CN1292069C - Hiv－1病毒tat－蛋白突变体 - Google Patents
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Abstract
本发明涉及用于制备野生型HIV-1病毒Tat蛋白的解毒免疫原性突变体的蛋白质突变方法的用途。本发明还涉及制备野生型HIV-1病毒Tat蛋白的解毒免疫原性突变体的方法，包括第一步制备野生型Tat蛋白质突变体、第二步筛选没有跨细胞活性但是核定位被改变的解毒突变体、以及第三步筛选能诱导同时针对所述突变体和野生Tat蛋白的抗体的免疫原性突变体。步骤二和三可以调换。
Description
本发明的一个主题是HIV-1病毒Tat-蛋白突变体，以及包含至少一种所述突变体的药物组合物，特别是包含至少一种所述突变体的疫苗。
HIV病毒是产生AIDS的病因物质。HIV属于人反转录病毒科(Retroviridae)慢病毒亚科。在两种HIV类型(HIV-1和HIV-2)中，HIV-1更具细胞病变能力且在全球尤其是西方国家中占主导地位。HIV-1感染伴随有被感染个体免疫系统早期功能障碍。
与其它反转录病毒一样，HIV-1具有编码病毒结构蛋白的基因。gag基因编码形成病毒粒子核心的蛋白质，包括p24抗原。pol基因编码负责反转录(反转录酶)及整合(整合酶)的酶。env基因编码外壳糖蛋白。然而HIV-1比其它反转录病毒更复杂，它包含其它六个涉及病毒基因表达调控的蛋白质的编码基因(tat、rev、nef、vif、vpr和vpu)。HIV-1基因组中还含有包括参与病毒基因表达的调控元件的5′和3′LTRs(长末端重复)。
在体内，Tat是HIV-1复制所必需的蛋白质。人们对Tat在转录中的功能已作了大量研究，现在非常清楚的是，Tat的主要作用之一是调控从5′LTR的转录。Tat是一种通过与其它细胞因子同时结合到TAR序列上来反式激活5′LTR的转录激活因子，从而导致病毒转录和延长的增加。LTR被Tat蛋白的反式激活对基因表达和病毒复制都是重要的。病毒启动子被Tat蛋白反式激活(17、18)使病毒信使RNAs得以大规模生产，这些信使RNA向细胞质的转移依赖于另一调控蛋白，Rev蛋白。Tat和Rev调控HIV-1的表达(7)。Tat蛋白由被HIV-1感染的细胞分泌。一旦达到细胞外，它就能被相邻的感染或未感染细胞内化(9、14)，从而能诱导对未感染T淋巴细胞激活状态的修饰。因此它直接涉及AIDS的发展以及可能涉及与AIDS有关的病理症状，例如卡波西肉瘤(Kaposi′s sarcoma)。
完整的Tat蛋白由101个氨基酸组成，残基1-72由第一个外显子编码，残基73-101由第二个外显子编码。Tat蛋白高度保守。在一些经传代培养得到的实验株系中存在一种不同于天然形式的具有86个氨基酸的截断形式。该截断形式是由在传代培养过程中向87位导入了一个终止密码子而产生的，但是在所研究的Tat蛋白中超过90％都保持101个氨基酸的构型。尽管氨基酸87-101不能大大促进离体(exvivo)增殖，但是它们在能复制的HIV-1天然分离物中的保守性却是它们生物学重要性的指征。具有101个氨基酸的HIV-1天然Tat蛋白由五个物理结构域组成，但是其作用分子机制还未得到完全阐明。简言之，这五个结构域描述于Jeang，K.T等人(18)的出版物。在该出版物中，结构域1对应于酸性氨基酸富集的氨基酸1-20，结构域2对应于半胱氨酸残基富集(7个半胱氨酸残基，其中6个极度保守)的氨基酸21-40，结构域3对应于氨基酸41-48且包含HIV-1、HIV-2和SIV共有的RKGLGI基元，结构域4对应于氨基酸49-72且包含一个碱性RKKRRQRRR基元，以及结构域5对应于氨基酸73-101且包含一个RGD基元。没有解释结构域1的作用。只显示出，该结构域中一个氨基酸的改变可以被良好耐受且不改变Tat蛋白的功能。所提出的一种假说是，结构域1可能涉及反式激活。改变结构域2的七个半胱氨酸中的六个抑制了Tat蛋白的功能。该结构域对于反式激活很重要。没有阐述结构域3的作用。结构域4赋予Tat结合TAR RNA的特性，而且它对于核定位以及Tat蛋白的跨细胞运输是重要的。结构域5也涉及Tat蛋白的跨细胞运输。
在后面的发明详述中，本发明人从ACH320.2A.2.1株的序列出发(NCBI accession no.U34604)，细化了Jeang，K.T等人的出版物(18)中给出的有关结构域的观点。因此，对于这一特定株系，本发明中的结构域1对应于氨基酸1-21(作用未阐明)，结构域2对应于氨基酸22-37(涉及反式激活)，结构域3对应于氨基酸38-48(作用未知)以及结构域5对应于氨基酸73-101(跨细胞运输)。在对应于氨基酸49-72的结构域4中，对结合TAR RNA、核定位以及Tat跨细胞运输来说重要的是肽49-57(18)。
全世界都在等待HIV-1疫苗的开发。在被HIV-1感染的病人中，只在未发展成AIDS的个体中检测到对Tat和Rev的免疫反应(26)。几项用Tat和/或Rev对SIV动物模型进行的接种研究显示出部分或完全预防感染保护(4-6，21)。然而，将这些方案直接应用于人类是不可能的。尤其已有显示，Tat在体外具有毒性效应(19、22)。这些毒性效应包括(i)对涉及凋亡的细胞信号失调(28、30)，(ii)免疫系统中部分基因表达的失调，例如编码白细胞介素-2的基因(29)，或编码I型主要组织相容性复合物(MHC)的基因(16)，和/或(iii)诱导血管形成(1、2、20)。因此Tat蛋白在用作疫苗抗原之前必须先解毒。一个小组选择通过化学灭活使Tat蛋白解毒(10)。然而，这样的灭活只能在以重组蛋白为抗原时实施。为了能够利用活或非活重组载体中核酸形式的Tat蛋白，只能设想采用遗传解毒作用。因此本发明人选择开发经定向诱变这一Tat蛋白解毒途径以使其用作疫苗蛋白亚单位和/或接种载体的一部分。
本发明涉及制备解毒免疫原性野生型Tat-蛋白突变体的蛋白质突变方法的用途。
野生型Tat蛋白“突变体”意指通过一个或多个氨基酸的取代或置换获得的突变体。
“解毒野生型Tat-蛋白突变体”意指不再具有以下毒性效应的Tat蛋白：
·当它由被感染细胞分泌时，由于具有通过与表面受体结合而诱导细胞信号传递、以及被未感染细胞内化并运输至靶细胞核的能力，因而对于未被HIV-1感染的细胞来说其具有外源形式的毒性；
·Tat蛋白以外源和内源形式定位于靶细胞核中并诱导对能够参与Tat蛋白反式激活特性或结构域5的细胞基因的表达调控。
“免疫原性野生型Tat-蛋白突变体”意指注射入模型动物后能够诱导抗体产生的突变体，这些抗体既能与Tat-蛋白突变体也能与野生型Tat蛋白反应。
本发明还涉及制备解毒免疫原性野生型Tat-蛋白突变体的方法，其特征在于它包括：
-制备野生型Tat-蛋白突变体的阶段，特别是通过使编码野生型Tat蛋白的核酸产生突变，
-筛选解毒突变体的阶段，该解毒突变体的特征在于无跨细胞活性且核定位发生改变、以及可选地无反式激活活性，和
-筛选免疫原性突变体的阶段，该免疫原性突变体的特征在于它们能诱导产生同时针对所述突变体和野生型Tat蛋白的抗体，
最后两个阶段的顺序可以调换。
无跨细胞活性也意味着无跨细胞运输，在已建立的细胞系中可通过LTR-报告基因结构中病毒启动子的未被激活进行检测，例如氯霉素乙酰基转移酶(CAT)的病毒启动子，氯霉素乙酰基转移酶的表达依赖于已建立的细胞系中的病毒启动子(LTR)(31)，并且在这之前将细胞与以外源方式产生的Tat-蛋白突变体接触，例如由不同于包含病毒LTR依赖性报告基因的细胞系的细胞系(32)产生的Tat-蛋白突变体。
核定位的改变可以定义为用编码野生型Tat-蛋白突变体的核酸转染的细胞其细胞质区室中Tat蛋白的存在，例如在转染后72小时内，可以在用编码Tat-蛋白突变体的核酸转染细胞系后通过这些基因产物的免疫标记技术用光学显微镜进行检测，也可以在转染后通过检测包含与自发荧光蛋白例如EGFP蛋白的编码基因融合的Tat突变体编码基因的核酸的翻译产物而进行检测(33，34)。
无反式激活活性对应于病毒启动子的未被激活，且用编码野生型Tat-蛋白突变体的核酸转染已建立的细胞系后可以通过报告基因的未表达来进行检测，报告基因例如是其在该细胞系中的表达依赖于病毒启动子(LTR)的氯霉素乙酰基转移酶(CAT)(31)。
本发明还涉及HIV-1病毒的解毒免疫原性Tat蛋白突变体，其特征在于它在野生型Tat蛋白的区域4和/或5中至少含有两个突变，且其中，当突变处于结构域4区域时它位于从氨基酸位置49至氨基酸位置57这部分中，且其中，当突变处于结构域5中时它或者位于RGD基元中或者在88-92区中，优选在位置89和/或92，突变是由一个氨基酸被另一氨基酸置换形成的突变。
一种根据本发明的有利突变体是如上述所定义的突变体，其特征在于它在区域4中至少含有一个突变。
本发明还涉及如上述所定义的突变体，其特征在于结构域4和/或5中的突变能带来以下特征中的至少一种：
-废除野生型Tat蛋白的跨细胞效应，
-改变野生型Tat蛋白的核定位。
本发明涉及如上述所定义的突变体，其特征在于它含有能够使野生型Tat蛋白反式激活活性丧失的附加突变。
因此可用作免疫接种抗原的Tat蛋白将满足下列大部分标准：
-废除Tat的跨细胞效应(结构域4和/或5)
-改变Tat的核定位(结构域4)
-丧失反式激活活性(结构域2)
-保持蛋白质的抗原性(至多4或5个突变，尽可能少的改变CTL表位)
根据一种有利实施方式，本发明涉及如上述所定义的突变体，其特征在于它在野生型Tat蛋白结构域4的N-末端区域中，特别是在从氨基酸位置49至氨基酸位置57这部分中，含有突变。
一种根据本发明的有利突变体是如上述所定义的突变体，其特征在于它在野生型Tat蛋白结构域4的N-末端区域从氨基酸位置49至氨基酸位置55这部分中含有突变。
一种根据本发明的有利突变体是如上述所定义的突变体，其特征在于它在野生型Tat蛋白结构域5中的下列至少一个区域内含有突变：
-RGD基元，
-区域88-92，优选在位置89和/或92。
一种根据本发明的有利突变体是如上述所定义的突变体，其特征在于它在野生型Tat蛋白结构域2中包含突变，特别是对任一半胱氨酸的置换，有利地是被丝氨酸置换。
本发明还涉及如上述所定义的突变体，其特征在于它含有以下至少一种突变：
-第27位的半胱氨酸被丝氨酸取代，
-第51位的赖氨酸被苏氨酸取代，
-第52位的精氨酸被亮氨酸取代，
-第55位的精氨酸被亮氨酸取代，
-第57位的精氨酸被亮氨酸取代，
-第79位的甘氨酸被丙氨酸取代，
-第89位的赖氨酸被亮氨酸取代，
-第92位的谷氨酸被谷氨酰胺取代。
本发明还涉及如上述所定义的突变体，其特征在于它选自具有下述两种突变的突变体，各突变用三联体表示：字母-数字-字母，其中的数字表示突变氨基酸的位置，数字前的字母对应突变所涉及的氨基酸，数字后的字母表示用于置换数字前氨基酸的氨基酸：
K51T-R52L (SEQ ID NO：2)
K51T-R55L (SEQ ID NO：3)
K51T-R57L (SEQ ID NO：4)
K51T-G79A (SEQ ID NO：5)
K51T-K89L (SEQ ID NO：6)
K51T-E92Q (SEQ ID NO：7)
R52L-R55L (SEQ ID NO：8)
R52L-R57L (SEQ ID NO：9)
R52L-G79A (SEQ ID NO：10)
R52L-K89L (SEQ ID NO：11)
R52L-E92Q (SEQ ID NO：12)
R55L-R57L (SEQ ID NO：13)
R55L-G79A (SEQ ID NO：14)
R55L-K89L (SEQ ID NO：15)
R55L-E92Q (SEQ ID NO：16)
R57L-G79A (SEQ ID NO：17)
R57L-K89L (SEQ ID NO：18)
R57L-E92Q (SEQ ID NO：19)
G79A-K89L (SEQ ID NO：20)
G79A-E92Q (SEQ ID NO：21)
K89L-E92Q (SEQ ID NO：22)
一种根据本发明的有利突变体是如上述所定义的突变体，其特征在于它选自下列突变体：
