CN116769046A - 含有热休克蛋白gp96的多肽、药物组合物和用途 - Google Patents

含有热休克蛋白gp96的多肽、药物组合物和用途 Download PDF

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CN116769046A
CN116769046A CN202210241991.7A CN202210241991A CN116769046A CN 116769046 A CN116769046 A CN 116769046A CN 202210241991 A CN202210241991 A CN 202210241991A CN 116769046 A CN116769046 A CN 116769046A
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王子豪
武岳
程放
鞠莹
李鑫
徐玉秀
梁振涛
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Foshan Rexiu Biotechnology Co ltd
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Abstract

本发明属于生物医药领域，涉及一种含有热休克蛋白gp96的多肽、药物组合物和用途。具体地，本发明涉及一种分离的多肽，其结构如下面的式I所示，其中：A表示gp96蛋白或gp96蛋白活性片段；B表示一个多肽片段，其结构如下面的式II所示，其中：m选自0、1、2、3、4、5，n选自1、2、3、4、5，r选自0、1、2、3、4、5，并且m和r不同时为0；B和A直接连接或通过连接片段连接。本发明的多肽能够显著地提高gp96蛋白的生物学活性，这对于靶向调节性T细胞的功能具有很大的提升，从而适合自身免疫性疾病的治疗，具有重要的临床应用价值。B——A式I(GGGGS)m‑(GGGGSC)n‑(GGGGS)r式II。

Description

含有热休克蛋白gp96的多肽、药物组合物和用途
技术领域
本发明属于生物医药领域，涉及一种含有热休克蛋白gp96的多肽、药物组合物和用途。
背景技术
调节性T细胞(Treg)在维持免疫稳态中发挥着关键作用，其中转录因子Foxp3决定了Treg细胞的发育和功能。foxp3基因缺陷可诱导人体Treg细胞功能障碍，从而导致免疫失调、多内分泌病、肠病和X染色体连锁综合征等一系列疾病的发生。Treg细胞功能缺陷或exTreg细胞发育与自身免疫性疾病呈正相关，如系统性红斑狼疮(SLE)、多发性硬化症(MS)、强直性脊柱炎(AS)等。
热休克蛋白(Heat shock protein，HSP)是一类在生物进化中高度保守且广泛存在于原核及真核生物中的蛋白质。HSP根据同源程度及分子量大小可分为HSP110、HSP90、HSP70、HSP60、HSP40、小分子HSP及泛素等多个亚家族。人类热休克蛋白90家族(HSP90)包括HSP90α,HSP90β,gp96(grp94)和Trap-1四个成员。gp96(GRP94)为内质网HSP90家族的代表，与细胞质HSP90高度同源，主要生物学功能有：分子伴侣，参与新合成蛋白的折叠与组装；与细胞内的其他肽类蛋白质尤其是变性蛋白的结合，参与细胞的抗损伤、修复和热耐受过程；参与蛋白质水解过程；结合抗原肽，加工提呈肿瘤抗原及维持细胞内环境稳定等作用；对细胞的生长、发育、分化及死亡具有一定的调节作用。
gp96由于具有显著的生物学活性，特别是在免疫学方面同时具有刺激特异性杀伤T细胞的作用和抑制调节T细胞的作用。研究表明，gp96可以通过调节性T细胞(Treg)和CD14+HLA-DR-/low抑制体内的免疫反应。这为治疗自身免疫类疾病提供了新的思路。然而，gp96的免疫学功能却有待进一步提高。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了一种分离的多肽。进一步地，所述分离的多肽形成二聚体形式的蛋白。本发明人惊奇地发现，该多肽或蛋白能够显著地提高gp96蛋白的生物学活性特别是免疫学功能。这对于靶向调节性T细胞的功能具有很大的提升，从而适合自身免疫性疾病的治疗，具有重要的临床应用价值。由此提供了下述发明：
本发明的一个方面涉及一种分离的多肽，其结构如下面的式I所示：
B——A
式I；
其中：
A表示gp96蛋白或gp96蛋白活性片段；
B表示一个多肽片段，其结构如下面的式II所示：
(GGGGS)m-(GGGGSC)n-(GGGGS)r
式II
，其中：
m选自0、1、2、3、4、5，
n选自1、2、3、4、5，
r选自0、1、2、3、4、5，
并且m和r不同时为0；
B和A直接连接或通过连接片段连接；
并且式I的N端或C端连接有或不连接蛋白纯化标签例如4-8个His或6个His。
在本发明的一些实施方式中，所述的分离的多肽，其中，式I的N端连接有或不连接蛋白纯化标签例如4-8个His或6个His。
在本发明的一些实施方式中，所述的分离的多肽，其中，式I的C端连接有或不连接蛋白纯化标签例如4-8个His或6个His。
在本发明的一些实施方式中，所述的分离的多肽，其中，A的氨基酸序列如SEQ IDNO:1所示。
MDDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAE(SEQ ID NO:1)
SEQ ID NO:1对应的编码核酸的序列如SEQ ID NO:2所示。
atggatgacgaggtggatgtggatggtaccgtggaggaggacttgggtaagagcagggagggtagccgcactgatgacgaggttgttcagcgtgaggaggaggctattcagttggacggtttgaacgcaagccagattagggagctgcgtgagaagagcgagaagttcgctttccaggctgaggtgaaccgcatgatgaagttgatcattaacagcttgtacaagaacaaggagatcttcctgagagagctgatctcaaacgcttccgacgccctggacaagatccgcctgatctccctgactgacgagaacgccctgtccggcaacgaagaactgaccgtgaaaatcaaatgcgacaaagaaaagaacctgctccacgttaccgataccggtgtgggcatgacccgcgaggagctcgtgaagaacctgggtaccatcgctaagagcggaacctcggaatttctgaacaagatgacagaagcccaagaagacggtcagtccacctccgagctgattggccagttcggtgtgggtttctactctgctttcctggtggctgacaaggtgatcgtgacctccaaacacaacaacgacacacaacacatctgggagtccgactccaacgaattttccgtgatcgccgacccgcgcggcaacactctgggtagaggtaccaccatcaccctcgtgctcaaagaggaggcctccgactacctcgaactggacaccatcaagaacctggtgaagaagtactcccaattcatcaacttccccatctacgtgtggagttccaagaccgaaaccgtggaagaacctatggaggaggaggaggccgctaaagaggaaaaggaggagtccgatgacgaggctgctgtcgaggaggaagaggaggagaagaagccgaagactaagaaggtggagaagacagtgtgggactgggagctgatgaacgacatcaagccaatctggcagaggccaagcaaggaggtggaggaggatgagtacaaggcattctacaagagcttcagcaaggagagcgacgacccgatggcttacattcacttcaccgctgagggagaggtgacattcaagagcatcctgttcgtgccaaccagcgctccccgcggtctgttcgacgagtacggtagcaagaagagcgattacatcaagctgtacgtccgccgcgtgttcatcacagatgacttccacgacatgatgcctaagtacctcaacttcgtgaagggtgtggtggactccgacgatctgcccctgaacgtttcccgcgagaccctgcagcaacataagctgctgaaggtgatccgtaaaaagctggtgcgcaagaccctcgacatgatcaaaaagatcgctgacgacaagtacaacgacaccttctggaaggaatttggcaccaacatcaaactgggtgtgatcgaggaccactccaaccgcaccagactggccaaactgctgagattccaatcctctcaccaccccaccgacatcacctccctggaccaatacgtggagcgcatgaaagagaaacaggacaaaatctacttcatggccggctcctcccgcaaagaagctgaatcctcccccttcgtcgaacgcctgctgaagaaaggttacgaggtcatctacctgaccgaacccgtggacgagtactgcatccaggcactgcctgagttcgacggtaagcgcttccagaacgtcgctaaggagggagtgaagttcgacgagagcgagaagaccaaggagtcccgcgaagctgtggagaaggagttcgagcccctgttgaactggatgaaggataaggctctgaaggacaagatcgagaaagcagtggtgtcccagaggctgaccgaaagcccctgtgctctggtggctagtcagtacggttggagcggtaacatggaaaggatcatgaaggcacaggcataccagaccggtaaggacattagtaccaactactacgcttcccagaaaaagaccttcgagattaacccccgccaccctctgatcagagacatgctgagacgcatcaaggaggacgaagacgacaaaaccgtcctcgacctggccgtggtgttgttcgagaccgccaccctccgcagcggctacttgctgcctgacaccaaagcgtacggcgacaggatcgagcgcatgctgaggctgagcctgaacatcgaccctgacgctaaggtggaggaggagcccgaggaagagccagaggagaccgcggaggacacaacagaggacactgagcaggacgaggacgaggagatggacgtgggaactgacgaggaagaggaaacagcaaaggagagcacagcagag(SEQ ID NO:2)
在本发明的一些实施方式中，所述的分离的多肽，其中：
m为0，n为1或2，并且r为1或2；
或者
m为1或2，n为1或2，并且r为1或2。
在本发明的一些实施方式中，所述的分离的多肽，其中，B的氨基酸序列如SEQ IDNOs:3-6中任一序列所示。
多肽片段1：GGGGSCGGGGS(SEQ ID NO:3)
多肽片段2：GGGGSGGGGSCGGGGS(SEQ ID NO:4)
多肽片段3：GGGGSCGGGGSCGGGGS(SEQ ID NO:5)
多肽片段4：GGGGSGGGGSCGGGGSCGGGGS(SEQ ID NO:6)
SEQ ID NOs:3-6对应的编码核酸的序列分别如SEQ ID NOs:7-10所示。
ggtggtggtggttcctgcggtggtggtggttcc(SEQ ID NO:7)
ggtggtggtggttccggtggtggtggttcctgcggtggtggtggttcc(SEQ ID NO:8)
ggtggtggtggttcctgcggtggtggtggttcctgcggtggtggtggttcc(SEQ ID NO:9)
ggtggtggtggttccggtggtggtggttcctgcggtggtggtggttcctgcggtggtggtggttcc(SEQ ID NO:10)
在本发明的一些实施方式中，所述的分离的多肽，其氨基酸序列如SEQ ID NOs:11-14中任一序列所示。
分离的多肽1：
MGGGGSCGGGGSMDDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAE(SEQ IDNO:11)
分离的多肽2：
MGGGGSGGGGSCGGGGSMDDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAE(SEQ ID NO:12)
分离的多肽3：
MGGGGSCGGGGSCGGGGSMDDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAE(SEQ ID NO:13)
分离的多肽4：
MGGGGSGGGGSCGGGGSCGGGGSMDDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAE(SEQ ID NO:14)
SEQ ID NOs:11-14对应的编码核酸的序列分别如SEQ ID NOs:15-18所示。
编码分离的多肽1的核酸：
