly Ser Thr Tyr Ile Phe Asp Arg His Gly His Ser Asn Thr Tyr
165 170 175
Tyr Ser Lys Tyr Asp Phe
180
<210>19
<211>546
<212>DNA
<213>鼠痘病毒菌株Naval
<220>
<221>基因
<222>(1)..(546)
<223>基因E184
<400>19
atgtataaaa aactaataac gtttttattt gtaataggtg cagtagcatc ttattcgaat 60
aatgagtaca ctccgtttaa taaacttagt gtaaaactgt atatagatgg agtagataat 120
atagaaaatt catatactga taataatgaa ttggtgttaa attttaaaga gtacacaatt 180
tctattatta cagagtcatg cgacgtcgga tttgattcca tagatataga tgttataaac 240
gactataaaa ttcttgatat gtataccatt gactcgtcta ccattcaacg cagaggtcac 300
acatgcaaaa tatctaccaa attatcatgc cattatgata agcaccctta tattcacaaa 360
tatgagggtg atgagcgaca atattctatt actgcagagg gaaaatgcta taaaggaata 420
aaatatgaaa taagtatgat gcacgatgat acgctattga gaaaacatac tcttaaaatt 480
ggatctactt atatattcga tcgccatgga catagtaata catattattc aaaatatgat 540
ttttaa 546
<210>20
<211>181
<212>PRT
<213>鼠痘病毒菌株Naval
<220>
<221>MISC_FEATURE
<222>(1)..(181)
<223>CTD-2
<400>20
Met Tyr Lys Lys Leu Ile Thr Phe Leu Phe Val Ile Gly Ala Val Ala
1 5 10 15
Ser Tyr Ser Asn Asn Glu Tyr Thr Pro Phe Asn Lys Leu Ser Val Lys
20 25 30
Leu Tyr Ile Asp Gly Val Asp Asn Ile Glu Asn Ser Tyr Thr Asp Asn
35 40 45
Asn Glu Leu Val Leu Asn Phe Lys Glu Tyr Thr Ile Ser Ile Ile Thr
50 55 60
Glu Ser Cys Asp Val Gly Phe Asp Ser Ile Asp Ile Asp Val Ile Asn
65 70 75 80
Asp Tyr Lys Ile Leu Asp Met Tyr Thr Ile Asp Ser Ser Thr Ile Gln
85 90 95
Arg Arg Gly His Thr Cys Lys Ile Ser Thr Lys Leu Ser Cys His Tyr
100 105 110
Asp Lys His Pro Tyr Ile His Lys Tyr Glu Gly Asp Glu Arg Gln Tyr
115 120 125
Ser Ile Thr Ala Glu Gly Lys Cys Tyr Lys Gly Ile Lys Tyr Glu Ile
130 135 140
Ser Met Met His Asp Asp Thr Leu Leu Arg Lys His Thr Leu Lys Ile
145 150 155 160
Gly Ser Thr Tyr Ile Phe Asp Arg His Gly His Ser Asn Thr Tyr Tyr
165 170 175
Ser Lys Tyr Asp Phe
180
<210>21
<211>480
<212>DNA
<213>牛痘病毒菌株Brighton Red
<220>
<221>C区
<222>(1)..(480)
<223>来自细胞因子应答修饰物D(CmrD)的CTD
<400>21
tcctttaaca gcatagatgt agaaattaat atgtatcctg ttaacaagac ctcttgtaat 60
tcgagtatag gaagtagcag taccatatca acttccgagt taacaattac tctaaaacat 120
gaggattgta ctactgtctt tattggagat tactattcag tcgttgataa actagcaact 180
tcaggtttct ttacaaacga taaagtacat caagacctca caacgcagtg caagattaat 240
ctagaaatca aatgtaattc tggaggagaa tctagacaac taacacccac gacgaaggta 300
tactttatgc ctcattcaga aacggtaact gtggtaggag actgtctctc taatctcgat 360
gtctatatag tatatgccaa tacggacgcg atatattccg acatggatgt cgtcgcttat 420
catactagtt atatactaaa tgttgatcat attccaccaa atgattgtga aagagattga 480
<210>22
<211>159
<212>PRT
<213>牛痘病毒菌株Brighton Red
<220>
<221>结构域
<222>(1)..(159)
<223>来自细胞因子应答修饰物D(CrmD)的CTD
<400>22
Ser Phe Asn Ser Ile Asp Val Glu Ile Asn Met Tyr Pro Val Asn Lys
1 5 10 15
Thr Ser Cys Asn Ser Ser Ile Gly Ser Ser Ser Thr Ile Ser Thr Ser
20 25 30