K51T-R55L (SEQ ID NO：3)
R52L-R55L (SEQ ID NO：8)
R52L-G79A (SEQ ID NO：10)
R55L-R57L (SEQ ID NO：13)
G79A-K89L (SEQ ID NO：20)
本发明还涉及如上述所定义的突变体，其特征在于它选自具有下述三种突变的突变体，各突变用三联体表示：字母-数字-字母，其中的数字表示突变氨基酸的位置，数字前的字母对应突变所涉及的氨基酸，数字后的字母表示用于置换数字前氨基酸的氨基酸：
C27S-K51T-R52L (SEQ ID NO：23)
C27S-K51T-R55L (SEQ ID NO：24)
C27S-K51T-R57L (SEQ ID NO：25)
C27S-K51T-G79A (SEQ ID NO：26)
C27S-K51T-K89L (SEQ ID NO：27)
C27S-K51T-E92Q (SEQ ID NO：28)
C27S-R52L-R55L (SEQ ID NO：29)
C27S-R52L-R57L (SEQ ID NO：30)
C27S-R52L-G79A (SEQ ID NO：31)
C27S-R52L-K89L (SEQ ID NO：32)
C27S-R52L-E92Q (SEQ ID NO：33)
C27S-R55L-R57L (SEQ ID NO：34)
C27S-R55L-G79A (SEQ ID NO：35)
C27S-R55L-K89L (SEQ ID NO：36)
C27S-R55L-E92Q (SEQ ID NO：37)
C27S-R57L-G79A (SEQ ID NO：38)
C27S-R57L-K89L (SEQ ID NO：39)
C27S-R57L-E92Q (SEQ ID NO：40)
C27S-G79A-K89L (SEQ ID NO：41)
C27S-G79A-E92Q (SEQ ID NO：42)
C27S-K89L-E92Q (SEQ ID NO：43)
本发明还涉及如上述所定义的突变体，其特征在于它选自下列突变体：
C27S-K51T-R55L (SEQ ID NO：24)
C27S-R52L-R55L (SEQ ID NO：29)
C27S-R52L-G79A (SEQ ID NO：31)
本发明还涉及如上述所定义的突变体，其特征在于它选自具有下述四种突变的突变体，各突变用三联体表示：字母-数字-字母，其中的数字表示突变氨基酸的位置，数字前的字母对应突变所涉及的氨基酸，数字后的字母表示用于置换数字前氨基酸的氨基酸：
C27S-K51T-R52L-G79A (SEQ ID NO：44)
C27S-K51T-R52L-K89L (SEQ ID NO：45)
C27S-K51T-R52L-E92Q (SEQ ID NO：46)
C27S-K51T-R55L-G79A (SEQ ID NO：47)
C27S-K51T-R55L-K89L (SEQ ID NO：48)
C27S-K51T-R55L-E92Q (SEQ ID NO：49)
C27S-K51T-R57L-G79A (SEQ ID NO：50)
C27S-K51T-R57L-K89L (SEQ ID NO：51)
C27S-K51T-R57L-E92Q (SEQ ID NO：52)
C27S-K51T-G79A-K89L (SEQ ID NO：53)
C27S-K51T-G79A-E92Q (SEQ ID NO：54)
C27S-K51T-K89L-E92Q (SEQ ID NO：55)
C27S-R52L-G79A-K89L (SEQ ID NO：56)
C27S-R52L-G79A-E92Q (SEQ ID NO：57)
C27S-R52L-K89L-E92Q (SEQ ID NO：58)
C27S-R52L-R55L-G79A (SEQ ID NO：59)
C27S-R52L-R55L-K89L (SEQ ID NO：60)
C27S-R52L-R55L-E92Q (SEQ ID NO：61)
C27S-R52L-R57L-G79A (SEQ ID NO：62)
C27S-R52L-R57L-K89L (SEQ ID NO：63)
C27S-R52L-R57L-E92Q (SEQ ID NO：64)
C27S-R55L-G79A-K89L (SEQ ID NO：65)
C27S-R55L-G79A-E92Q (SEQ ID NO：66)
C27S-R55L-K89L-E92Q (SEQ ID NO：67)
C27S-R55L-R57L-G79A (SEQ ID NO：68)
C27S-R55L-R57L-K89L (SEQ ID NO：69)
C27S-R55L-R57L-E92Q (SEQ ID NO：70)
C27S-R57L-G79A-K89L (SEQ ID NO：71)
C27S-R57L-G79A-E92Q (SEQ ID NO：72)
C27S-R57L-K89L-E92Q (SEQ ID NO：73)
C27S-G79A-K89L-E92Q (SEQ ID NO：74)
一种根据本发明的有利突变体是如上述所定义的突变体，其特征在于它选自下列突变体：
C27S-K51T-R55L-G79A (SEQ ID NO：47)
C27S-K51T-R55L-K89L (SEQ ID NO：48)
C27S-K51T-R55L-E92Q (SEQ ID NO：49)
C27S-R52L-R55L-G79A (SEQ ID NO：59)
本发明还涉及如上述所定义的突变体，其特征在于它选自具有下述五种突变的突变体，各突变用三联体表示：字母-数字-字母，其中的数字表示突变氨基酸的位置，数字前的字母对应突变所涉及的氨基酸，数字后的字母表示用于置换数字前氨基酸的氨基酸：
C27S-K51T-G79A-K89L-E92Q (SEQ ID NO：75)
C27S-K51T-R52L-R55L-G79A (SEQ ID NO：76)
C27S-K51T-R52L-R55L-K89L (SEQ ID NO：77)
C27S-K51T-R52L-R55L-E92Q (SEQ ID NO：78)
C27S-K51T-R52L-R57L-G79A (SEQ ID NO：79)
C27S-K51T-R52L-R57L-K89L (SEQ ID NO：80)
C27S-K51T-R52L-R57L-E92Q (SEQ ID NO：81)
C27S-K51T-R52L-G79A-K89L (SEQ ID NO：82)
C27S-K51T-R52L-G79A-E92Q (SEQ ID NO：83)
C27S-K51T-R52L-K89L-E92Q (SEQ ID NO：84)
C27S-K51T-R55L-R57L-G79A (SEQ ID NO：85)
C27S-K51T-R55L-R57L-K89L (SEQ ID NO：86)
C27S-K51T-R55L-R57L-E92Q (SEQ ID NO：87)
C27S-K51T-R55L-G79A-K89L (SEQ ID NO：88)
C27S-K51T-R55L-G79A-E92Q (SEQ ID NO：89)
C27S-K51T-R55L-K89L-E92Q (SEQ ID NO：90)
C27S-K51T-R57L-G79A-K89L (SEQ ID NO：91)
C27S-K51T-R57L-G79A-E92Q (SEQ ID NO：92)
C27S-K51T-R57L-K89L-E92Q (SEQ ID NO：93)
C27S-R52L-R55L-R57L-G79A (SEQ ID NO：94)
C27S-R52L-R55L-R57L-K89L (SEQ ID NO：95)
C27S-R52L-R55L-R57L-E92Q (SEQ ID NO：96)
C27S-R52L-R55L-G79A-K89L (SEQ ID NO：97)
C27S-R52L-R55L-G79A-E92Q (SEQ ID NO：98)
C27S-R52L-R55L-K89L-E92Q (SEQ ID NO：99)
C27S-R52L-R57L-G79A-K89L (SEQ ID NO：100)
C27S-R52L-R57L-G79A-E92Q (SEQ ID NO：101)
C27S-R52L-R57L-K89L-E92Q (SEQ ID NO：102)
C27S-R52L-G79A-K89L-E92Q (SEQ ID NO：103)
C27S-R55L-R57L-G79A-K89L (SEQ ID NO：104)
C27S-R55L-R57L-G79A-E92Q (SEQ ID NO：105)
C27S-R55L-R57L-K89L-E92Q (SEQ ID NO：106)
C27S-R55L-G79A-K89L-E92Q (SEQ ID NO：107)
C27S-R57L-G79A-K89L-E92Q (SEQ ID NO：108)
一种根据本发明的有利突变体是如上述所定义的突变体，其特征在于它选自下列突变体：
C27S-K51T-R55L-G79A-K89L (SEQ ID NO：88)
C27S-K51T-R55L-G79A-E92Q (SEQ ID NO：89)
本发明还涉及编码如上述所定义突变体之一的核苷酸序列。
本发明还涉及用本发明的核苷酸序列转染的细胞系。
本发明还涉及针对如上述所定义突变体的抗体，其不识别野生型蛋白质结构域D1。
这样的抗体通过，例如在Elisa检测中检测并除去与对应于Tat结构域D1的肽有亲和力的抗体而进行选择，其中该肽至少包含序列EPVDPKLEPWKHPGS(残基2-16)。
根据本发明的抗体识别或不识别野生型蛋白质。
一种根据本发明的有利抗体类别包括识别野生型蛋白质的如上述所定义的抗体。
根据本发明的抗体是多克隆或单克隆抗体。
上述多克隆抗体通过用至少一种根据本发明的突变体免疫动物获得，之后通过取所述动物的血清样品、从血清中的其它成分中分离出所述抗体，特别是通过过柱亲和层析，回收纯化形式的抗体，其中柱中固定有被抗体特异识别的抗原，特别是根据本发明的突变体。
根据本发明的单克隆抗体可以通过杂交瘤技术获得，下文重述了其原理。
第一阶段中，用根据本发明的突变体免疫接种动物，通常为小鼠(或在免疫接种框架中体外培养的细胞)，之后其B淋巴细胞就能够产生针对所述突变体的抗体。之后将这些产抗体淋巴细胞与“永生”骨髓瘤细胞(实施例中为鼠科)融合以产生杂交瘤。之后从如此获得的异源细胞混合物中选择能够产生特定抗体并无限增殖的细胞。各杂交瘤以克隆形式增殖，各自导致单克隆抗体的产生，这些单克隆抗体对本发明突变体的识别特性可以用例如ELISA、一或二维的免疫转移、免疫荧光、或用biocaptor进行检测。随后纯化如此选择的单克隆抗体，特别是根据上述亲和层析技术。
本发明还涉及药物组合物，特别是疫苗，它包含作为活性成分的至少一种如上述所定义的突变体或至少一种如上述所定义的核苷酸序列或至少一种如上述所定义抗体以及药学适宜载体，其中核酸位于如上述所定义突变体之一的组成型表达所必需的元件控制之下。
当然，本领域技术人员易于决定用作药物组合物功能组分的突变体的量。
本发明还涉及含有至少一种如上述所定义突变体、或至少一种如上述所定义抗体的检测和/或定量HIV-1病毒的诊断组合物。
当然，本领域技术人员易于决定用作诊断技术所用功能的突变体的量。
本发明还涉及检测和定量取自能被HIV-1感染的个体的生物样品中HIV-1病毒的方法，例如血浆、血清或组织，其特征在于它包括下列阶段：
-在预定条件下将所述生物样品与含有如上述所定义突变体或如上述所定义抗体的诊断组合物接触，如果需要的话该预定条件允许上述定义的突变体与针对野生型Tat蛋白的抗体之间、或上述定义的抗体与野生型Tat蛋白之间形成抗体/抗原复合物，和
-用任何适当的方法检测和/或定量所述复合物的形成。
检测和/或定量病毒的方法用本领域技术人员熟知的标准技术完成，例如印迹法，所谓的三明治技术和竞争技术。
