CN114891118B - 抗原pla2r-thsd7a-nell-1融合蛋白及其产品和用途 - Google Patents

抗原pla2r-thsd7a-nell-1融合蛋白及其产品和用途 Download PDF

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CN114891118B
CN114891118B CN202210457146.3A CN202210457146A CN114891118B CN 114891118 B CN114891118 B CN 114891118B CN 202210457146 A CN202210457146 A CN 202210457146A CN 114891118 B CN114891118 B CN 114891118B
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江水清
姜德华
黄文喜
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Guangzhou Kangrun Biotechnology Co ltd
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Abstract

本发明涉及生物技术领域，提供了一种抗原PLA2R‑THSD7A‑NELL‑1融合蛋白及其产品和应用。该融合蛋白包括PLA2R的3段抗原显性表位序列、THSD7A和NELL‑1的N端优势表位序列，片段之间通过Linker连接构成线性片段。该抗原避免了现有技术中PLA2R和THSD7A检测抗原存在空间位阻的问题，显著降低了阳性漏检率，准确性和特异性得到有效提高，同时抗原的稳定性较好。以该融合蛋白作为抗原的试剂或试剂盒具有检测灵敏、准确度高的特点。

Description

抗原PLA2R-THSD7A-NELL-1融合蛋白及其产品和用途
技术领域
本发明涉及生物技术领域，特别是涉及一种抗原PLA2R-THSD7A-NELL-1融合蛋白及其产品和用途。
背景技术
膜性肾病(membranous nephropathy，MN)是一种自身免疫性肾小球疾病，特点是形成原位免疫复合物，沉积在肾小球足细胞上皮下对肾小球结构进行破坏。有研究表明，磷脂酶A2受体(phospholipase A2 receptor，PLA2R)和I型血小板反应蛋白7A(THSD7A)能作为靶抗原在肾脏小球内沉积并与肾脏循环中的抗PLA2R抗体形成新生免疫抗原-抗体复合物，即导致了特发性膜性肾病(70～75％)的发生。膜性肾病按病因又可分为特发性膜性肾病(idiopathic membranous nephropathy，IMN)和继发性膜性肾病(secondarymembranous nephropathy，SMN)，但75～80％的患者被诊断为IMN。
PLA2R是一种存在于正常人足细胞中的跨膜糖蛋白。PLA2R有N型和M型两种，但人肾脏组织中主要表达的是M型PLA2R，且已确定M型PLA2R是自身抗体的主要靶抗原。M型PLA2R编码区长4392bp，编码1464个氨基酸，编码蛋白的相对分子质量为180kDa。PLA2R属于甘露醇受体及C型外源性凝集素超家族的一员，主要由短胞内端(C端)、长胞外端和一个跨膜域组成。胞外结构包含10个结构域，其中包括半胱氨酸富含域(cysteine-rich domain，CysR)、8个C型凝集素样结构域(C-type lectin-like domains，CTLDs)和纤维链接蛋白样Ⅱ型结构域(fibronectin type II domain，FNII)。另外，Dong等以HEK293F制备重组蛋白rPLA2R，冷冻电镜观察结果显示，M型PLA2R可以分为三组：CTLD1～CTLD2、CTLD4～CTLD5和CTLD7～CTLD8，且蛋白在酸性条件下，结构稳定，蛋白柔性较高。PLA2R可以在足细胞膜表面持续表达，Maryline等发现，3个靠近PLA2R N末端的结构域，均可作为抗PLA2R抗体识别的优势表位。虽然去糖基化后仍保持免疫原性，但以还原剂处理后，抗体针对PIA2R的反应性消失。有研究表明，PLA2R主要位于人类2号染色体q23-q24区，最先存在于脑组织，后发现在不同动物的肺、肝、肾等多种脏器中也有表达，且2号染色体上的PLA2R基因编码的遗传变异与膜性肾病相关。但是，单独检测血清PLA2R抗体对IMN诊断的精确度不能满足要求。