atgggtggtggtggttcctgcggtggtggtggttccatggatgacgaggtggatgtggatggtaccgtggaggaggacttgggtaagagcagggagggtagccgcactgatgacgaggttgttcagcgtgaggaggaggctattcagttggacggtttgaacgcaagccagattagggagctgcgtgagaagagcgagaagttcgctttccaggctgaggtgaaccgcatgatgaagttgatcattaacagcttgtacaagaacaaggagatcttcctgagagagctgatctcaaacgcttccgacgccctggacaagatccgcctgatctccctgactgacgagaacgccctgtccggcaacgaagaactgaccgtgaaaatcaaatgcgacaaagaaaagaacctgctccacgttaccgataccggtgtgggcatgacccgcgaggagctcgtgaagaacctgggtaccatcgctaagagcggaacctcggaatttctgaacaagatgacagaagcccaagaagacggtcagtccacctccgagctgattggccagttcggtgtgggtttctactctgctttcctggtggctgacaaggtgatcgtgacctccaaacacaacaacgacacacaacacatctgggagtccgactccaacgaattttccgtgatcgccgacccgcgcggcaacactctgggtagaggtaccaccatcaccctcgtgctcaaagaggaggcctccgactacctcgaactggacaccatcaagaacctggtgaagaagtactcccaattcatcaacttccccatctacgtgtggagttccaagaccgaaaccgtggaagaacctatggaggaggaggaggccgctaaagaggaaaaggaggagtccgatgacgaggctgctgtcgaggaggaagaggaggagaagaagccgaagactaagaaggtggagaagacagtgtgggactgggagctgatgaacgacatcaagccaatctggcagaggccaagcaaggaggtggaggaggatgagtacaaggcattctacaagagcttcagcaaggagagcgacgacccgatggcttacattcacttcaccgctgagggagaggtgacattcaagagcatcctgttcgtgccaaccagcgctccccgcggtctgttcgacgagtacggtagcaagaagagcgattacatcaagctgtacgtccgccgcgtgttcatcacagatgacttccacgacatgatgcctaagtacctcaacttcgtgaagggtgtggtggactccgacgatctgcccctgaacgtttcccgcgagaccctgcagcaacataagctgctgaaggtgatccgtaaaaagctggtgcgcaagaccctcgacatgatcaaaaagatcgctgacgacaagtacaacgacaccttctggaaggaatttggcaccaacatcaaactgggtgtgatcgaggaccactccaaccgcaccagactggccaaactgctgagattccaatcctctcaccaccccaccgacatcacctccctggaccaatacgtggagcgcatgaaagagaaacaggacaaaatctacttcatggccggctcctcccgcaaagaagctgaatcctcccccttcgtcgaacgcctgctgaagaaaggttacgaggtcatctacctgaccgaacccgtggacgagtactgcatccaggcactgcctgagttcgacggtaagcgcttccagaacgtcgctaaggagggagtgaagttcgacgagagcgagaagaccaaggagtcccgcgaagctgtggagaaggagttcgagcccctgttgaactggatgaaggataaggctctgaaggacaagatcgagaaagcagtggtgtcccagaggctgaccgaaagcccctgtgctctggtggctagtcagtacggttggagcggtaacatggaaaggatcatgaaggcacaggcataccagaccggtaaggacattagtaccaactactacgcttcccagaaaaagaccttcgagattaacccccgccaccctctgatcagagacatgctgagacgcatcaaggaggacgaagacgacaaaaccgtcctcgacctggccgtggtgttgttcgagaccgccaccctccgcagcggctacttgctgcctgacaccaaagcgtacggcgacaggatcgagcgcatgctgaggctgagcctgaacatcgaccctgacgctaaggtggaggaggagcccgaggaagagccagaggagaccgcggaggacacaacagaggacactgagcaggacgaggacgaggagatggacgtgggaactgacgaggaagaggaaacagcaaaggagagcacagcagag(SEQ ID NO:15)
编码分离的多肽2的核酸：
atgggtggtggtggttccggtggtggtggttcctgcggtggtggtggttccatggatgacgaggtggatgtggatggtaccgtggaggaggacttgggtaagagcagggagggtagccgcactgatgacgaggttgttcagcgtgaggaggaggctattcagttggacggtttgaacgcaagccagattagggagctgcgtgagaagagcgagaagttcgctttccaggctgaggtgaaccgcatgatgaagttgatcattaacagcttgtacaagaacaaggagatcttcctgagagagctgatctcaaacgcttccgacgccctggacaagatccgcctgatctccctgactgacgagaacgccctgtccggcaacgaagaactgaccgtgaaaatcaaatgcgacaaagaaaagaacctgctccacgttaccgataccggtgtgggcatgacccgcgaggagctcgtgaagaacctgggtaccatcgctaagagcggaacctcggaatttctgaacaagatgacagaagcccaagaagacggtcagtccacctccgagctgattggccagttcggtgtgggtttctactctgctttcctggtggctgacaaggtgatcgtgacctccaaacacaacaacgacacacaacacatctgggagtccgactccaacgaattttccgtgatcgccgacccgcgcggcaacactctgggtagaggtaccaccatcaccctcgtgctcaaagaggaggcctccgactacctcgaactggacaccatcaagaacctggtgaagaagtactcccaattcatcaacttccccatctacgtgtggagttccaagaccgaaaccgtggaagaacctatggaggaggaggaggccgctaaagaggaaaaggaggagtccgatgacgaggctgctgtcgaggaggaagaggaggagaagaagccgaagactaagaaggtggagaagacagtgtgggactgggagctgatgaacgacatcaagccaatctggcagaggccaagcaaggaggtggaggaggatgagtacaaggcattctacaagagcttcagcaaggagagcgacgacccgatggcttacattcacttcaccgctgagggagaggtgacattcaagagcatcctgttcgtgccaaccagcgctccccgcggtctgttcgacgagtacggtagcaagaagagcgattacatcaagctgtacgtccgccgcgtgttcatcacagatgacttccacgacatgatgcctaagtacctcaacttcgtgaagggtgtggtggactccgacgatctgcccctgaacgtttcccgcgagaccctgcagcaacataagctgctgaaggtgatccgtaaaaagctggtgcgcaagaccctcgacatgatcaaaaagatcgctgacgacaagtacaacgacaccttctggaaggaatttggcaccaacatcaaactgggtgtgatcgaggaccactccaaccgcaccagactggccaaactgctgagattccaatcctctcaccaccccaccgacatcacctccctggaccaatacgtggagcgcatgaaagagaaacaggacaaaatctacttcatggccggctcctcccgcaaagaagctgaatcctcccccttcgtcgaacgcctgctgaagaaaggttacgaggtcatctacctgaccgaacccgtggacgagtactgcatccaggcactgcctgagttcgacggtaagcgcttccagaacgtcgctaaggagggagtgaagttcgacgagagcgagaagaccaaggagtcccgcgaagctgtggagaaggagttcgagcccctgttgaactggatgaaggataaggctctgaaggacaagatcgagaaagcagtggtgtcccagaggctgaccgaaagcccctgtgctctggtggctagtcagtacggttggagcggtaacatggaaaggatcatgaaggcacaggcataccagaccggtaaggacattagtaccaactactacgcttcccagaaaaagaccttcgagattaacccccgccaccctctgatcagagacatgctgagacgcatcaaggaggacgaagacgacaaaaccgtcctcgacctggccgtggtgttgttcgagaccgccaccctccgcagcggctacttgctgcctgacaccaaagcgtacggcgacaggatcgagcgcatgctgaggctgagcctgaacatcgaccctgacgctaaggtggaggaggagcccgaggaagagccagaggagaccgcggaggacacaacagaggacactgagcaggacgaggacgaggagatggacgtgggaactgacgaggaagaggaaacagcaaaggagagcacagcagag(SEQ ID NO:16)
编码分离的多肽3的核酸：
atgggtggtggtggttcctgcggtggtggtggttcctgcggtggtggtggttccatggatgacgaggtggatgtggatggtaccgtggaggaggacttgggtaagagcagggagggtagccgcactgatgacgaggttgttcagcgtgaggaggaggctattcagttggacggtttgaacgcaagccagattagggagctgcgtgagaagagcgagaagttcgctttccaggctgaggtgaaccgcatgatgaagttgatcattaacagcttgtacaagaacaaggagatcttcctgagagagctgatctcaaacgcttccgacgccctggacaagatccgcctgatctccctgactgacgagaacgccctgtccggcaacgaagaactgaccgtgaaaatcaaatgcgacaaagaaaagaacctgctccacgttaccgataccggtgtgggcatgacccgcgaggagctcgtgaagaacctgggtaccatcgctaagagcggaacctcggaatttctgaacaagatgacagaagcccaagaagacggtcagtccacctccgagctgattggccagttcggtgtgggtttctactctgctttcctggtggctgacaaggtgatcgtgacctccaaacacaacaacgacacacaacacatctgggagtccgactccaacgaattttccgtgatcgccgacccgcgcggcaacactctgggtagaggtaccaccatcaccctcgtgctcaaagaggaggcctccgactacctcgaactggacaccatcaagaacctggtgaagaagtactcccaattcatcaacttccccatctacgtgtggagttccaagaccgaaaccgtggaagaacctatggaggaggaggaggccgctaaagaggaaaaggaggagtccgatgacgaggctgctgtcgaggaggaagaggaggagaagaagccgaagactaagaaggtggagaagacagtgtgggactgggagctgatgaacgacatcaagccaatctggcagaggccaagcaaggaggtggaggaggatgagtacaaggcattctacaagagcttcagcaaggagagcgacgacccgatggcttacattcacttcaccgctgagggagaggtgacattcaagagcatcctgttcgtgccaaccagcgctccccgcggtctgttcgacgagtacggtagcaagaagagcgattacatcaagctgtacgtccgccgcgtgttcatcacagatgacttccacgacatgatgcctaagtacctcaacttcgtgaagggtgtggtggactccgacgatctgcccctgaacgtttcccgcgagaccctgcagcaacataagctgctgaaggtgatccgtaaaaagctggtgcgcaagaccctcgacatgatcaaaaagatcgctgacgacaagtacaacgacaccttctggaaggaatttggcaccaacatcaaactgggtgtgatcgaggaccactccaaccgcaccagactggccaaactgctgagattccaatcctctcaccaccccaccgacatcacctccctggaccaatacgtggagcgcatgaaagagaaacaggacaaaatctacttcatggccggctcctcccgcaaagaagctgaatcctcccccttcgtcgaacgcctgctgaagaaaggttacgaggtcatctacctgaccgaacccgtggacgagtactgcatccaggcactgcctgagttcgacggtaagcgcttccagaacgtcgctaaggagggagtgaagttcgacgagagcgagaagaccaaggagtcccgcgaagctgtggagaaggagttcgagcccctgttgaactggatgaaggataaggctctgaaggacaagatcgagaaagcagtggtgtcccagaggctgaccgaaagcccctgtgctctggtggctagtcagtacggttggagcggtaacatggaaaggatcatgaaggcacaggcataccagaccggtaaggacattagtaccaactactacgcttcccagaaaaagaccttcgagattaacccccgccaccctctgatcagagacatgctgagacgcatcaaggaggacgaagacgacaaaaccgtcctcgacctggccgtggtgttgttcgagaccgccaccctccgcagcggctacttgctgcctgacaccaaagcgtacggcgacaggatcgagcgcatgctgaggctgagcctgaacatcgaccctgacgctaaggtggaggaggagcccgaggaagagccagaggagaccgcggaggacacaacagaggacactgagcaggacgaggacgaggagatggacgtgggaactgacgaggaagaggaaacagcaaaggagagcacagcagag(SEQ ID NO:17)
编码分离的多肽4的核酸：
atgggtggtggtggttccggtggtggtggttcctgcggtggtggtggttcctgcggtggtggtggttccatggatgacgaggtggatgtggatggtaccgtggaggaggacttgggtaagagcagggagggtagccgcactgatgacgaggttgttcagcgtgaggaggaggctattcagttggacggtttgaacgcaagccagattagggagctgcgtgagaagagcgagaagttcgctttccaggctgaggtgaaccgcatgatgaagttgatcattaacagcttgtacaagaacaaggagatcttcctgagagagctgatctcaaacgcttccgacgccctggacaagatccgcctgatctccctgactgacgagaacgccctgtccggcaacgaagaactgaccgtgaaaatcaaatgcgacaaagaaaagaacctgctccacgttaccgataccggtgtgggcatgacccgcgaggagctcgtgaagaacctgggtaccatcgctaagagcggaacctcggaatttctgaacaagatgacagaagcccaagaagacggtcagtccacctccgagctgattggccagttcggtgtgggtttctactctgctttcctggtggctgacaaggtgatcgtgacctccaaacacaacaacgacacacaacacatctgggagtccgactccaacgaattttccgtgatcgccgacccgcgcggcaacactctgggtagaggtaccaccatcaccctcgtgctcaaagaggaggcctccgactacctcgaactggacaccatcaagaacctggtgaagaagtactcccaattcatcaacttccccatctacgtgtggagttccaagaccgaaaccgtggaagaacctatggaggaggaggaggccgctaaagaggaaaaggaggagtccgatgacgaggctgctgtcgaggaggaagaggaggagaagaagccgaagactaagaaggtggagaagacagtgtgggactgggagctgatgaacgacatcaagccaatctggcagaggccaagcaaggaggtggaggaggatgagtacaaggcattctacaagagcttcagcaaggagagcgacgacccgatggcttacattcacttcaccgctgagggagaggtgacattcaagagcatcctgttcgtgccaaccagcgctccccgcggtctgttcgacgagtacggtagcaagaagagcgattacatcaagctgtacgtccgccgcgtgttcatcacagatgacttccacgacatgatgcctaagtacctcaacttcgtgaagggtgtggtggactccgacgatctgcccctgaacgtttcccgcgagaccctgcagcaacataagctgctgaaggtgatccgtaaaaagctggtgcgcaagaccctcgacatgatcaaaaagatcgctgacgacaagtacaacgacaccttctggaaggaatttggcaccaacatcaaactgggtgtgatcgaggaccactccaaccgcaccagactggccaaactgctgagattccaatcctctcaccaccccaccgacatcacctccctggaccaatacgtggagcgcatgaaagagaaacaggacaaaatctacttcatggccggctcctcccgcaaagaagctgaatcctcccccttcgtcgaacgcctgctgaagaaaggttacgaggtcatctacctgaccgaacccgtggacgagtactgcatccaggcactgcctgagttcgacggtaagcgcttccagaacgtcgctaaggagggagtgaagttcgacgagagcgagaagaccaaggagtcccgcgaagctgtggagaaggagttcgagcccctgttgaactggatgaaggataaggctctgaaggacaagatcgagaaagcagtggtgtcccagaggctgaccgaaagcccctgtgctctggtggctagtcagtacggttggagcggtaacatggaaaggatcatgaaggcacaggcataccagaccggtaaggacattagtaccaactactacgcttcccagaaaaagaccttcgagattaacccccgccaccctctgatcagagacatgctgagacgcatcaaggaggacgaagacgacaaaaccgtcctcgacctggccgtggtgttgttcgagaccgccaccctccgcagcggctacttgctgcctgacaccaaagcgtacggcgacaggatcgagcgcatgctgaggctgagcctgaacatcgaccctgacgctaaggtggaggaggagcccgaggaagagccagaggagaccgcggaggacacaacagaggacactgagcaggacgaggacgaggagatggacgtgggaactgacgaggaagaggaaacagcaaaggagagcacagcagag(SEQ ID NO:18)
本发明的另一方面涉及一种分离的蛋白，其为本发明中任一项所述的分离的多肽的二聚体。
本发明的再一方面涉及一种分离的多核苷酸，其编码本发明中任一项所述的分离的多肽。
本发明的再一方面涉及一种核酸构建体，其含有本发明的多核苷酸；
优选地，所述核酸构建体为重组载体；
优选地，所述重组载体为重组表达载体；
优选地，所述重组载体为重组昆虫病毒表达载体；
优选地，所述重组昆虫病毒表达载体为可在细菌和昆虫细胞之间转移和扩增的病毒穿梭质粒；
优选地，所述病毒穿梭质粒为杆状病毒穿梭质粒。
本发明的再一方面涉及一种转化的细胞，其含有本发明的多核苷酸，或者本发明的核酸构建体；
优选地，所述转化的细胞为转化的昆虫细胞，优选为转化的Sf9细胞。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的分离的多肽或者本发明的分离的蛋白，以及至少一种药学上可接受的辅料。
本发明的再一方面涉及本发明中任一项所述的分离的多肽或者本发明的分离的蛋白在制备治疗或预防自身免疫性疾病的药物中的用途；
优选地，所述自身免疫性疾病选自系统性红斑狼疮、1型糖尿病、类风湿性关节炎、强直性脊柱炎、多发性硬化症、银屑病、白癜风、红皮病、炎症性肠病、溃疡性结肠炎、克罗恩病、重症肌无力、自身免疫性肝炎、自身免疫性肾炎、渐冻症和多发性肌炎。
本发明的分离的多肽或者分离的蛋白的免疫学功能包括但不限于选自下列的一种或多种：
1)诱导调节性T细胞活化；
2)在受试者中治疗自身免疫性疾病(例如系统性红斑狼疮、1型糖尿病、类风湿性关节炎、强直性脊柱炎)或缓解自身免疫性疾病的一种或多种症状；
3)在受试者中降低抗双链DNA抗体的水平；
4)在受试者中降低尿蛋白的水平；
5)在受试者中降低血糖。
发明的有益效果
与现有技术相比，本发明的优化后的热休克蛋白gp96具有显著的免疫学功能。本发明的蛋白诱导调节性T细胞活化的能力明显提高，具有更优的免疫免疫调节活性，从而大幅提高了治疗自身免疫性疾病的效果。因此，本发明的蛋白特别适用于自身免疫性疾病的治疗，具有重大的临床价值。
附图说明
图1：显示非还原SDS-PAGE电泳分析检测gp96和gp96-plus1-4分子量结果。从左侧起，第一泳道为分子量标记，第二泳道为gp96，第三至六泳道为gp96-plus1-4，考马斯亮蓝染色。
图2A：100μg剂量的gp96-plus1-4和gp96蛋白免疫小鼠后的调节性T细胞(Tregs)百分比(％CD3+CD4+CD25+Foxp3+Tregs/CD3+CD4+T细胞)(*,P<0.05；****,p<0.0001)。
图2B：300μg剂量的gp96-plus1-4和gp96蛋白免疫小鼠后的调节性T细胞(Tregs)百分比(％CD3+CD4+CD25+Foxp3+Tregs/CD3+CD4+T细胞)(*,P<0.05；****,p<0.0001)。
图2C：500μg剂量的gp96-plus1-4和gp96蛋白免疫小鼠后的调节性T细胞(Tregs)百分比(％CD3+CD4+CD25+Foxp3+Tregs/CD3+CD4+T细胞)(*,P<0.05；****,p<0.0001)。
图3：不同融合蛋白gp96-plus和gp96蛋白免疫Lyn(-/-)小鼠后小鼠血清中抗dsDNA抗体的水平(*,P<0.05；***,p<0.001)。
图4：不同融合蛋白gp96-plus和gp96蛋白免疫Lyn(-/-)小鼠后小鼠尿液中蛋白的水平(*,P<0.05；**,p<0.01)。
图5：不同融合蛋白gp96-plus和gp96蛋白免疫后NOD小鼠治疗模型血液中的血糖水平(**,p<0.01)。
图6：不同融合蛋白gp96-plus和gp96蛋白免疫后诱导性小鼠关节炎症模型中的类风湿性炎症指数检测结果(**,p<0.01)。
图7：不同融合蛋白gp96-plus和gp96蛋白免疫后诱导性小鼠强直性脊柱炎模型中的外周关节炎症指数检测结果(**,p<0.01)。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
以下实施例中所涉及的实验材料来源如下：
雌性Lyn(-/-)敲除鼠系统性红斑狼疮模型参见如下文献：HibbsML,etal.MultipledefectsintheimmunesystemofLyn-deficientmice,culminatinginautoimmunedisease.Cell(1995)83(2):301–11；YuCC,YenTS,LowellCA,DeFrancoAL.Lupus-likekidneydiseaseinmicedeficientintheSrcfamilytyrosinekinasesLynandFyn.CurrBiol.2001Jan9；11(1):34-8.
雌性NOD/LTJ小鼠参见如下文献：TerryL.etal.ITheNonobeseDiabeticMouseasaModelofAutoimmuneDiabetes:ImmuneDysregulationGetstheNOD.Immunity,December,1997，Vol.7,727-738；MichelleSolomon,NoraSarvetnick,etal.ThePathogenesisofDiabetesintheNODMouse.Immunology,2004，84:239-64.