本发明还涉及至少一种如上述所定义的突变体或至少一种如上述所定义的抗体的用途，用于体外诊断生物样本或样品中HIV-1病毒。
本发明还涉及至少一种如上述所定义的突变体或至少一种如上述所定义的抗体的用途，用于制备疫苗组合物。
发明人因此指出，为上述用途必须在Tat蛋白结构域4产生至少一个突变和/或在Tat蛋白结构域5产生至少一个突变。他们通过定向诱变得到了Tat-蛋白突变体，之后根据这些突变体的特性进行选择。保留的突变体选自具有以下至少一个突变的突变体：K51T(结构域4中第51位的赖氨酸被苏氨酸取代)，R52L(结构域4中第52位的精氨酸被亮氨酸取代)，R55L(结构域4中第55位的精氨酸被亮氨酸取代)，R57L(结构域4中第57位的精氨酸被亮氨酸取代)，G79A(结构域5中第79位的甘氨酸被丙氨酸取代)，K89L(结构域5中第89位的赖氨酸被亮氨酸取代)和E92Q(结构域5中第92位的谷氨酸被谷氨酰胺取代)。上述及后述所有氨基酸位置根据ACH320.2A.2.1株的101个氨基酸完整序列给出。本发明的一个主题是上述突变体。但是本发明还涉及在Tat蛋白结构域4中具有两个突变的突变体。这些“双重”突变体选自突变体K51T-R55L、R52L-R55L、R52L-G79A、R55L-R57L和G79A-K89L。发明人之后显示，通过将结构域4中的这些双重突变与结构域2中的附加突变C27S(用丝氨酸取代半胱氨酸)组合，他们获得了令人非常满意的结果。因此，本发明还包括选自“三重”突变体C27S-K51T-R55L、C27S-R52L-R55L和C27S-R52L-G79A的突变体。优选所选突变体为突变体C27S-K51T-R55L。最后，他们证实组合有结构域2中至少一个突变、结构域4中至少两个突变以及结构域5中至少一个突变的“四重”突变体对获得非毒性Tat蛋白具有极端良好的表现。该“四重”突变体选自突变体C27S-K51T-R55L-G79A，C27S-K51T-R55L-K89L，C27S-K51T-R55L-E92Q和C27S-R52L-R55L-G79A。优选所选突变体为突变体C27S-K51T-R55L-G79A。
附图说明
图1是PCR定点诱变技术的图形表示。
图2示出毒株ACH320.2A.2.1 Tat蛋白的蛋白质序列(SEQ IDNO：1)与本发明突变Tat蛋白的蛋白质序列的比较。
图3a和3b是表示ACH320.2A.2.1或其突变体反式激活能力的图表。各构建体的结果是两个独立试验的平均值。图3a中，NT(未转染)柱表示LTR-CAT构建体的基线活性。
y-轴表示反式激活的倍增因子。
图4a和4b表示ACH320.2A.2.1构建体或其突变体的细胞内定位。
图5表示ACH320.2A.2.1构建体或其突变体的转导能力。所示数值为两个独立试验的平均值。测定pEGFP的反式激活百分数，通过计算该百分数的平均值+3SD(标准差)来确定分隔线。
白柱对应293T/HL3T1共培养，黑柱对应HL3T1细胞转染。
y-轴对应相对于野生型ACH320.2A.2.1的反式激活百分数。
图6表示另一种筛选表达本发明突变体的细胞系的方法(描述于实施例5)。
表1代表用于Tat PCR定向诱变的寡核苷酸。
实施例1：构建编码突变Tat蛋白的突变DNA。
用商业试剂盒(Clontech)和表1中所述的核苷酸引物使一段含有306个碱基对的cDNA片段产生突变，该cDNA片段对应于分离的HIV-1 ACH320.2A.2.1野生型Tat基因的两个外显子。PCR定点诱变的原理描述于图1。
如图1所示，从野生型Tat基因的cDNA出发，用一个位于末端(E5′或E3′)的末端引物和一个位于基因内部且带有所需突变的引物(分别为M3′和M5′)进行两次独立的PCR(第一个PCR循环)。然后以等摩尔混合两个PCR产物，并用5′端包含EcoRI限制位点和3′端包含SaII限制位点的末端引物进行第二个PCR循环。由此获得了在所需位点产生突变的cDNA。
该操作原理用于所有突变体，除了K89L和E92Q。对于后者，所要突变的核苷酸几乎位于cDNA的一个末端，这样可以直接进行半巢式PCR定向诱变，在第一个PCR循环中分别用以下引物对：E5′/K89L(M3′)和E5′/E92Q(M5′)。
用下列引物进行包含两个突变的单次诱变以产生双重突变体R52L-R55L：
R52L-R55L(M5′)
5′-GGCAGGAAGCTTAGACAGCTGCGAAGATC-3′
R52L-R55L(M3′)
5′-GATCTTCGCAGCTGTCTAAGCTTCTTCCTGCC-3′
以突变体G79A的cDNA为模板(matrix)、R52L(M5′)/R52L(M3′)为引物对进行PCR定向诱变得到双重突变体R52L-G79A。
以突变体G79A的cDNA为模板、E5′/K89L(M3′)为第一个PCR循环的引物对进行半巢式PCR产生双重突变体G79A-K89L。
以双重突变体K51T-R55L的cDNA为模板、C27S(M5′)/C27S(M3′)为引物对进行PCR定向诱变得到三重突变体C27S-K51T-R55L(STL)。
以突变体R52L-G79A的cDNA为模板、C27S(M5′)/C27S(M3′)为引物对进行PCR定向诱变得到三重突变体C27S-R52L-G79A。
用STL的cDNA作为模板以及引物G79A(M5′)/G79A(M3′)进行诱变产生四重突变体C27S-K51T-R55L-G79A(STLA)。
所有的第一个PCR循环都用0.5μg质粒和0.29ng/ml各自引物在下列条件下进行：1×94℃5′1×[94℃2′50℃2′72℃4′]25×[94℃1′50℃1′72℃4′]1×72℃5′。
引物对EcoRI/SalI用于所有PCR定向诱变的第二个循环，除了突变体R52L、R55L和双突变体R52L-R55L，对它们用引物E6854替换引物SalI 3′。在所有例子中第二个循环都用与第一个循环相同的条件进行，其中使用0.5μl第一个循环的5′和3′PCR产物(图1)。形成一个323碱基对的条带，之后根据生产者的说明将其结合于pCR2，1-Topo(Invitrogen，K4500-40)质粒以产生pCR-TEX构建体。
用DyeTerminator(商标)测序混合物在377X自动测序仪(Applied Biosystems)上自动测序后选择阳性克隆。用MacVector 7.0软件(Oxford Molecular)分析序列。在所有测序的构建体中，全都仅含所需突变，除了一个衍生自PCR-R55L产物的克隆，其在位置51表现出一个附加突变K-＞T。因此保存该双重突变体K51T-R55L作另外的分析，并用引物K51T(M5′)和K51T(M3′)产生单突变体K51T。
将pCR-TEX构建体的EcoRI-SalI或EcoRI-EcoRI片段亚克隆进真核载体pEGFP-C2(Clontech)，其中Tat突变体在C末端与EGFP(增强的绿色荧光蛋白)融合。以前显示，EGFP与Tat融合既不改变Tat的反式激活能力，也不改变其细胞定位(25)。根据标准的分子生物学技术(23)在大肠杆菌(Escherichia coli，E.Coli)DH5α中完成克隆。通过对阳性克隆自动测序筛选并获得pEGFP-TEX构建体，其中融合蛋白的表达受巨细胞病毒(CMV)启动子的控制。所选择的阳性克隆的氨基酸序列示于图2。扩增这些克隆的DNA，并按照生产者的说明用Nucléobond AX试剂盒(商标)(Macherey-Nagel)纯化。
实施例2：Tat突变体的反式激活能力。
为了研究EGFP-Tat融合蛋白的反式激活能力，使用了细胞系HL3T1。该细胞系是HeLa细胞的一个用氯霉素乙酰基转移酶(CAT)基因稳定转染的衍生物，该氯霉素乙酰基转移酶依赖于病毒HIV-1启动子(LTR)(8)。在6孔板中接种2.5×105HL3T1细胞一天后，用2μg pEGFP-TEX构建体按照生产者推荐的程序用Exgen 500试剂盒(由Euromedex销售)转染细胞。培养48至72小时后，用胰蛋白酶消化细胞并借助荧光显微镜估测被转染细胞的量。在100μl Tris0.01M-EDTA 1nM-NaCl 150mM(TEN)中酶解1.5×103的荧光细胞，并将其经过一个冻/融阶段后于65℃下处理20分钟。之后根据前人的描述(24)在相提取测试中测定CAT活性。简言之，37℃下将70μl裂解物与130μl CAT反应混合物(Tris-HCl pH＝7.5 150mM，EDTA0.2nM，NaCl30mM，丁酰辅酶A0.3mg/ml，丙三醇3％，D-苏-[二氯乙酰-l-14C]氯霉素0.08μCi)孵育2小时。之后用400μl体积/体积比为2∶1的朴日斯烷(2，6，10，14-四甲基十五烷)与二甲苯的混合物抽提反应混合物。用闪烁计数器测定300μl所得有机相的放射活性。
图3a和3b显示，没有一个单突变能够单独完全破坏ACH320.2A.2.1Tat蛋白的反式激活活性，除了突变体C27S。突变体R55L并不非常显著地改变ACH320.2A.2.1 Tat的反式激活活性。但是该突变与突变K51T或R52L之一组合则显示出对Tat反式激活活性非常显著的抑制。该抑制是三重和四重突变体STL和STLA的总和。
实施例3：Tat-EGFP融合蛋白的细胞内定位。
碱性Tat结构域负责Tat的核定位。确信所构建的某些突变影响该碱性结构域。同样，发明人想要鉴定影响Tat细胞内定位的突变。在显微镜玻片上接种2.5×105HL3T1细胞后，用2μg各pEGFP-TEX构建体按照生产者推荐的程序用Exgen 500试剂盒(由Euromedex销售)转染细胞。1、2或3天后回收玻片，并在用Axioplan 2(商标)荧光显微镜(Zeiss)观察前用4％多聚甲醛将其固定。如图4a或b(A和B)中所述以及已有的描述(25)，野生型Tat-EGFP融合蛋白在培养3天后显示核定位。单一Tat突变不影响该定位(图4a，C至H)，双重突变R52L-R55L(图4a，I)、R52L-G79A(图4b，C)、G79A-K89L(图4b，D)、和三重突变体C27S-R52L-G79A(图4b，E)也不影响。然而，K51T-R55L组合或包含它的多重突变体(STL，STLA)在第三天显示Tat蛋白-EGFP的核定位和细胞质定位(图4a，J to L)，而在转染后第一和第二天信号严格限制于核。因此，看来Tat的核定位信号似乎是不连续的且至少包含残基K51和R55，但是不含R52。
实施例4：Tat突变体的跨细胞活性。
不同的研究显示，碱性Tat结构域中的碱性残基总数对由被感染细胞分泌而存在于胞外介质中的Tat被未感染细胞内化的能力(这一现象也称作转导)起作用。发明人评估了在碱性结构域中含有突变的构建体的转导能力。用生产细胞和效应细胞进行了共培养试验。采用磷酸钙技术(23)以3μg pEGFP-TEX构建体转染293T细胞。转染后24小时，将转染的293T细胞用胰蛋白酶消化，并在100μM氯喹存在下与2.5×105HL3T1细胞再共培养48小时。之后根据前人所述，收集细胞并在评估CAT活性前于TEN中裂解。由于该系统同时依赖于转导效果和反式激活活性，因此发明人着手于这样一个前提，也就是，与标准化阳性对照相比，直接转染后测定的活性与共培养后测定的活性之间的显著差异反映了转导能力的急剧变化。这就是为什么所有数据都以分离的ACH320.2A.2.1的野生型蛋白的反式激活活性百分数表示的原因，以及为什么比较各构建体获自HL3T1细胞转染和293T/HL3T1细胞共培养的数据的原因。如图5所示，R55L突变并不显著改变蛋白质的转导能力。突变R52L显著降低蛋白质的转导能力(用该构建体转染的HL3T1细胞和与表达pEGFP-TEX-R52L的293T细胞共培养的HL3T1细胞间的CAT活性降低5倍)。正如共培养后测定的CAT活性背景噪音所示，双重突变体R52L-R55L显示转导能力的完全丢失。用R52L和R52L-R55L突变体获得的结果都暗示，Tat的转导能力与碱性结构域中精氨酸残基的数量有关(27)。看来对于Tat的转导能力残基R55似乎不及残基R52重要。因此，精氨酸残基的定位可能也在整个转导机制中起作用。
实施例5：克隆用本发明核苷酸序列转染的细胞系。