Glu Leu Thr Ile Thr Leu Lys His Glu Asp Cys Thr Thr Val Phe Ile
35 40 45
Gly Asp Tyr Tyr Ser Val Val Asp Lys Leu Ala Thr Ser Gly Phe Phe
50 55 60
Thr Asn Asp Lys Val His Gln Asp Leu Thr Thr Gln Cys Lys Ile Asn
65 70 75 80
Leu Glu Ile Lys Cys Asn Ser Gly Gly Glu Ser Arg Gln Leu Thr Pro
85 90 95
Thr Thr Lys Val Tyr Phe Met Pro His Ser Glu Thr Val Thr Val Val
100 105 110
Gly Asp Cys Leu Ser Asn Leu Asp Val Tyr Ile Val Tyr Ala Asn Thr
115 120 125
Asp Ala Ile Tyr Ser Asp Met Asp Val Val Ala Tyr His Thr Ser Tyr
130 135 140
Ile Leu Asn Val Asp His Ile Pro Pro Asn Asp Cys Glu Arg Asp
145 150 155
<210>23
<211>477
<212>DNA
<213>鼠痘病毒菌株Naval
<220>
<221>C区
<222>(1)..(477)
<223>来自细胞因子应答修饰物D(CrmD)的CTD
<400>23
tcctttaaca gcatagatgt agaaattaat atgtatcctg ttaacaagac ctcttgtaat 60
tcgagtatag gaagcagcag taccatatca acttccgagt taacaattac tctaacacat 120
gaggattgta ctcctgtctt tattggagat tactattcag tcgttgataa actagcaact 180
tcaggtttct ttacaaacga taaagtacat caagacctca caacgcagtg caagattaat 240
ctagaaatca aatgtaattc tggaagagaa tctagacaac taacacccac gacgaaggta 300
taccttatgc ctcattcaga aacggtaact gtggtaggag actgtctctc taatctcgat 360
gtctatatag tatatgccaa tacggacgcg atatattccg acatggacgt cgtcgcgtat 420
catactagtt atatactaaa tgttgatcat attccaccaa atgattgtga aagagat 477
<210>24
<211>159
<212>PRT
<213>鼠痘病毒菌株Naval
<220>
<221>结构域
<222>(1)..(159)
<223>来自细胞因子应答修饰物D(CrmD)的CTD
<400>24
ser Phe Asn Ser Ile Asp Val Glu Ile Asn Met Tyr Pro Val Asn Lys
1 5 10 15
Thr Ser Cys Asn Ser Ser Ile Gly Ser Ser Ser Thr Ile Ser Thr Ser
20 25 30
Glu Leu Thr Ile Thr Leu Thr His Glu Asp Cys Thr Pro Val Phe Ile
35 40 45
Gly Asp Tyr Tyr Ser Val Val Asp Lys Leu Ala Thr Ser Gly Phe Phe
50 55 60
Thr Asn Asp Lys Val His Gln Asp Leu Thr Thr Gln Cys Lys Ile Asn
65 70 75 80
Leu Glu Ile Lys Cys Asn Ser Gly Arg Glu Ser Arg Gln Leu Thr Pro
85 90 95
Thr Thr Lys Val Tyr Leu Met Pro His Ser Glu Thr Val Thr Val Val
100 105 110
Gly Asp Cys Leu Ser Asn Leu Asp Val Tyr Ile Val Tyr Ala Asn Thr
115 120 125
Asp Ala Ile Tyr Ser Asp Met Asp Val Val Ala Tyr His Thr Ser Tyr
130 135 140
Ile Leu Asn Val Asp His Ile Pro Pro Asn Asp Cys Glu Arg Asp
145 150 155
<210>25
<211>546
<212>DNA
<213>牛痘病毒菌株Brighton Red
<220>
<221>C区
<222>(1)..(546)
<223>细胞因子应答修饰物B(CrmB)的CTD
<400>25
acctttaact atatcgatgt ggaaattaat ctgtatcccg tcaacgacac atcgtgtact 60
cggacgacca ctaccggtct cagtgaatcc atctcaactt cggaactaac gattactatg 120
aatcataaag actgcgatcc cgtctttcgt aatggatact tctccgttct taatgaggta 180
gcaacttcag ggttctttac aggacaaaat agatatcaga atatttcaaa ggtatgcact 240
ctgaatttcg agattaaatg taataacaaa gattcttatt cttcctccaa acagttaacg 300
aaaacaaaga atgatgacga ctccatcatg ccgcattcgg aatcggtaac tctagtgggc 360
gactgtctat ccagcgtcga catctatata ctatatagta ataccaatac tcaagactac 420
gaaactgata caatctctta tcatgtgggt aatgttctcg atgtcgatag ccatatgccc 480