Sf9细胞购自Invitrogen公司，目录号：11496-015；
pFastBacTM1质粒购自Invitrogen公司，产品目录号：10359-016；
DH10BacTM感受态细胞购自Invitrogen公司，产品目录号：10361-012；
Insect-XPRESSTMProtein-freeInsectCellsmediumwithL-Glutamine购自LONZA公司，产品目录号：12-730Q；
超滤管购自MerckMillipore公司，产品目录号：UFC905096；
ELISA试剂盒购自eBioscience公司，产品目录号：BMS614INST；
Ni亲和层析预装柱购自阿拉丁公司，产品目录号：N5289-01；
Superdex20010/300GL分子筛层析柱购自GE公司，产品目录号：17517501；
大肠杆菌DH10Bac感受态细胞购自北京原平皓生物技术有限公司，产品目录号：CL108-01。
实施例1：gp96-plus1-4蛋白的制备
1、重组质粒的构建
将热休克蛋白gp96(SEQ ID NO:1)的N端分别与4个不同长度的片段(SEQ ID NOs:3-6)经人工碱基合成串联连接在一起(金斯瑞生物科技股份有限公司)，再将目的片段连接至昆虫细胞表达载体Pfastbac1中，构建重组表达载体，命名为Pfastbac1-gp96-plus。将重组载体分别转化至DH10BacTM感受态细胞，经重组筛选得到重组杆粒DNA。在以上各蛋白的C端添加6xHis标签。
2、gp96-plus蛋白的表达
2.1将重组杆粒DNA转染贴壁Sf9细胞(每8×105个Sf9细胞约转染2μg重组质粒；转染过程中，转染试剂为CellfectinIIreagent(购自Lifetechnologies，产品目录号：10362-100))，27℃孵育72h，离心，上清液即为P1代病毒。
2.2将Sf9细胞悬浮液1(含8×106个Sf9细胞)27℃培养1～5h，得到贴壁培养细胞；然后向所述贴壁培养细胞中加入P1代病毒(剂量为0.05～0.1MOI)，27℃孵育72h，4000rpm离心5min，上清液即为P2代病毒。
2.3向Sf9细胞悬浮液2(含8×106个Sf9细胞)中加入P2代病毒(剂量为0.05～0.1MOI)，27℃、100～120rpm培养72h，4000rpm离心5min，上清液即为P3代病毒。
3、gp96-plus蛋白的纯化。
3.1向300ml的Sf9细胞悬浮液3(含Sf9细胞2～4×106个/ml)中加入P3代病毒(剂量为0.05MOI)，27℃、100～120rpm培养72～96h，得到悬浮液。
3.2取所述悬浮液，7000rpm离心20min，获得上清液。
3.3取所述上清液，经0.22mm滤膜过滤，获得上样液。
3.4将所述上样液上样于Ni亲和层析柱。上样流速控制在1ml/min，然后用不含咪唑的Tris-HCl缓冲液冲洗，再用含有咪唑的Tris-HCl缓冲液洗脱并收获洗脱液。将洗脱液用截留分子量为50KD的超滤管进行超滤换液得到重组gp96-plus蛋白置换液。
3.5将所述置换液上样于HiTrap-QSepharose离子交换层析柱(流速为1ml/min)，然后先用5ml的pH7.5、200mM的PBS缓冲液冲洗(流速为1ml/min)；再用10ml的pH7.5、300mM的PBS缓冲液冲洗(流速为1ml/min)；最后用3ml的pH7.5、600mM的PBS缓冲液冲洗(流速为1ml/min)，收集过柱后溶液并采用截留分子量为50KD的超滤管进行超滤浓缩，得到1ml左右的浓缩液。所述浓缩液即含有重组热休克蛋白gp96，分别命名为gp96-plus1至gp96-plus4。采用BCA法测定蛋白溶液中的蛋白浓度，最后分装，蛋白浓度为1mg/ml，贮存于-80℃。gp96-plus1至gp96-plus4用于下面的实验。
4、非还原SDS-PAGE电泳分析
将gp96-plus1-4蛋白和全长gp96的溶液进行非还原SDS-PAGE电泳分析，实验结果见图1。
结果表明，gp96的溶液在分子量96kDa位置显示条带，gp96-plus1-4溶液在分子量190kDa位置显示条带，说明gp96-plus1-4四种蛋白为二聚体形式。蛋白纯度大于95％。
实施例2：gp96-plus1-4蛋白诱导小鼠调节性T细胞活性的评价
本实施例考察单独gp96蛋白(SEQ ID NO:1)、gp96-plus1(gp96与多肽片段1连接，SEQ ID NO:11)、gp96-plus2(gp96与多肽片段2连接，SEQ ID NO:12)、gp96-plus3(gp96与多肽片段3连接，SEQ ID NO:13)、gp96-plus4(gp96与多肽片段4连接，SEQ ID NO:14)蛋白诱导小鼠产生调节性T细胞的情况。
1、小鼠分组免疫
将30只六周龄体重为14～16g的C57BL/6小鼠(购自北京维通利华实验动物技术有限公司)随机分成gp96处理组、gp96-plus1处理组、gp96-plus2处理组、gp96-plus3处理组、gp96-plus4处理组和对照组(每组5只小鼠)，分别进行如下处理：
gp96-plus1处理组至gp96-plus4处理组：小鼠生长至第9周龄，腹部皮下分别注射实施例1制备获得的经纯化的gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4的溶液，记为实验第1天；实验第8天后，再次腹部皮下注射实施例1制备获得的经纯化的gp96-plus1的溶液；实验第22天，再次腹部皮下分别注射实施例1制备获得的经纯化的gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4的溶液。每次注射剂量分别为100μg、300μg和500μg蛋白/只小鼠。
gp96处理组：小鼠生长至第9周龄，腹部皮下注射实施例1制备的经纯化的gp96，记为实验第1天；实验第8天后，再次腹部皮下注射实施例1制备的经纯化的gp96的溶液；实验第22天，再次腹部皮下注射实施例1制备的全长gp96的溶液。每次注射剂量为100μg、300μg和500μg gp96蛋白/只小鼠。
对照组：实验第1天腹部皮下注射pH7.2、0.01mol/LPBS缓冲液，实验第8天，再次腹部皮下注射pH7.2、0.01mol/LPBS缓冲液；实验第22天，再次腹部皮下注射pH7.2、0.01mol/LPBS缓冲液。每次注射PBS剂量均为100μl/只、300μg/只或500μl/只。
2、免疫效果评价
在第25天处死小鼠，取小鼠脾脏分离制备小鼠脾脏淋巴细胞，利用流式细胞仪分析小鼠的调节性T细胞(Tregs)百分比(％CD3+CD4+CD25+Foxp3+Tregs/CD3+CD4+T细胞)。调节性T细胞的分离和检测方法详见XinghuiLi,etal.2013.InductionofregulatoryTcellsbyhigh-dosegp96supp ressesmurineliverimmunehyperactivation.PLoSOne.8(7):e68997。
结果如图2A至图2C所示。
结果显示，gp96和gp96-plus蛋白免疫小鼠后的调节性T细胞(Tregs)百分比。结果表明，gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4蛋白免疫组调节性T细胞水平明显高于gp96免疫组(P<0.0001)，而且gp96-plus3、gp96-plus4蛋白高于gp96-plus1、gp96-plus2蛋白处理小鼠的调节性T细胞百分比。
结果还显示，注射剂量100μg和500μg gp96-plus的小鼠的调节性T细胞水平也明显高于gp96治疗组，说明注射剂量/只小鼠为100μg、300μg或500μg gp96-plus对小鼠调节性T细胞增加效果相对于gp96均有明显的提高；300μg的效果优于100μg，但是500μg和300μg的结果接近。
实施例3：gp96-plus1-4蛋白在治疗系统性红斑狼疮中的应用
1、小鼠分组免疫
将60只六周龄体重为14～16g的雌性Lyn(-/-)小鼠随机分成gp96-plus1组、gp96-plus2组、gp96-plus3组、gp96-plus4组及gp96免疫组和对照组(PBS)(每组10只)，分别进行如下处理：小鼠生长至第7周龄、第8周龄、第10周龄、第12周龄、第14周龄、第16周龄时，分别在小鼠腹部皮下注射实施例1制备的经纯化的gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4、gp96、PBS，每次注射剂量/只小鼠为300μg。
小鼠生长至20周龄时，眼眶取血200μl。室温静置30min，3000rpm离心20min，取上清。
2、抗双链DNA抗体滴度的分析
血清中抗双链DNA抗体的水平采用酶联免疫吸附(ELISA)方法测定，步骤如下：
2.1向96孔酶标板(购自Nunc,RochesterNY,USA)中加入2.5μg/ml的小牛胸腺dsDNA标准品(购自SigmaAldrich,USA，货号：D8899-1MG)，每孔100μl。4℃包被过夜。
2.2加入2％牛血清白蛋白(购自Invitrogen,USA)100μl，37℃孵育60分钟。
2.3加入血清样本50μl(1：50比例稀释)和抗dsDNA抗体标准品(购自ChemiconInternational,USA，clone号：16-13)，37℃孵育60分钟。
2.4弃去孔内液体，每孔加入200μl的1×洗涤液(PBST，PBS按0.1％加入Triton-X100)，静置30秒，甩干，重复洗涤5次。最后在吸水纸上拍干。
2.5加入HRP标记的抗小鼠二抗(购自中杉金桥，1：5000稀释)，每孔50μl。37℃孵育60分钟。
2.6弃去孔内液体，每孔加入200μl的1×洗涤液，静置30秒，甩干，重复洗涤5次。最后在吸水纸上拍干。
2.7每孔加入100μl底物(1×TMBELISASubstrateSolution，购自eBioscience，货号：00-4201-56)。37℃孵育15分钟。
2.8弃去孔内液体，每孔加入50μl终止液(2MH2SO4)，混匀，酶标仪上450nm波长下读数。测定应在加终止液后15分钟以内进行。
3、尿蛋白的测定
尿蛋白检测试剂盒购自南京建成生物工程研究所(货号：C035-2)
3.1取三个管，分别标记为空白管，标准管和测定管。空白管加入双蒸水0.05ml，CBB试剂3.0ml。标准管加入563mg/L蛋白标准液0.05ml，CBB试剂3.0ml。测定管加入尿液样本0.05ml，CBB试剂3.0ml。
3.2充分混匀，放置5分钟。分光光度计用波长595nm检测各管的吸光度。
尿蛋白浓度(mg/L)＝(测定OD值-空白OD值)/(标准OD值-空白OD值)×标准品浓度
4、效果评价
小鼠的抗双链DNA抗体检测结果见图3。结果表明，注射300μg gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4治疗小鼠的抗双链DNA抗体滴度显著小于对照组(PBS)(P<0.001)，同时也明显低于gp96治疗组(P<0.001)，这说明gp96-plus治疗系统性红斑狼疮的效果优于单独gp96。另外，gp96-plus3、gp96-plus4的疗效优于gp96-plus1、gp96-plus2。
小鼠的尿蛋白检测结果见图4。
结果表明，gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4蛋白治疗小鼠的尿蛋白水平显著低于对照组(PBS)(P<0.001)，同时也明显低于gp96治疗组(P<0.01)，这说明gp96-plus治疗系统性红斑狼疮的效果优于gp96。另外，gp96-plus3、gp96-plus4的疗效优于gp96-plus1、gp96-plus2。
实施例4：gp96-plus1-4蛋白在治疗1型糖尿病中的应用
1、小鼠免疫
将60只六周龄体重为14～16g的NOD小鼠治疗模型随机分成gp96-plus1组、gp96-plus2组、gp96-plus3组、gp96-plus4组和gp96免疫组和对照组(PBS)(每组10只)，分别进行如下处理：小鼠生长至第14周龄、第15周龄、第17周龄、第19周龄、第21周龄时，分别在小鼠腹部皮下注射实施例1制备的gp96-plus1-4、全长gp96或PBS，每次注射剂量/只小鼠为300μg。
从最后一次免疫后的第1周开始监测小鼠血糖值，每周检测1次。取小鼠尾静脉血1滴，用血糖仪(德国罗氏公司ACCU-Performa)参照说明书测定血糖值。
2、效果评价
小鼠血糖浓度检测结果见图5。
结果显示，gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4蛋白免疫后小鼠的血糖浓度随着时间变化而逐渐降低，最终浓度显著低于对照组(PBS)(P<0.001)，同时所有gp96-plus免疫组小鼠血糖水平也明显低于gp96免疫组(P<0.01)。
上述结果表明，用gp96-plus免疫小鼠，能够有效地治疗小鼠的T1D，其治疗效果优于gp96。
实施例5：gp96-plus1-4蛋白在治疗诱导性小鼠关节炎症中的应用
1、小鼠分组免疫
选取60只雄性DBA/1小鼠(北京维通利华实验动物技术有限公司)进行CIA造模。将10mg牛Ⅱ型胶原溶于0.01mmol/L的醋酸溶液5mL，4℃摇床过夜，取5mL含2mg/mL的完全弗氏佐剂等体积混合，充分乳化制得抗原乳剂，并置于冰箱4℃保存备用。首次免疫于小鼠尾根部皮下注射抗原乳剂0.1mL，造模第21天使用0.1mLⅡ型胶原和不完全弗氏佐剂混合的乳剂进行加强免疫一次，第28天左右筛选出60只发病情况相近的小鼠，随机分成gp96-plus1组、gp96-plus2组、gp96-plus3组、gp96-plus4组、gp96免疫组和对照组(PBS)(每组10只)，分组完成后第0、1、2、3、4、5、6、7、8或9周分别在小鼠腹部皮下注射实施例1制备的蛋白gp96-plus1-4、gp96或PBS，每次注射剂量为300μg/只小鼠。
指标检测，关节炎指数:小鼠给药后第0、7、14、21、28、35、42、49、56和63天给予关节炎症指数评分，评分标准：0分:没有明显的关节红肿；1分：踝关节或趾关节轻度红肿；2分：从趾关节到踝关节轻度红肿；3分：从趾关节到踝关节中度红胀；4分：严重的踝关节到整个足掌的红肿，每只小鼠最高评分16分。
2、效果评价
小鼠关节炎症指数结果见图6。
结果显示，gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4蛋白免疫后小鼠的关节炎症指数显著低于模型组(P<0.001)，同时也低于gp96治疗组(P<0.05)，同时gp96-plus3、gp96-plus4治疗组的关节炎症指数低于gp96-plus1、gp96-plus2治疗组。此外，足掌厚度的检测结果显示，gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4蛋白免疫后小鼠的足掌厚度显著低于模型组(P<0.001)，同时也低于gp96治疗组(P<0.01)。
注射gp96-plus剂量为100μg或500μg的小鼠治疗类风湿也得到类似的治疗结果。上述结果表明，用gp96-plus免疫小鼠，能够有效地治疗小鼠的诱导性关节炎症，其治疗效果优于gp96。
实施例6：gp96-plus1-4蛋白在治疗诱导性强直性脊柱炎中的应用
1、小鼠分组免疫
选取60只小鼠六月龄的雌性BALB/c小鼠进行蛋白聚糖(PG)诱导造模，诱导小鼠强制性脊柱炎。将100μg软骨蛋白聚糖(购自Sigma，货号：P5864)与1g gDDA佐剂(购自Avanti，货号：890810p)混合在200μL的生理盐水中，在第0、21和42天注射入小鼠的腹腔。第49天左右筛选出60只发病情况相近的小鼠，随机分成gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4蛋白、gp96免疫组、对照组(PBS)(每组10只)，分组完成后第0、1、2、3、4、5、6、7、8、9周分别在小鼠腹部皮下注射实施例1制备的gp96-plus1-4蛋白、gp96或PBS，每次注射剂量为300μg/只小鼠。
指标检测：小鼠给药后每周进行外周关节炎的记录，连续7周。外周关节炎指数评分标准：无任何炎性肿胀为0分，一个脚趾受累表现肿胀为1分，大于一个脚趾受累为2分，足趾僵直失去关节功能为3分，踝关节肿胀僵直为4分，对模型小鼠的四肢进行评分，总评分在0-16之间。
2、效果评价
结果如图7所示。
结果显示，gp96-plus1、gp96-plus2、gp96-plus3、gp96-plus4蛋白免疫后强直性脊柱炎模型小鼠的外周关节炎指数显著低于模型组(P<0.001)，同时也低于单独gp96治疗组(P<0.01)。
上述结果表明，用上述gp96-plus免疫小鼠，能够有效地治疗小鼠的诱导性强直性脊柱炎，其治疗效果优于单独gp96。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
SEQUENCE LISTING
<110> 佛山热休生物技术有限公司
<120> 含有热休克蛋白gp96的多肽、药物组合物和用途
<130> IDC220002
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 779
<212> PRT
<213> Artificial Sequence
<220>
<223> gp96蛋白
<400> 1
Met Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu Glu Asp Leu Gly
1 5 10 15
Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val Val Gln Arg Glu
20 25 30
Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser Gln Ile Arg Glu
35 40 45
Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala Glu Val Asn Arg
50 55 60
Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn Lys Glu Ile Phe
65 70 75 80
Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu Asp Lys Ile Arg
85 90 95
Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly Asn Glu Glu Leu
100 105 110
Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu Leu His Val Thr
115 120 125
Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val Lys Asn Leu Gly
130 135 140
Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn Lys Met Thr Glu
145 150 155 160
Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile Gly Gln Phe Gly
165 170 175
Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys Val Ile Val Thr
180 185 190
Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu Ser Asp Ser Asn
195 200 205
Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr Leu Gly Arg Gly
210 215 220
Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser Asp Tyr Leu Glu
225 230 235 240
Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser Gln Phe Ile Asn
245 250 255
Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr Val Glu Glu Pro
260 265 270
Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu Glu Ser Asp Asp
275 280 285
Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys Pro Lys Thr Lys
290 295 300
Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met Asn Asp Ile Lys
305 310 315 320
Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu Asp Glu Tyr Lys
325 330 335
Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp Pro Met Ala Tyr
340 345 350
Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys Ser Ile Leu Phe
355 360 365
Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu Tyr Gly Ser Lys
370 375 380
Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val Phe Ile Thr Asp
385 390 395 400
Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe Val Lys Gly Val
405 410 415
Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg Glu Thr Leu Gln
420 425 430
Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu Val Arg Lys Thr
435 440 445
Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr Asn Asp Thr Phe
450 455 460
Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val Ile Glu Asp His
465 470 475 480
Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe Gln Ser Ser His
485 490 495
His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val Glu Arg Met Lys
500 505 510
Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser Ser Arg Lys Glu
515 520 525
Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu
530 535 540
Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu
545 550 555 560
Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val
565 570 575
Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu
580 585 590
Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys
595 600 605
Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro
610 615 620
Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg
625 630 635 640
Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn
645 650 655
Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His Pro
660 665 670
Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys
675 680 685
Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg
690 695 700
Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu
705 710 715 720
Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu
725 730 735
Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu
740 745 750
Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val Gly Thr Asp Glu
755 760 765
Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu
770 775
<210> 2
<211> 2337
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码gp96的核酸
<400> 2
atggatgacg aggtggatgt ggatggtacc gtggaggagg acttgggtaa gagcagggag 60
ggtagccgca ctgatgacga ggttgttcag cgtgaggagg aggctattca gttggacggt 120
ttgaacgcaa gccagattag ggagctgcgt gagaagagcg agaagttcgc tttccaggct 180
gaggtgaacc gcatgatgaa gttgatcatt aacagcttgt acaagaacaa ggagatcttc 240
ctgagagagc tgatctcaaa cgcttccgac gccctggaca agatccgcct gatctccctg 300
actgacgaga acgccctgtc cggcaacgaa gaactgaccg tgaaaatcaa atgcgacaaa 360
gaaaagaacc tgctccacgt taccgatacc ggtgtgggca tgacccgcga ggagctcgtg 420
aagaacctgg gtaccatcgc taagagcgga acctcggaat ttctgaacaa gatgacagaa 480
gcccaagaag acggtcagtc cacctccgag ctgattggcc agttcggtgt gggtttctac 540
tctgctttcc tggtggctga caaggtgatc gtgacctcca aacacaacaa cgacacacaa 600
cacatctggg agtccgactc caacgaattt tccgtgatcg ccgacccgcg cggcaacact 660
ctgggtagag gtaccaccat caccctcgtg ctcaaagagg aggcctccga ctacctcgaa 720
ctggacacca tcaagaacct ggtgaagaag tactcccaat tcatcaactt ccccatctac 780
gtgtggagtt ccaagaccga aaccgtggaa gaacctatgg aggaggagga ggccgctaaa 840
gaggaaaagg aggagtccga tgacgaggct gctgtcgagg aggaagagga ggagaagaag 900
ccgaagacta agaaggtgga gaagacagtg tgggactggg agctgatgaa cgacatcaag 960
ccaatctggc agaggccaag caaggaggtg gaggaggatg agtacaaggc attctacaag 1020
agcttcagca aggagagcga cgacccgatg gcttacattc acttcaccgc tgagggagag 1080
gtgacattca agagcatcct gttcgtgcca accagcgctc cccgcggtct gttcgacgag 1140
tacggtagca agaagagcga ttacatcaag ctgtacgtcc gccgcgtgtt catcacagat 1200
gacttccacg acatgatgcc taagtacctc aacttcgtga agggtgtggt ggactccgac 1260
gatctgcccc tgaacgtttc ccgcgagacc ctgcagcaac ataagctgct gaaggtgatc 1320
cgtaaaaagc tggtgcgcaa gaccctcgac atgatcaaaa agatcgctga cgacaagtac 1380
aacgacacct tctggaagga atttggcacc aacatcaaac tgggtgtgat cgaggaccac 1440
tccaaccgca ccagactggc caaactgctg agattccaat cctctcacca ccccaccgac 1500
atcacctccc tggaccaata cgtggagcgc atgaaagaga aacaggacaa aatctacttc 1560
atggccggct cctcccgcaa agaagctgaa tcctccccct tcgtcgaacg cctgctgaag 1620
aaaggttacg aggtcatcta cctgaccgaa cccgtggacg agtactgcat ccaggcactg 1680
cctgagttcg acggtaagcg cttccagaac gtcgctaagg agggagtgaa gttcgacgag 1740
agcgagaaga ccaaggagtc ccgcgaagct gtggagaagg agttcgagcc cctgttgaac 1800
tggatgaagg ataaggctct gaaggacaag atcgagaaag cagtggtgtc ccagaggctg 1860
accgaaagcc cctgtgctct ggtggctagt cagtacggtt ggagcggtaa catggaaagg 1920
atcatgaagg cacaggcata ccagaccggt aaggacatta gtaccaacta ctacgcttcc 1980
cagaaaaaga ccttcgagat taacccccgc caccctctga tcagagacat gctgagacgc 2040
atcaaggagg acgaagacga caaaaccgtc ctcgacctgg ccgtggtgtt gttcgagacc 2100
gccaccctcc gcagcggcta cttgctgcct gacaccaaag cgtacggcga caggatcgag 2160
cgcatgctga ggctgagcct gaacatcgac cctgacgcta aggtggagga ggagcccgag 2220
gaagagccag aggagaccgc ggaggacaca acagaggaca ctgagcagga cgaggacgag 2280
gagatggacg tgggaactga cgaggaagag gaaacagcaa aggagagcac agcagag 2337
<210> 3
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> 多肽片段1
<400> 3
Gly Gly Gly Gly Ser Cys Gly Gly Gly Gly Ser
1 5 10
<210> 4
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> 多肽片段2
<400> 4
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Gly Gly Gly Gly Ser
1 5 10 15
<210> 5
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> 多肽片段3
<400> 5
Gly Gly Gly Gly Ser Cys Gly Gly Gly Gly Ser Cys Gly Gly Gly Gly
1 5 10 15
Ser
<210> 6
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> 多肽片段4
<400> 6
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Gly Gly Gly Gly Ser
1 5 10 15
Cys Gly Gly Gly Gly Ser
20
<210> 7
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码多肽片段1的核酸序列
<400> 7
ggtggtggtg gttcctgcgg tggtggtggt tcc 33
<210> 8
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码多肽片段2的核酸序列
<400> 8
ggtggtggtg gttccggtgg tggtggttcc tgcggtggtg gtggttcc 48
<210> 9
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码多肽片段3的核酸序列
<400> 9
ggtggtggtg gttcctgcgg tggtggtggt tcctgcggtg gtggtggttc c 51
<210> 10
<211> 66
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码多肽片段4的核酸序列
<400> 10
ggtggtggtg gttccggtgg tggtggttcc tgcggtggtg gtggttcctg cggtggtggt 60
ggttcc 66
<210> 11
<211> 791
<212> PRT
<213> Artificial Sequence
<220>
<223> 分离的多肽1
<400> 11
Met Gly Gly Gly Gly Ser Cys Gly Gly Gly Gly Ser Met Asp Asp Glu
1 5 10 15
Val Asp Val Asp Gly Thr Val Glu Glu Asp Leu Gly Lys Ser Arg Glu
20 25 30
Gly Ser Arg Thr Asp Asp Glu Val Val Gln Arg Glu Glu Glu Ala Ile
35 40 45
Gln Leu Asp Gly Leu Asn Ala Ser Gln Ile Arg Glu Leu Arg Glu Lys
50 55 60
Ser Glu Lys Phe Ala Phe Gln Ala Glu Val Asn Arg Met Met Lys Leu
65 70 75 80
Ile Ile Asn Ser Leu Tyr Lys Asn Lys Glu Ile Phe Leu Arg Glu Leu
85 90 95
Ile Ser Asn Ala Ser Asp Ala Leu Asp Lys Ile Arg Leu Ile Ser Leu
100 105 110
Thr Asp Glu Asn Ala Leu Ser Gly Asn Glu Glu Leu Thr Val Lys Ile
115 120 125
Lys Cys Asp Lys Glu Lys Asn Leu Leu His Val Thr Asp Thr Gly Val
130 135 140
Gly Met Thr Arg Glu Glu Leu Val Lys Asn Leu Gly Thr Ile Ala Lys
145 150 155 160
Ser Gly Thr Ser Glu Phe Leu Asn Lys Met Thr Glu Ala Gln Glu Asp
165 170 175
Gly Gln Ser Thr Ser Glu Leu Ile Gly Gln Phe Gly Val Gly Phe Tyr
180 185 190
Ser Ala Phe Leu Val Ala Asp Lys Val Ile Val Thr Ser Lys His Asn
195 200 205
Asn Asp Thr Gln His Ile Trp Glu Ser Asp Ser Asn Glu Phe Ser Val
210 215 220
Ile Ala Asp Pro Arg Gly Asn Thr Leu Gly Arg Gly Thr Thr Ile Thr
225 230 235 240
Leu Val Leu Lys Glu Glu Ala Ser Asp Tyr Leu Glu Leu Asp Thr Ile
245 250 255
Lys Asn Leu Val Lys Lys Tyr Ser Gln Phe Ile Asn Phe Pro Ile Tyr
260 265 270
Val Trp Ser Ser Lys Thr Glu Thr Val Glu Glu Pro Met Glu Glu Glu
275 280 285
Glu Ala Ala Lys Glu Glu Lys Glu Glu Ser Asp Asp Glu Ala Ala Val
290 295 300
Glu Glu Glu Glu Glu Glu Lys Lys Pro Lys Thr Lys Lys Val Glu Lys
305 310 315 320
Thr Val Trp Asp Trp Glu Leu Met Asn Asp Ile Lys Pro Ile Trp Gln
325 330 335
Arg Pro Ser Lys Glu Val Glu Glu Asp Glu Tyr Lys Ala Phe Tyr Lys
340 345 350
Ser Phe Ser Lys Glu Ser Asp Asp Pro Met Ala Tyr Ile His Phe Thr
355 360 365
Ala Glu Gly Glu Val Thr Phe Lys Ser Ile Leu Phe Val Pro Thr Ser
370 375 380
Ala Pro Arg Gly Leu Phe Asp Glu Tyr Gly Ser Lys Lys Ser Asp Tyr
385 390 395 400
Ile Lys Leu Tyr Val Arg Arg Val Phe Ile Thr Asp Asp Phe His Asp
405 410 415
Met Met Pro Lys Tyr Leu Asn Phe Val Lys Gly Val Val Asp Ser Asp
420 425 430
Asp Leu Pro Leu Asn Val Ser Arg Glu Thr Leu Gln Gln His Lys Leu