许多有关HIV-1 Tat蛋白对细胞基因调控的功能试验涉及使用组成型表达该蛋白质的细胞系。因此发明人建立了表达不同Tat-蛋白突变体的细胞系。为此，将HeLa细胞以2.5×105细胞每孔接种于6孔板，之后于第二天以2μg编码各种Tat突变体的DNA用试剂Exgen 500(由Euromedex销售)转染。为对这些转染细胞进行生物克隆，细胞转染3天后用胰蛋白酶消化并计数，之后以3至30个细胞每孔的浓度、每次转染3至5个96孔板的比率(每次转染的总数为288至480孔)接种于平底96孔板。之后在500μg/ml geneticin(Geneticin硫酸盐，Gibco-BRL)存在下于96-孔板中培养15天。15天后，将细胞仍然存活并显著增殖的孔视为阳性。在标准方法中，当来自相同转染的各96孔板中含有少于10个阳性孔每板，则认为该生物克隆是成功的。之后在geneticin存在下将每次转染的3至15个阳性孔增殖六代以获得充足的用于冷冻的细胞量。第六代时，用免疫转移(蛋白质印迹)验证各克隆的Tat的表达。
用免疫转移证实了如此产生的细胞系中的Tat表达之后，发明人使用了含有依赖于病毒HIV启动子(LTR-CAT构建体)的CAT基因的质粒构建体，以转染由此获得的不同的细胞系克隆。从而有可能显示，该细胞系中Tat突变体的稳定表达不改变它们的反式激活活性(图6)。
实施例6：位置58的突变体
以ACH.320.2A.2.1株野生型Tat基因的cDNA为模板、S58A(M5′)/S58A(M3′)为引物对获得单突变体S58A。然而，存在其它的在位置58天然带有丙氨酸且具有功能性(核定位、反式激活等)Tat所有特性的其它HIV-1株，例如HXB2株。ACH320.2A.2.1株上这样的突变S58A不改变蛋白质的行为且不可能实现Tat解毒。
| 末端引物，第一个循环 | 序列 |
| E5′ | 5′-GAA TTC ATG GAG CCA GTA GAT C-3′ |
| E3′ | 5′-AGA TCT CTA ATC GAC CGG ATC-3′ |
| 末端引物，第二个循环 | |
| EcoR I | 5′-AAA GAA TTC ATG GAG CCA GTA GAT CC-3′ |
| E6854 | 5′-AAA GAT CTC TAA TCG ACC GGA TCT GTCTCT GTC TC-3′ |
| Sal I | 5′-AAG TCG ACC TAA TCG ACC GGA TCT GTCTCT GTC TC-3′ |
| 中间引物 | |
| W11F(M5′) | 5′-CCA GTA GAT CCT AAA CTA GAG CCC TTCAAG CAT CCA G-3′ |
| C27S(M5′) | 5′-ACA ATT GCT ATT CGA AAA AGT G-3′ |
| C27S(M3′) | 5′-CAC TTT TTC GAA TAG CAA TTG T-3′ |
| K50R(M5′) | 5′-ATC TCA TAT GGC AGG CGG AAG-3′ |
| K50R(M3′) | 5′-CTT CCG CCT GCC ATA TGA GAT-3′ |
| K51T(M5′) | 5′-GGC AGG AAG ACC CGG AGA CAG C-3′ |
| K51T(M3′) | 5′-GCT GTC TCC GGG TCT TCC TGC C-3′ |
| R52L(M5′) | 5′-GGC AGG AAG AAG CTT AGA CAG CGA CGAAGATC-3′ |
| R52L(M3′) | 5′-GAT CTT CGT CGC TGT CTA AGC TTC TTCCTG CC-3′ |
| R55L(M5′) | 5′-GGC AGG AAG AAG CGG AGA CAG CTG CGAAGATC-3′ |
| R55L(M3′) | 5′-GAT CTT CGC AGC TGT CTC CGC TTC TTCCTG CC-3′ |
| R57L(M5′) | 5′-GAC AGC GAC GAC TAT CTC CTC AAG AC-3′ |
| R57L(M3′) | 5′-GTC TTG AGG AGA TAG TCG TCG CTG TC-3′ |
| G79A(M5′) | 5′-CAG CCC CGA GCG GAT CCG ACA GG-3′ |
| G79A(M3′) | 5′-CCT GTC GGA TCC GCT CGG GGC TG-3′ |
| K89L(M3′) | 5′-CTG TCT CTG TCT CTC TCT CCA CCT TAAGCT TCG ATT CC-3′ |
| E92Q(M3′) | 5′-CTG TCT CTG TCT CTC TTT GCA CCT TCTTCT TCG AAT CC-3′ |
| R52L-R55L(M5′) | 5′-GGC AGG AAG AAG CTT AGA CAG CTG CGAAGA TC-3′ |
| R52L-R55L(M3′) | 5′-GAT CTT CGC AGC TGT CTA AGC TTC TTCCTG CC-3′ |
| R55L-R57L(M5′) | 5′-GAA GCG GAG ACA GCT GCG ACT ATC TCCTCA AGA C-3′ |
| R55L-R57L(M3′) | 5′-GTC TTG AGG AGA TAG TCG CAG CTG TCTCCG CTT C-3′ |
| S58A(M5′) | 5′-GAC AGC GAC GAA GAG CAC CTC AAG ACAGT-3′ |
| S58A(M3′) | 5′-ACT GTC TTG AGG TGC TCT TCG TCG CTG TC-3′ |
表1
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Arg Lys Thr Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>4
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体K51T-R57L
<400>4
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp 65 70
75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>5
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体K51T-G79A
<400>5
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>6
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体K51T-K89L
<400>6
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>7
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体K51T-E92Q
<400>7
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>8
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R52L-R55L
<400>8
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>9
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R52L-R57L
<400>9
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>10
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R52L-G79A
<400>10
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>11
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R52L-K89L
<400>11
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>12
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R52L-E92Q
<400>12
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>13
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R55L-R57L
<400>13
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>14
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R55L-G79A
<400>14
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>15
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R55L-K89L
<400>15
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>16
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R55L-E92Q
<400>16
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>17
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R57L-G79A
<400>17
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>18
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R57L-K89L
<400>18
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>19
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体R57L-E92Q
<400>19
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>20
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体G79A-K89L
<400>20
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>21
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体G79A-E92Q
<400>21
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>22
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体K89L-E92Q
<400>22
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>23
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L
<400>23