ggtaggtgcg atacacataa actgattact aattccaatt cccagtatcc cacccacttt 540
ttatag 546
<210>26
<211>181
<212>PRT
<213>牛痘病毒菌株Brighton Red
<220>
<221>结构域
<222>(1)..(181)
<223>细胞因子应答修饰物B(CrmB)的CTD
<400>26
Thr Phe Asn Tyr Ile Asp Val Glu Ile Asn Leu Tyr Pro Val Asn Asp
1 5 10 15
Thr Ser Cys Thr Arg Thr Thr Thr Thr Gly Leu Ser Glu Ser Ile Ser
20 25 30
Thr Ser Glu Leu Thr Ile Thr Met Asn His Lys Asp Cys Asp Pro Val
35 40 45
Phe Arg Asn Gly Tyr Phe Ser Val Leu Asn Glu Val Ala Thr Ser Gly
50 55 60
Phe Phe Thr Gly Gln Asn Arg Tyr Gln Asn Ile Ser Lys Val Cys Thr
65 70 75 80
Leu Asn Phe Glu Ile Lys Cys Asn Asn Lys Asp Ser Tyr Ser Ser Ser
85 90 95
Lys Gln Leu Thr Lys Thr Lys Asn Asp Asp Asp Ser Ile Met Pro His
100 105 110
Ser Glu Ser Val Thr Leu Val Gly Asp Cys Leu Ser Ser Val Asp Ile
115 120 125
Tyr Ile Leu Tyr Ser Asn Thr Asn Thr Gln Asp Tyr Glu Thr Asp Thr
130 135 140
Ile Ser Tyr His Val Gly Asn Val Leu Asp Val Asp Ser His Met Pro
145 150 155 160
Gly Arg Cys Asp Thr His Lys Leu Ile Thr Asn Ser Asn Ser Gln Tyr
165 170 175
Pro Thr His Phe Leu
180
<210>27
<211>528
<212>DNA
<213>天花主要病毒菌株Bangladesh
<220>
<221>C区
<222>(1)..(528)
<223>细胞因子应答修饰物B(CrmB)的CTD
<400>27
acatttaact atatcgatgt ggaaattaca ctgtatccag ttaacgacac atcgtgtact 60
cggacgacca ctaccggtct cagcgaatcc atcttaacgt cggaactaac tattactatg 120
aatcatacag attgcaatcc cgtatttcgt gaggaatact tctctgtcct taataaggta 180
gcaacttcag gattttttac aggagaaaat agatatcaaa atatttcaaa ggtgtgtact 240
ttaaattttg agattaaatg taataacaaa ggttcttcct tcaaacagct aacgaaagca 300
aagaatgatg acggtatgat gtcgcattcg gagacggtaa ctctagcggg tgactgtcta 360
tctagcgtcg acatctatat actatatagt aataccaatg ctcaagacta cgaaactgat 420
acaatctctt atcgtgtggg taatgttctc gatgatgata gccatatgcc cggtagttgc 480
aatatacata aaccgatcac taattccaaa cccacccgct ttttatag 528
<210>28
<211>175
<212>PRT
<213>天花主要病毒菌株Bangladesh
<220>
<221>结构域
<222>(1)..(175)
<223>细胞因子应答修饰物B(CrmB)的CTD
<400>28
Thr Phe Asn Tyr Ile Asp Val Glu Ile Thr Leu Tyr Pro Val Asn Asp
1 5 10 15
Thr Ser Cys Thr Arg Thr Thr Thr Thr Gly Leu Ser Glu Ser Ile Leu
20 25 30
Thr Ser Glu Leu Thr Ile Thr Met Asn His Thr Asp Cys Asn Pro Val
35 40 45
Phe Arg Glu Glu Tyr Phe Ser Val Leu Asn Lys Val Ala Thr Ser Gly
50 55 60
Phe Phe Thr Gly Glu Asn Arg Tyr Gln Asn Ile Ser Lys Val Cys Thr
65 70 75 80
Leu Asn Phe Glu Ile Lys Cys Asn Asn Lys Gly Ser Ser Phe Lys Gln
85 90 95
Leu Thr Lys Ala Lys Asn Asp Asp Gly Met Met Ser His Ser Glu Thr
100 105 110
Val Thr Leu Ala Gly Asp Cys Leu Ser Ser Val Asp Ile Tyr Ile Leu
115 120 125
Tyr Ser Asn Thr Asn Ala Gln Asp Tyr Glu Thr Asp Thr Ile Ser Tyr
130 135 140
Arg Val Gly Asn Val Leu Asp Asp Asp Ser His Met Pro Gly Ser Cys
145 150 155 160
Asn Ile His Lys Pro Ile Thr Asn Ser Lys Pro Thr Arg Phe Leu
165 170 175