435 440 445
Leu Lys Val Ile Arg Lys Lys Leu Val Arg Lys Thr Leu Asp Met Ile
450 455 460
Lys Lys Ile Ala Asp Asp Lys Tyr Asn Asp Thr Phe Trp Lys Glu Phe
465 470 475 480
Gly Thr Asn Ile Lys Leu Gly Val Ile Glu Asp His Ser Asn Arg Thr
485 490 495
Arg Leu Ala Lys Leu Leu Arg Phe Gln Ser Ser His His Pro Thr Asp
500 505 510
Ile Thr Ser Leu Asp Gln Tyr Val Glu Arg Met Lys Glu Lys Gln Asp
515 520 525
Lys Ile Tyr Phe Met Ala Gly Ser Ser Arg Lys Glu Ala Glu Ser Ser
530 535 540
Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu Val Ile Tyr Leu
545 550 555 560
Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu Pro Glu Phe Asp
565 570 575
Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val Lys Phe Asp Glu
580 585 590
Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu Lys Glu Phe Glu
595 600 605
Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys Asp Lys Ile Glu
610 615 620
Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro Cys Ala Leu Val
625 630 635 640
Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg Ile Met Lys Ala
645 650 655
Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn Tyr Tyr Ala Ser
660 665 670
Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His Pro Leu Ile Arg Asp
675 680 685
Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys Thr Val Leu Asp
690 695 700
Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg Ser Gly Tyr Leu
705 710 715 720
Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu Arg Met Leu Arg
725 730 735
Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu Glu Glu Pro Glu
740 745 750
Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu Asp Thr Glu Gln
755 760 765
Asp Glu Asp Glu Glu Met Asp Val Gly Thr Asp Glu Glu Glu Glu Thr
770 775 780
Ala Lys Glu Ser Thr Ala Glu
785 790
<210> 12
<211> 796
<212> PRT
<213> Artificial Sequence
<220>
<223> 分离的多肽2
<400> 12
Met Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Gly Gly Gly Gly
1 5 10 15
Ser Met Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu Glu Asp Leu
20 25 30
Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val Val Gln Arg
35 40 45
Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser Gln Ile Arg
50 55 60
Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala Glu Val Asn
65 70 75 80
Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn Lys Glu Ile
85 90 95
Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu Asp Lys Ile
100 105 110
Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly Asn Glu Glu
115 120 125
Leu Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu Leu His Val
130 135 140
Thr Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val Lys Asn Leu
145 150 155 160
Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn Lys Met Thr
165 170 175
Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile Gly Gln Phe
180 185 190
Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys Val Ile Val
195 200 205
Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu Ser Asp Ser
210 215 220
Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr Leu Gly Arg
225 230 235 240
Gly Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser Asp Tyr Leu
245 250 255
Glu Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser Gln Phe Ile
260 265 270
Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr Val Glu Glu
275 280 285
Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu Glu Ser Asp
290 295 300
Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys Pro Lys Thr
305 310 315 320
Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met Asn Asp Ile
325 330 335
Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu Asp Glu Tyr
340 345 350
Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp Pro Met Ala
355 360 365
Tyr Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys Ser Ile Leu
370 375 380
Phe Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu Tyr Gly Ser
385 390 395 400
Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val Phe Ile Thr
405 410 415
Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe Val Lys Gly
420 425 430
Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg Glu Thr Leu
435 440 445
Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu Val Arg Lys
450 455 460
Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr Asn Asp Thr
465 470 475 480
Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val Ile Glu Asp
485 490 495
His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe Gln Ser Ser
500 505 510
His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val Glu Arg Met
515 520 525
Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser Ser Arg Lys
530 535 540
Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr
545 550 555 560
Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala
565 570 575
Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly
580 585 590
Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val
595 600 605
Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu
610 615 620
Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser
625 630 635 640
Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu
645 650 655
Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr
660 665 670
Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His
675 680 685
Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp
690 695 700
Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu
705 710 715 720
Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile
725 730 735
Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val
740 745 750
Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr
755 760 765
Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val Gly Thr Asp
770 775 780
Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu
785 790 795
<210> 13
<211> 797
<212> PRT
<213> Artificial Sequence
<220>
<223> 分离的多肽3
<400> 13
Met Gly Gly Gly Gly Ser Cys Gly Gly Gly Gly Ser Cys Gly Gly Gly
1 5 10 15
Gly Ser Met Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu Glu Asp
20 25 30
Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val Val Gln
35 40 45
Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser Gln Ile
50 55 60
Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala Glu Val
65 70 75 80
Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn Lys Glu
85 90 95
Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu Asp Lys
100 105 110
Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly Asn Glu
115 120 125
Glu Leu Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu Leu His
130 135 140
Val Thr Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val Lys Asn
145 150 155 160
Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn Lys Met
165 170 175
Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile Gly Gln
180 185 190
Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys Val Ile
195 200 205
Val Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu Ser Asp
210 215 220
Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr Leu Gly
225 230 235 240
Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser Asp Tyr
245 250 255
Leu Glu Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser Gln Phe
260 265 270
Ile Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr Val Glu
275 280 285
Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu Glu Ser
290 295 300
Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys Pro Lys
305 310 315 320
Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met Asn Asp
325 330 335
Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu Asp Glu
340 345 350
Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp Pro Met
355 360 365
Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys Ser Ile
370 375 380
Leu Phe Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu Tyr Gly
385 390 395 400
Ser Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val Phe Ile
405 410 415
Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe Val Lys
420 425 430
Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg Glu Thr
435 440 445
Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu Val Arg
450 455 460
Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr Asn Asp
465 470 475 480
Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val Ile Glu
485 490 495
Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe Gln Ser
500 505 510
Ser His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val Glu Arg
515 520 525
Met Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser Ser Arg
530 535 540
Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly
545 550 555 560
Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln
565 570 575
Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu
580 585 590
Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala
595 600 605
Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala
610 615 620
Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu