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>24
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R55L
<400>24
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>25
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R57L
<400>25
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>26
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-G79A
<400>26
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>27
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-K89L
<400>27
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>28
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-E92Q
<400>28
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>29
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R55L
<400>29
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>30
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R57L
<400>30
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>31
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-G79A
<400>31
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>32
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-K89L
<400>32
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>33
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-E92Q
<400>33
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>34
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-R57L
<400>34
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr 50
55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>35
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-G79A
<400>35
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>36
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-K89L
<400>36
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>37
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-E92Q
<400>37
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>38
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R57L-G79A
<400>38
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>39
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R57L-K89L
<400>39
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>40
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R57L-E92Q
<400>40
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>41
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-G79A-K89L
<400>41
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>42
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-G79A-E92Q
<400>42
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>43
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K89L-E92Q
<400>43
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>44
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-G79A
<400>44
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>45
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-K89L
<400>45
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>46
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-E92Q
<400>46
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>47
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R55L-G79A
<400>47
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>48
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R55L-K89L
<400>48
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>49
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R55L-E92Q
<400>49
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>50
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R57L-G79A
<400>50
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>51
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R57L-K89L
<400>51
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>52
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R57L-E92Q
<400>52
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>53
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-G79A-K89L
<400>53
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>54
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-G79A-E92Q
<400>54
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>55
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-K89L-E92Q
<400>55
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>56
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-G79A-K89L
<400>56
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>57
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-G79A-E92Q
<400>57
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>58
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-K89L-E92Q
<400>58
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>59
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R55L-G79A
<400>59
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>60
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R55L-K89L
<400>60
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>61
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R55L-E92Q
<400>61
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>62
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R57L-G79A
<400>62
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>63
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R57L-K89L
<400>63
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>64
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R57L-E92Q
<400>64
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>65
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-G79A-K89L
<400>65
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>66
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-G79A-E92Q
<400>66
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>67
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-K89L-E92Q
<400>67
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>68
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-R57L-G79A
<400>68
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>69
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-R57L-K89L
<400>69
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>70
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-R57L-E92Q
<400>70
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>71
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R57L-G79A-K89L
<400>71
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>72
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R57L-G79A-E92Q
<400>72
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>73
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R57L-K89L-E92Q
<400>73
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>74
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-G79A-K89L-E92Q
<400>74
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>75
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-G79A-K89L-E92Q
<400>75
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp 100
<210>76
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-R55L-G79A
<400>76
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>77
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-R55L-K89L
<400>77
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>78
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-R55L-E92Q
<400>78
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>79
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-R57L-G79A
<400>79
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>80
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-R57L-K89L
<400>80
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>81
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-R57L-E92Q
<400>81
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>82
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-G79A-K89L
<400>82
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>83
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-G79A-E92Q
<400>83
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>84
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R52L-K89L-E92Q
<400>84
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>85
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R55L-R57L-G79A
<400>85
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>86
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R55L-R57L-K89L
<400>86
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>87
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R55L-R57L-E92Q
<400>87
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>88
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R55L-G79A-K89L
<400>88
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>89
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R55L-G79A-E92Q
<400>89
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>90
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R55L-K89L-E92Q
<400>90
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>91
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R57L-G79A-K89L
<400>91
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp 65 70
75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>92
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R57L-G79A-E92Q
<400>92
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>93
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-K51T-R57L-K89L-E92Q
<400>93
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Thr Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>94
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R55L-R57L-G79A
<400>94
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>95
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R55L-R57L-K89L
<400>95
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>96
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R55L-R57L-E92Q
<400>96
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>97
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R55L-G79A-K89L
<400>97
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>98
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R55L-G79A-E92Q
<400>98
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>99
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R55L-K89L-E92Q
<400>99
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>100
<211>101<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R57L-G79A-K89L
<400>100
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>101
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R57L-G79A-E92Q
<400>101
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>102
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-R57L-K89L-E92Q