625 630 635 640
Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met
645 650 655
Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser
660 665 670
Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg
675 680 685
His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp
690 695 700
Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr
705 710 715 720
Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg
725 730 735
Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys
740 745 750
Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr
755 760 765
Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val Gly Thr
770 775 780
Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu
785 790 795
<210> 14
<211> 802
<212> PRT
<213> Artificial Sequence
<220>
<223> 分离的多肽4
<400> 14
Met Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Gly Gly Gly Gly
1 5 10 15
Ser Cys Gly Gly Gly Gly Ser Met Asp Asp Glu Val Asp Val Asp Gly
20 25 30
Thr Val Glu Glu Asp Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp
35 40 45
Asp Glu Val Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu
50 55 60
Asn Ala Ser Gln Ile Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala
65 70 75 80
Phe Gln Ala Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu
85 90 95
Tyr Lys Asn Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser
100 105 110
Asp Ala Leu Asp Lys Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala
115 120 125
Leu Ser Gly Asn Glu Glu Leu Thr Val Lys Ile Lys Cys Asp Lys Glu
130 135 140
Lys Asn Leu Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg Glu
145 150 155 160
Glu Leu Val Lys Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu
165 170 175
Phe Leu Asn Lys Met Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser
180 185 190
Glu Leu Ile Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val
195 200 205
Ala Asp Lys Val Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His
210 215 220
Ile Trp Glu Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg
225 230 235 240
Gly Asn Thr Leu Gly Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu
245 250 255
Glu Ala Ser Asp Tyr Leu Glu Leu Asp Thr Ile Lys Asn Leu Val Lys
260 265 270
Lys Tyr Ser Gln Phe Ile Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys
275 280 285
Thr Glu Thr Val Glu Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu
290 295 300
Glu Lys Glu Glu Ser Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu
305 310 315 320
Glu Lys Lys Pro Lys Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp
325 330 335
Glu Leu Met Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu
340 345 350
Val Glu Glu Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu
355 360 365
Ser Asp Asp Pro Met Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val
370 375 380
Thr Phe Lys Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg Gly Leu
385 390 395 400
Phe Asp Glu Tyr Gly Ser Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val
405 410 415
Arg Arg Val Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr
420 425 430
Leu Asn Phe Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn
435 440 445
Val Ser Arg Glu Thr Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg
450 455 460
Lys Lys Leu Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp
465 470 475 480
Asp Lys Tyr Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys
485 490 495
Leu Gly Val Ile Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu
500 505 510
Leu Arg Phe Gln Ser Ser His His Pro Thr Asp Ile Thr Ser Leu Asp
515 520 525
Gln Tyr Val Glu Arg Met Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met
530 535 540
Ala Gly Ser Ser Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg
545 550 555 560
Leu Leu Lys Lys Gly Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp
565 570 575
Glu Tyr Cys Ile Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln
580 585 590
Asn Val Ala Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys
595 600 605
Glu Ser Arg Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp
610 615 620
Met Lys Asp Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser
625 630 635 640
Gln Arg Leu Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly
645 650 655
Trp Ser Gly Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr
660 665 670
Gly Lys Asp Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe
675 680 685
Glu Ile Asn Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile
690 695 700
Lys Glu Asp Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu
705 710 715 720
Phe Glu Thr Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys
725 730 735
Ala Tyr Gly Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile
740 745 750
Asp Pro Asp Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu
755 760 765
Thr Ala Glu Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu
770 775 780
Met Asp Val Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr
785 790 795 800
Ala Glu
<210> 15
<211> 2373
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码分离的多肽1的核酸
<400> 15
atgggtggtg gtggttcctg cggtggtggt ggttccatgg atgacgaggt ggatgtggat 60
ggtaccgtgg aggaggactt gggtaagagc agggagggta gccgcactga tgacgaggtt 120
gttcagcgtg aggaggaggc tattcagttg gacggtttga acgcaagcca gattagggag 180
ctgcgtgaga agagcgagaa gttcgctttc caggctgagg tgaaccgcat gatgaagttg 240
atcattaaca gcttgtacaa gaacaaggag atcttcctga gagagctgat ctcaaacgct 300
tccgacgccc tggacaagat ccgcctgatc tccctgactg acgagaacgc cctgtccggc 360
aacgaagaac tgaccgtgaa aatcaaatgc gacaaagaaa agaacctgct ccacgttacc 420
gataccggtg tgggcatgac ccgcgaggag ctcgtgaaga acctgggtac catcgctaag 480
agcggaacct cggaatttct gaacaagatg acagaagccc aagaagacgg tcagtccacc 540
tccgagctga ttggccagtt cggtgtgggt ttctactctg ctttcctggt ggctgacaag 600
gtgatcgtga cctccaaaca caacaacgac acacaacaca tctgggagtc cgactccaac 660
gaattttccg tgatcgccga cccgcgcggc aacactctgg gtagaggtac caccatcacc 720
ctcgtgctca aagaggaggc ctccgactac ctcgaactgg acaccatcaa gaacctggtg 780
aagaagtact cccaattcat caacttcccc atctacgtgt ggagttccaa gaccgaaacc 840
gtggaagaac ctatggagga ggaggaggcc gctaaagagg aaaaggagga gtccgatgac 900
gaggctgctg tcgaggagga agaggaggag aagaagccga agactaagaa ggtggagaag 960
acagtgtggg actgggagct gatgaacgac atcaagccaa tctggcagag gccaagcaag 1020
gaggtggagg aggatgagta caaggcattc tacaagagct tcagcaagga gagcgacgac 1080
ccgatggctt acattcactt caccgctgag ggagaggtga cattcaagag catcctgttc 1140
gtgccaacca gcgctccccg cggtctgttc gacgagtacg gtagcaagaa gagcgattac 1200
atcaagctgt acgtccgccg cgtgttcatc acagatgact tccacgacat gatgcctaag 1260
tacctcaact tcgtgaaggg tgtggtggac tccgacgatc tgcccctgaa cgtttcccgc 1320
gagaccctgc agcaacataa gctgctgaag gtgatccgta aaaagctggt gcgcaagacc 1380
ctcgacatga tcaaaaagat cgctgacgac aagtacaacg acaccttctg gaaggaattt 1440
ggcaccaaca tcaaactggg tgtgatcgag gaccactcca accgcaccag actggccaaa 1500
ctgctgagat tccaatcctc tcaccacccc accgacatca cctccctgga ccaatacgtg 1560
gagcgcatga aagagaaaca ggacaaaatc tacttcatgg ccggctcctc ccgcaaagaa 1620
gctgaatcct cccccttcgt cgaacgcctg ctgaagaaag gttacgaggt catctacctg 1680
accgaacccg tggacgagta ctgcatccag gcactgcctg agttcgacgg taagcgcttc 1740
cagaacgtcg ctaaggaggg agtgaagttc gacgagagcg agaagaccaa ggagtcccgc 1800
gaagctgtgg agaaggagtt cgagcccctg ttgaactgga tgaaggataa ggctctgaag 1860
gacaagatcg agaaagcagt ggtgtcccag aggctgaccg aaagcccctg tgctctggtg 1920
gctagtcagt acggttggag cggtaacatg gaaaggatca tgaaggcaca ggcataccag 1980
accggtaagg acattagtac caactactac gcttcccaga aaaagacctt cgagattaac 2040
ccccgccacc ctctgatcag agacatgctg agacgcatca aggaggacga agacgacaaa 2100
accgtcctcg acctggccgt ggtgttgttc gagaccgcca ccctccgcag cggctacttg 2160
ctgcctgaca ccaaagcgta cggcgacagg atcgagcgca tgctgaggct gagcctgaac 2220
atcgaccctg acgctaaggt ggaggaggag cccgaggaag agccagagga gaccgcggag 2280
gacacaacag aggacactga gcaggacgag gacgaggaga tggacgtggg aactgacgag 2340
gaagaggaaa cagcaaagga gagcacagca gag 2373
<210> 16
<211> 2388
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码分离的多肽2的核酸
<400> 16
atgggtggtg gtggttccgg tggtggtggt tcctgcggtg gtggtggttc catggatgac 60
gaggtggatg tggatggtac cgtggaggag gacttgggta agagcaggga gggtagccgc 120
actgatgacg aggttgttca gcgtgaggag gaggctattc agttggacgg tttgaacgca 180
agccagatta gggagctgcg tgagaagagc gagaagttcg ctttccaggc tgaggtgaac 240
cgcatgatga agttgatcat taacagcttg tacaagaaca aggagatctt cctgagagag 300
ctgatctcaa acgcttccga cgccctggac aagatccgcc tgatctccct gactgacgag 360
aacgccctgt ccggcaacga agaactgacc gtgaaaatca aatgcgacaa agaaaagaac 420
ctgctccacg ttaccgatac cggtgtgggc atgacccgcg aggagctcgt gaagaacctg 480
ggtaccatcg ctaagagcgg aacctcggaa tttctgaaca agatgacaga agcccaagaa 540
gacggtcagt ccacctccga gctgattggc cagttcggtg tgggtttcta ctctgctttc 600
ctggtggctg acaaggtgat cgtgacctcc aaacacaaca acgacacaca acacatctgg 660
gagtccgact ccaacgaatt ttccgtgatc gccgacccgc gcggcaacac tctgggtaga 720
ggtaccacca tcaccctcgt gctcaaagag gaggcctccg actacctcga actggacacc 780
atcaagaacc tggtgaagaa gtactcccaa ttcatcaact tccccatcta cgtgtggagt 840