<400>102
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>103
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R52L-G79A-K89L-E92Q
<400>103
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Leu Arg Gln Arg Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>104
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-R57L-G79A-K89L
<400>104
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Glu Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>105
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-R57L-G79A-E92Q
<400>105
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>106
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-R57L-K89L-E92Q
<400>106
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Gly Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>107
<211>101
<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R55L-G79A-K89L-E92Q
<400>107
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Leu Arg Arg Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
<210>108
<211>101<212>PRT
<213>人工序列
<220>
<223>Tat蛋白突变体C27S-R57L-G79A-K89L-E92Q
<400>108
Met Glu Pro Val Asp Pro Lys Leu Glu Pro Trp Lys His Pro Gly Ser
1 5 10 15
Gln Pro Lys Thr Ala Cys Asn Asn Cys Tyr Ser Lys Lys Cys Cys Phe
20 25 30
His Cys Gln Val Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser Tyr Gly
35 40 45
Arg Lys Lys Arg Arg Gln Arg Arg Leu Ser Pro Gln Asp Ser Glu Thr
50 55 60
His Gln Val Ser Leu Ser Lys Gln Pro Ala Ser Gln Pro Arg Ala Asp
65 70 75 80
Pro Thr Gly Pro Lys Glu Ser Lys Leu Lys Val Gln Arg Glu Thr Glu
85 90 95
Thr Asp Pro Val Asp
100
76
Claims (29)
1.HIV-1病毒Tat蛋白的解毒免疫原性突变体，其特征在于它在野生型Tat蛋白的结构域4含有至少一个突变，在结构域4和/或5中至少含有其它突变，且其中，当突变处于结构域4时它位于从氨基酸位置49至氨基酸位置57这部分中，且其中，当突变处于结构域5中时，它或者位于RGD基元中或者在88-92区中，突变是由一个氨基酸被另一氨基酸置换形成的突变。
2.根据权利要求1的突变体，其特征在于当所述突变在结构域5的88-92区中时，它在位置89和/或92。
3.根据权利要求1或2的突变体，其特征在于结构域4和/或5中的突变能带来以下特征中的至少一种：
-废除野生型Tat蛋白的跨细胞效应，
-改变野生型Tat蛋白的核定位。
4.根据权利要求1或2的突变体，其特征在于它含有能够使野生型Tat蛋白反式激活活性丧失的附加突变。
5.根据权利要求1或2的突变体，其特征在于它在野生型Tat蛋白结构域4的N-末端区域从氨基酸位置49至氨基酸位置55这部分中含有突变。
6.根据权利要求1或2的突变体，其特征在于它在野生型Tat蛋白结构域2中包含突变。
7.根据权利要求6的突变体，其特征在于在野生型Tat蛋白结构域2中的突变是对任一半胱氨酸的置换。
8.根据权利要求6的突变体，其特征在于在野生型Tat蛋白结构域2中的突变是任一半胱氨酸被丝氨酸的置换。
9.根据权利要求1或2的突变体，其特征在于它含有以下至少一种突变：
-第27位的半胱氨酸被丝氨酸取代，
-第51位的赖氨酸被苏氨酸取代，
-第52位的精氨酸被亮氨酸取代，
-第55位的精氨酸被亮氨酸取代，
-第57位的精氨酸被亮氨酸取代，
-第79位的甘氨酸被丙氨酸取代，
-第89位的赖氨酸被亮氨酸取代，
-第92位的谷氨酸被谷氨酰胺取代。
10.根据权利要求1或2的突变体，其特征在于它选自下述具有两种突变的突变体，各突变用三联体表示：字母-数字-字母，其中的数字表示突变氨基酸的位置，数字前的字母对应突变所涉及的氨基酸，数字后的字母表示用于置换数字前氨基酸的氨基酸：
K51T-R52L (SEQ ID NO：2)
K51T-R55L (SEQ ID NO：3)
K51T-R57L (SEQ ID NO：4)
K51T-G79A (SEQ ID NO：5)
K51T-K89L (SEQ ID NO：6)
K51T-E92Q (SEQ ID NO：7)
R52L-R55L (SEQ ID NO：8)
R52L-R57L (SEQ ID NO：9)
R52L-G79A (SEQ ID NO：10)
R52L-K89L (SEQ ID NO：11)
R52L-E92Q (SEQ ID NO：12)
R55L-R57L (SEQ ID NO：13)
R55L-G79A (SEQ ID NO：14)
R55L-K89L (SEQ ID NO：15)
R55L-E92Q (SEQ ID NO：16)
R57L-G79A (SEQ ID NO：17)
R57L-K89L (SEQ ID NO：18)
R57L-E92Q (SEQ ID NO：19)。
11.根据权利要求10的突变体，其特征在于它选自下列突变体：
K51T-R55L (SEQ ID NO：3)
R52L-R55L (SEQ ID NO：8)
R52L-G79A (SEQ ID NO：10)
R55L-R57L (SEQ ID NO：13)。
12.根据权利要求1或2的突变体，其特征在于它选自下述具有三种突变的突变体，各突变用三联体表示：字母-数字-字母，其中的数字表示突变氨基酸的位置，数字前的字母对应突变所涉及的氨基酸，数字后的字母表示用于置换数字前氨基酸的氨基酸：
C27S-K51T-R52L (SEQ ID NO：23)
C27S-K51T-R55L (SEQ ID NO：24)
C27S-K51T-R57L (SEQ ID NO：25)
C27S-K51T-G79A (SEQ ID NO：26)
C27S-K51T-K89L (SEQ ID NO：27)
C27S-K51T-E92Q (SEQ ID NO：28)
C27S-R52L-R55L (SEQ ID NO：29)
C27S-R52L-R57L (SEQ ID NO：30)
C27S-R52L-G79A (SEQ ID NO：31)
C27S-R52L-K89L (SEQ ID NO：32)
C27S-R52L-E92Q (SEQ ID NO：33)
C27S-R55L-R57L (SEQ ID NO：34)
C27S-R55L-G79A (SEQ ID NO：35)
C27S-R55L-K89L (SEQ ID NO：36)
C27S-R55L-E92Q (SEQ ID NO：37)
C27S-R57L-G79A (SEQ ID NO：38)
C27S-R57L-K89L (SEQ ID NO：39)
C27S-R57L-E92Q (SEQ ID NO：40)。
13.根据权利要求12的突变体，其特征在于它选自下列突变体：
C27S-K51T-R55L (SEQ ID NO：24)
C27S-R52L-R55L (SEQ ID NO：29)
C27S-R52L-G79A (SEQ ID NO：31)。
14.根据权利要求1或2的突变体，其特征在于它选自下述具有四种突变的突变体，各突变用三联体表示：字母-数字-字母，其中的数字表示突变氨基酸的位置，数字前的字母对应突变所涉及的氨基酸，数字后的字母表示用于置换数字前氨基酸的氨基酸：
C27S-K51T-R52L-G79A (SEQ ID NO：44)
C27S-K51T-R52L-K89L (SEQ ID NO：45)
C27S-K51T-R52L-E92Q (SEQ ID NO：46)
C27S-K51T-R55L-G79A (SEQ ID NO：47)
C27S-K51T-R55L-K89L (SEQ ID NO：48)
C27S-K51T-R55L-E92Q (SEQ ID NO：49)
C27S-K51T-R57L-G79A (SEQ ID NO：50)
C27S-K51T-R57L-K89L (SEQ ID NO：51)
C27S-K51T-R57L-E92Q (SEQ ID NO：52)
C27S-K51T-G79A-K89L (SEQ ID NO：53)
C27S-K51T-G79A-E92Q (SEQ ID NO：54)
C27S-K51T-K89L-E92Q (SEQ ID NO：55)
C27S-R52L-G79A-K89L (SEQ ID NO：56)
C27S-R52L-G79A-E92Q (SEQ ID NO：57)
C27S-R52L-K89L-E92Q (SEQ ID NO：58)
C27S-R52L-R55L-G79A (SEQ ID NO：59)
C27S-R52L-R55L-K89L (SEQ ID NO：60)
C27S-R52L-R55L-E92Q (SEQ ID NO：61)
C27S-R52L-R57L-G79A (SEQ ID NO：62)
C27S-R52L-R57L-K89L (SEQ ID NO：63)
C27S-R52L-R57L-E92Q (SEQ ID NO：64)
C27S-R55L-G79A-K89L (SEQ ID NO：65)
C27S-R55L-G79A-E92Q (SEQ ID NO：66)
C27S-R55L-K89L-E92Q (SEQ ID NO：67)
C27S-R55L-R57L-G79A (SEQ ID NO：68)
C27S-R55L-R57L-K89L (SEQ ID NO：69)
C27S-R55L-R57L-E92Q (SEQ ID NO：70)
C27S-R57L-G79A-K89L (SEQ ID NO：71)
C27S-R57L-G79A-E92Q (SEQ ID NO：72)
C27S-R57L-K89L-E92Q (SEQ ID NO：73)。
15.根据权利要求14的突变体，其特征在于它选自下列突变体：
C27S-K51T-R55L-G79A (SEQ ID NO：47)
C27S-K51T-R55L-K89L (SEQ ID NO：48)
C27S-K51T-R55L-E92Q (SEQ ID NO：49)
C27S-R52L-R55L-G79A (SEQ ID NO：59)。
16.根据权利要求1或2的突变体，其特征在于它选自下述具有五种突变的突变体，各突变用三联体表示：字母-数字-字母，其中的数字表示突变氨基酸的位置，数字前的字母对应突变所涉及的氨基酸，数字后的字母表示用于置换数字前氨基酸的氨基酸：
C27S-K51T-G79A-K89L-E92Q (SEQ ID NO：75)
C27S-K51T-R52L-R55L-G79A (SEQ ID NO：76)
C27S-K51T-R52L-R55L-K89L (SEQ ID NO：77)
C27S-K51T-R52L-R55L-E92Q (SEQ ID NO：78)
C27S-K51T-R52L-R57L-G79A (SEQ ID NO：79)
C27S-K51T-R52L-R57L-K89L (SEQ ID NO：80)
C27S-K51T-R52L-R57L-E92Q (SEQ ID NO：81)
C27S-K51T-R52L-G79A-K89L (SEQ ID NO：82)
C27S-K51T-R52L-G79A-E92Q (SEQ ID NO：83)
C27S-K51T-R52L-K89L-E92Q (SEQ ID NO：84)
C27S-K51T-R55L-R57L-G79A (SEQ ID NO：85)
C27S-K51T-R55L-R57L-K89L (SEQ ID NO：86)
C27S-K51T-R55L-R57L-E92Q (SEQ ID NO：87)
C27S-K51T-R55L-G79A-K89L (SEQ ID NO：88)
C27S-K51T-R55L-G79A-E92Q (SEQ ID NO：89)
C27S-K51T-R55L-K89L-E92Q (SEQ ID NO：90)
C27S-K51T-R57L-G79A-K89L (SEQ ID NO：91)
C27S-K51T-R57L-G79A-E92Q (SEQ ID NO：92)
C27S-K51T-R57L-K89L-E92Q (SEQ ID NO：93)
C27S-R52L-R55L-R57L-G79A (SEQ ID NO：94)
C27S-R52L-R55L-R57L-K89L (SEQ ID NO：95)
C27S-R52L-R55L-R57L-E92Q (SEQ ID NO：96)
C27S-R52L-R55L-G79A-K89L (SEQ ID NO：97)
C27S-R52L-R55L-G79A-E92Q (SEQ ID NO：98)
C27S-R52L-R55L-K89L-E92Q (SEQ ID NO：99)
C27S-R52L-R57L-G79A-K89L (SEQ ID NO：100)
C27S-R52L-R57L-G79A-E92Q (SEQ ID NO：101)
C27S-R52L-R57L-K89L-E92Q (SEQ ID NO：102)
C27S-R52L-G79A-K89L-E92Q (SEQ ID NO：103)
C27S-R55L-R57L-G79A-K89L (SEQ ID NO：104)
C27S-R55L-R57L-G79A-E92Q (SEQ ID NO：105)
C27S-R55L-R57L-K89L-E92Q (SEQ ID NO：106)
C27S-R55L-G79A-K89L-E92Q (SEQ ID NO：107)
C27S-R57L-G79A-K89L-E92Q (SEQ ID NO：108)。
17.根据权利要求16的突变体，其特征在于它选自下列突变体：
C27S-K51T-R55L-G79A-K89L (SEQ ID NO：88)
C27S-K51T-R55L-G79A-E92Q (SEQ ID NO：89)。
18.编码根据权利要求1至17任一项的突变体之一的核苷酸序列。
19.用根据权利要求18的核苷酸序列转染的细胞系。
20.针对根据权利要求1至17任一项的突变体之一的抗体，其不识别野生型蛋白质结构域1。
21.根据权利要求20的抗体，其识别野生型蛋白质。
22.根据权利要求20的抗体，其不识别野生型蛋白质。
23.药物组合物，它包含作为活性成分的至少一种根据权利要求1至17任一项的突变体或至少一种根据权利要求18的核苷酸序列或至少一种根据权利要求20-22任一项的抗体以及药学适宜载体，其中的核苷酸序列位于权利要求1-17任一项的突变体之一的组成型表达所必需的元件控制之下。
24.根据权利要求23的药物组合物，其是疫苗。
25.含有至少一种如权利要求1至17任一项中所定义的突变体、或至少一种根据权利要求20至22任一项的抗体的检测和/或定量HIV-1病毒的诊断组合物。
26.检测和/或定量取自能被HIV-1感染的个体的生物样品中HIV-1病毒的方法，其特征在于该方法包括下列阶段：
-在预定条件下将所述生物样品与含有如权利要求1至17任一项所定义的突变体或如权利要求20至22任一项所定义的抗体的诊断组合物接触，其中该预定条件允许上述定义的突变体与针对野生型Tat蛋白的抗体之间、或上述定义的抗体与野生型Tat蛋白之间形成抗体/抗原复合物，和
-用适当的手段检测和/或定量所述复合物的形成。
27.根据权利要求26的方法，其中所述生物样品选自血浆、血清或组织。
28.至少一种如权利要求1至17任一项所定义的突变体或至少一种根据权利要求20至22任一项的抗体在体外检测生物样本或样品中HIV-1病毒中的用途。
29.至少一种如权利要求1至17任一项所定义的突变体或至少一种根据权利要求20至22任一项的抗体在制备疫苗组合物中的用途。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0200319A FR2834717B1 (fr) | 2002-01-11 | 2002-01-11 | Mutants de la proteine tat du virus vih-1 |
| FR02/00319 | 2002-01-11 |
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| Publication Number | Publication Date |
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| CN1615364A CN1615364A (zh) | 2005-05-11 |
| CN1292069C true CN1292069C (zh) | 2006-12-27 |
Family
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| Application Number | Title | Priority Date | Filing Date |
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| CNB038020726A Expired - Fee Related CN1292069C (zh) | 2002-01-11 | 2003-01-09 | Hiv－1病毒tat－蛋白突变体 |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US20050164164A1 (zh) |
| EP (1) | EP1463819A1 (zh) |
| CN (1) | CN1292069C (zh) |
| AU (1) | AU2003214299A1 (zh) |
| FR (1) | FR2834717B1 (zh) |
| WO (1) | WO2003057885A1 (zh) |
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| FR2868318B1 (fr) | 2004-04-01 | 2012-11-16 | Commissariat Energie Atomique | Antigene tat stabilise et ses applications pour la vaccination anti-vih |
| CN107759698A (zh) * | 2017-09-18 | 2018-03-06 | 广东工业大学 | 一种egfp‑cta2‑tat融合蛋白在制备荧光探针中的应用 |
| CN111778269B (zh) * | 2020-06-11 | 2022-05-17 | 军事科学院军事医学研究院军事兽医研究所 | 抗h5n1病毒入胞抗体ptd-3f及其应用 |
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| US5474921A (en) * | 1993-10-15 | 1995-12-12 | Merck & Co., Inc. | Expression and purification of phosphoinositide-specific phospholipase C-γ |
| AU5135896A (en) * | 1995-03-06 | 1996-10-02 | Baylor College Of Medicine | A double transdominant fusion gene and protein |
| IT1297090B1 (it) * | 1997-12-01 | 1999-08-03 | Barbara Ensoli | Tat di hiv-1 o suoi derivati, da soli od in combinazione, a scopo vaccinale, profilattico e terapeutico, contro l'aids i tumori e le |
| FR2792204B1 (fr) * | 1999-04-13 | 2001-07-13 | Centre Nat Rech Scient | Vaccin anti-vih-1 comprenant tout en partie de la proteine tat de vih-1 |
-
2002
- 2002-01-11 FR FR0200319A patent/FR2834717B1/fr not_active Expired - Fee Related
-
2003
- 2003-01-09 WO PCT/FR2003/000051 patent/WO2003057885A1/fr not_active Application Discontinuation
- 2003-01-09 AU AU2003214299A patent/AU2003214299A1/en not_active Abandoned
- 2003-01-09 CN CNB038020726A patent/CN1292069C/zh not_active Expired - Fee Related
- 2003-01-09 US US10/501,223 patent/US20050164164A1/en not_active Abandoned
- 2003-01-09 EP EP03709864A patent/EP1463819A1/fr not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| WO2003057885A1 (fr) | 2003-07-17 |
| US20050164164A1 (en) | 2005-07-28 |
| EP1463819A1 (fr) | 2004-10-06 |
| AU2003214299A1 (en) | 2003-07-24 |
| FR2834717A1 (fr) | 2003-07-18 |
| FR2834717B1 (fr) | 2004-12-10 |
| CN1615364A (zh) | 2005-05-11 |
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