tccaagaccg aaaccgtgga agaacctatg gaggaggagg aggccgctaa agaggaaaag 900
gaggagtccg atgacgaggc tgctgtcgag gaggaagagg aggagaagaa gccgaagact 960
aagaaggtgg agaagacagt gtgggactgg gagctgatga acgacatcaa gccaatctgg 1020
cagaggccaa gcaaggaggt ggaggaggat gagtacaagg cattctacaa gagcttcagc 1080
aaggagagcg acgacccgat ggcttacatt cacttcaccg ctgagggaga ggtgacattc 1140
aagagcatcc tgttcgtgcc aaccagcgct ccccgcggtc tgttcgacga gtacggtagc 1200
aagaagagcg attacatcaa gctgtacgtc cgccgcgtgt tcatcacaga tgacttccac 1260
gacatgatgc ctaagtacct caacttcgtg aagggtgtgg tggactccga cgatctgccc 1320
ctgaacgttt cccgcgagac cctgcagcaa cataagctgc tgaaggtgat ccgtaaaaag 1380
ctggtgcgca agaccctcga catgatcaaa aagatcgctg acgacaagta caacgacacc 1440
ttctggaagg aatttggcac caacatcaaa ctgggtgtga tcgaggacca ctccaaccgc 1500
accagactgg ccaaactgct gagattccaa tcctctcacc accccaccga catcacctcc 1560
ctggaccaat acgtggagcg catgaaagag aaacaggaca aaatctactt catggccggc 1620
tcctcccgca aagaagctga atcctccccc ttcgtcgaac gcctgctgaa gaaaggttac 1680
gaggtcatct acctgaccga acccgtggac gagtactgca tccaggcact gcctgagttc 1740
gacggtaagc gcttccagaa cgtcgctaag gagggagtga agttcgacga gagcgagaag 1800
accaaggagt cccgcgaagc tgtggagaag gagttcgagc ccctgttgaa ctggatgaag 1860
gataaggctc tgaaggacaa gatcgagaaa gcagtggtgt cccagaggct gaccgaaagc 1920
ccctgtgctc tggtggctag tcagtacggt tggagcggta acatggaaag gatcatgaag 1980
gcacaggcat accagaccgg taaggacatt agtaccaact actacgcttc ccagaaaaag 2040
accttcgaga ttaacccccg ccaccctctg atcagagaca tgctgagacg catcaaggag 2100
gacgaagacg acaaaaccgt cctcgacctg gccgtggtgt tgttcgagac cgccaccctc 2160
cgcagcggct acttgctgcc tgacaccaaa gcgtacggcg acaggatcga gcgcatgctg 2220
aggctgagcc tgaacatcga ccctgacgct aaggtggagg aggagcccga ggaagagcca 2280
gaggagaccg cggaggacac aacagaggac actgagcagg acgaggacga ggagatggac 2340
gtgggaactg acgaggaaga ggaaacagca aaggagagca cagcagag 2388
<210> 17
<211> 2391
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码分离的多肽3的核酸
<400> 17
atgggtggtg gtggttcctg cggtggtggt ggttcctgcg gtggtggtgg ttccatggat 60
gacgaggtgg atgtggatgg taccgtggag gaggacttgg gtaagagcag ggagggtagc 120
cgcactgatg acgaggttgt tcagcgtgag gaggaggcta ttcagttgga cggtttgaac 180
gcaagccaga ttagggagct gcgtgagaag agcgagaagt tcgctttcca ggctgaggtg 240
aaccgcatga tgaagttgat cattaacagc ttgtacaaga acaaggagat cttcctgaga 300
gagctgatct caaacgcttc cgacgccctg gacaagatcc gcctgatctc cctgactgac 360
gagaacgccc tgtccggcaa cgaagaactg accgtgaaaa tcaaatgcga caaagaaaag 420
aacctgctcc acgttaccga taccggtgtg ggcatgaccc gcgaggagct cgtgaagaac 480
ctgggtacca tcgctaagag cggaacctcg gaatttctga acaagatgac agaagcccaa 540
gaagacggtc agtccacctc cgagctgatt ggccagttcg gtgtgggttt ctactctgct 600
ttcctggtgg ctgacaaggt gatcgtgacc tccaaacaca acaacgacac acaacacatc 660
tgggagtccg actccaacga attttccgtg atcgccgacc cgcgcggcaa cactctgggt 720
agaggtacca ccatcaccct cgtgctcaaa gaggaggcct ccgactacct cgaactggac 780
accatcaaga acctggtgaa gaagtactcc caattcatca acttccccat ctacgtgtgg 840
agttccaaga ccgaaaccgt ggaagaacct atggaggagg aggaggccgc taaagaggaa 900
aaggaggagt ccgatgacga ggctgctgtc gaggaggaag aggaggagaa gaagccgaag 960
actaagaagg tggagaagac agtgtgggac tgggagctga tgaacgacat caagccaatc 1020
tggcagaggc caagcaagga ggtggaggag gatgagtaca aggcattcta caagagcttc 1080
agcaaggaga gcgacgaccc gatggcttac attcacttca ccgctgaggg agaggtgaca 1140
ttcaagagca tcctgttcgt gccaaccagc gctccccgcg gtctgttcga cgagtacggt 1200
agcaagaaga gcgattacat caagctgtac gtccgccgcg tgttcatcac agatgacttc 1260
cacgacatga tgcctaagta cctcaacttc gtgaagggtg tggtggactc cgacgatctg 1320
cccctgaacg tttcccgcga gaccctgcag caacataagc tgctgaaggt gatccgtaaa 1380
aagctggtgc gcaagaccct cgacatgatc aaaaagatcg ctgacgacaa gtacaacgac 1440
accttctgga aggaatttgg caccaacatc aaactgggtg tgatcgagga ccactccaac 1500
cgcaccagac tggccaaact gctgagattc caatcctctc accaccccac cgacatcacc 1560
tccctggacc aatacgtgga gcgcatgaaa gagaaacagg acaaaatcta cttcatggcc 1620
ggctcctccc gcaaagaagc tgaatcctcc cccttcgtcg aacgcctgct gaagaaaggt 1680
tacgaggtca tctacctgac cgaacccgtg gacgagtact gcatccaggc actgcctgag 1740
ttcgacggta agcgcttcca gaacgtcgct aaggagggag tgaagttcga cgagagcgag 1800
aagaccaagg agtcccgcga agctgtggag aaggagttcg agcccctgtt gaactggatg 1860
aaggataagg ctctgaagga caagatcgag aaagcagtgg tgtcccagag gctgaccgaa 1920
agcccctgtg ctctggtggc tagtcagtac ggttggagcg gtaacatgga aaggatcatg 1980
aaggcacagg cataccagac cggtaaggac attagtacca actactacgc ttcccagaaa 2040
aagaccttcg agattaaccc ccgccaccct ctgatcagag acatgctgag acgcatcaag 2100
gaggacgaag acgacaaaac cgtcctcgac ctggccgtgg tgttgttcga gaccgccacc 2160
ctccgcagcg gctacttgct gcctgacacc aaagcgtacg gcgacaggat cgagcgcatg 2220
ctgaggctga gcctgaacat cgaccctgac gctaaggtgg aggaggagcc cgaggaagag 2280
ccagaggaga ccgcggagga cacaacagag gacactgagc aggacgagga cgaggagatg 2340
gacgtgggaa ctgacgagga agaggaaaca gcaaaggaga gcacagcaga g 2391
<210> 18
<211> 2406
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码分离的多肽4的核酸
<400> 18
atgggtggtg gtggttccgg tggtggtggt tcctgcggtg gtggtggttc ctgcggtggt 60
ggtggttcca tggatgacga ggtggatgtg gatggtaccg tggaggagga cttgggtaag 120
agcagggagg gtagccgcac tgatgacgag gttgttcagc gtgaggagga ggctattcag 180
ttggacggtt tgaacgcaag ccagattagg gagctgcgtg agaagagcga gaagttcgct 240
ttccaggctg aggtgaaccg catgatgaag ttgatcatta acagcttgta caagaacaag 300
gagatcttcc tgagagagct gatctcaaac gcttccgacg ccctggacaa gatccgcctg 360
atctccctga ctgacgagaa cgccctgtcc ggcaacgaag aactgaccgt gaaaatcaaa 420
tgcgacaaag aaaagaacct gctccacgtt accgataccg gtgtgggcat gacccgcgag 480
gagctcgtga agaacctggg taccatcgct aagagcggaa cctcggaatt tctgaacaag 540
atgacagaag cccaagaaga cggtcagtcc acctccgagc tgattggcca gttcggtgtg 600
ggtttctact ctgctttcct ggtggctgac aaggtgatcg tgacctccaa acacaacaac 660
gacacacaac acatctggga gtccgactcc aacgaatttt ccgtgatcgc cgacccgcgc 720
ggcaacactc tgggtagagg taccaccatc accctcgtgc tcaaagagga ggcctccgac 780
tacctcgaac tggacaccat caagaacctg gtgaagaagt actcccaatt catcaacttc 840
cccatctacg tgtggagttc caagaccgaa accgtggaag aacctatgga ggaggaggag 900
gccgctaaag aggaaaagga ggagtccgat gacgaggctg ctgtcgagga ggaagaggag 960
gagaagaagc cgaagactaa gaaggtggag aagacagtgt gggactggga gctgatgaac 1020
gacatcaagc caatctggca gaggccaagc aaggaggtgg aggaggatga gtacaaggca 1080
ttctacaaga gcttcagcaa ggagagcgac gacccgatgg cttacattca cttcaccgct 1140
gagggagagg tgacattcaa gagcatcctg ttcgtgccaa ccagcgctcc ccgcggtctg 1200
ttcgacgagt acggtagcaa gaagagcgat tacatcaagc tgtacgtccg ccgcgtgttc 1260
atcacagatg acttccacga catgatgcct aagtacctca acttcgtgaa gggtgtggtg 1320
gactccgacg atctgcccct gaacgtttcc cgcgagaccc tgcagcaaca taagctgctg 1380
aaggtgatcc gtaaaaagct ggtgcgcaag accctcgaca tgatcaaaaa gatcgctgac 1440
gacaagtaca acgacacctt ctggaaggaa tttggcacca acatcaaact gggtgtgatc 1500
gaggaccact ccaaccgcac cagactggcc aaactgctga gattccaatc ctctcaccac 1560
cccaccgaca tcacctccct ggaccaatac gtggagcgca tgaaagagaa acaggacaaa 1620
atctacttca tggccggctc ctcccgcaaa gaagctgaat cctccccctt cgtcgaacgc 1680
ctgctgaaga aaggttacga ggtcatctac ctgaccgaac ccgtggacga gtactgcatc 1740
caggcactgc ctgagttcga cggtaagcgc ttccagaacg tcgctaagga gggagtgaag 1800
ttcgacgaga gcgagaagac caaggagtcc cgcgaagctg tggagaagga gttcgagccc 1860
ctgttgaact ggatgaagga taaggctctg aaggacaaga tcgagaaagc agtggtgtcc 1920
cagaggctga ccgaaagccc ctgtgctctg gtggctagtc agtacggttg gagcggtaac 1980
atggaaagga tcatgaaggc acaggcatac cagaccggta aggacattag taccaactac 2040
tacgcttccc agaaaaagac cttcgagatt aacccccgcc accctctgat cagagacatg 2100
ctgagacgca tcaaggagga cgaagacgac aaaaccgtcc tcgacctggc cgtggtgttg 2160
ttcgagaccg ccaccctccg cagcggctac ttgctgcctg acaccaaagc gtacggcgac 2220
aggatcgagc gcatgctgag gctgagcctg aacatcgacc ctgacgctaa ggtggaggag 2280
gagcccgagg aagagccaga ggagaccgcg gaggacacaa cagaggacac tgagcaggac 2340
gaggacgagg agatggacgt gggaactgac gaggaagagg aaacagcaaa ggagagcaca 2400
gcagag 2406

Claims (10)

1.一种分离的多肽，其结构如下面的式I所示：
B——A
式I；
其中：
A表示gp96蛋白；
B表示一个多肽片段，其结构如下面的式II所示：
(GGGGS)m-(GGGGSC)n-(GGGGS)r
式II，
其中：
m选自0、1、2、3、4、5，
n选自1、2、3、4、5，
r选自0、1、2、3、4、5，
并且m和r不同时为0；
B和A直接连接或通过连接片段连接；
并且式I的N端或C端连接有或不连接蛋白纯化标签例如4-8个His或6个His。
2.根据权利要求1所述的分离的多肽，其中，A的氨基酸序列如SEQ ID NO:1所示。
3.根据权利要求1至2中任一权利要求所述的分离的多肽，其中：
m为0，n为1或2，并且r为1或2；
或者
m为1或2，n为1或2，并且r为1或2；
优选地，B的氨基酸序列如SEQ ID NOs:3-6中任一序列所示。
4.根据权利要求1至3中任一权利要求所述的分离的多肽，其氨基酸序列如SEQ IDNOs:11-14中任一序列所示。
5.一种分离的蛋白，其为权利要求1至4中任一权利要求所述的分离的多肽的二聚体。
6.一种分离的多核苷酸，其编码权利要求1至4中任一权利要求所述的分离的多肽。
7.一种核酸构建体，其含有权利要求6所述的多核苷酸；
优选地，所述核酸构建体为重组载体；
优选地，所述重组载体为重组表达载体；
优选地，所述重组载体为重组昆虫病毒表达载体；
优选地，所述重组昆虫病毒表达载体为可在细菌和昆虫细胞之间转移和扩增的病毒穿梭质粒；
优选地，所述病毒穿梭质粒为杆状病毒穿梭质粒。
8.一种转化的细胞，其含有权利要求6所述的多核苷酸，或者权利要求7所述的核酸构建体；
优选地，所述转化的细胞为转化的昆虫细胞，优选为转化的Sf9细胞。
9.一种药物组合物，其包含权利要求1至4中任一权利要求所述的分离的多肽或者权利要求5所述的分离的蛋白，以及至少一种药学上可接受的辅料。
10.权利要求1至4中任一权利要求所述的分离的多肽或者权利要求5所述的分离的蛋白在制备治疗或预防自身免疫性疾病的药物中的用途；
优选地，所述自身免疫性疾病选自系统性红斑狼疮、1型糖尿病、类风湿性关节炎、强直性脊柱炎、多发性硬化症、银屑病、白癜风、红皮病、炎症性肠病、溃疡性结肠炎、克罗恩病、重症肌无力、自身免疫性肝炎、自身免疫性肾炎、渐冻症和多发性肌炎。
CN202210241991.7A 2022-03-11 2022-03-11 含有热休克蛋白gp96的多肽、药物组合物和用途 Pending CN116769046A (zh)

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