THSD7A蛋白是2014年在原发性MN患者中发现的靶细胞抗原，其是一种已知的位于细胞外的I型跨膜蛋白，这个250kDa的糖蛋白已经被发现含有多个包含抗原表位的区域，这些区域是免疫系统的目标。THSD7A主要表达于PLA2R阴性的患者体内。有研究显示表达THSD7A的肾小球上皮细胞发生了明显的细胞骨架重排列，细胞形态发生改变，并在抗体与细胞膜结合时发生局部粘连。Nicola M通过实验证明人和兔抗THSD7A通过改变抗体直接致病在足突细胞结构中，应力纤维的形成增加局部粘连信号的激活。这些都表明，抗THSD7A抗体可能直接破坏足突细胞的完整性，导致细胞损伤和蛋白尿。在现有研究中，基本上在2.0～13.6％的IMN患者中存在THSD7A。一项研究中，12例患者(2％)出现THSD7A相关特发性膜性肾病，占PLA2R阴性患者的16％(64例中的10例)。
Larissa Seifert等通过对来自THSD7A相关IMN患者的31份血清样本进行了Western和Native Blotting检测，确定了THSD7A的细胞外拓扑结构为21个血栓蛋白I型结构域串联，其中，28份(90％)血清样本识别多个抗原决定基域，由第一和第二N末端结构域组成的复合物被31例患者血清样本中的27例(87％)识别。
临床上用于膜性肾病检测的方法主要为酶联免疫吸附测定(ELISA)，该商业化试剂盒所用的靶抗原多为PLA2R的胞外域的重组蛋白。单纯用PLA2R为靶抗原进行膜性肾病检测会出现一定的漏检。
Nel样分子I型(neural epidermal growth factor-like 1protein，NELL-1)是一种外分泌型糖蛋白，可在人类胚胎肾细胞中表达，充当细胞外基质成分。2020年，SETHI等在《Kidney International》上发表最新研究发现，NELL-1在经肾脏活检明确的PLA2R和THSD7A阴性MN患者血清和肾小球中过表达。IF共聚焦显微镜显示NELL-1和IgG共定位于肾小球基底膜。同时检测到NELL-1循环抗体的存在，表明NELL-1可能是IgG的靶抗原，并由此推测NELL-1和抗NELL-1与MN密切相关。
而将PLA2R、THSD7A以及NELL-1的基因序列提取出来并通过线性相连拼接后的融合蛋白容易造成空间位阻，特异位点被阻挡，特异性又变差。
因此，针对现有技术不足，提供一种抗原PLA2R-THSD7A-NELL-1融合蛋白及其产品和应用以克服现有技术不足甚为必要。
发明内容
本发明的目的在于避免现有技术的不足之处而提供一种抗原PLA2R-THSD7A-NELL-1融合蛋白及其产品和应用，克服蛋白拼接存在空间位阻的问题，提高检测试剂盒的灵敏度和特异性。
本发明的目的通过以下技术措施实现。
提供一种抗原PLA2R-THSD7A-NELL-1融合蛋白，包括PLA2R蛋白片段、THSD7A蛋白片段和NELL-1蛋白片段，三者之间通过连接肽Linker(GGGGSGGGS)n连接构成线性片段，n为1-5的整数。
PLA2R蛋白片段含有氨基酸序列为SEQ ID NO.1-SEQ ID NO.3的3段蛋白片段，PLA2R蛋白的3段优势抗原肽片段，具有PLA2R蛋白的免疫显性表位。各相邻表位之间通过连接肽Linker(GGGGSGGGS)n进行连接。
THSD7A蛋白片段的氨基酸序列为SEQ ID NO.5，其具有THSD7A蛋白N端优势的免疫显性表位。
NELL-1蛋白片段的氨基酸序列为SEQ ID NO.6，其具有NELL-1蛋白N端优势的免疫显性表位－TSPN结构域。
连接肽为(GGGGSGGGS)n，其中，n为1-5的整数，且PLA2R蛋白片段、THSD7A蛋白片段和NELL-1蛋白片段两两之间的连接肽相同或不同。
优选的，上述的抗原PLA2R-THSD7A-NELL-1融合蛋白，连接方式为如下任意一种，
(1)THSD7A蛋白片段-连接肽-PLA2R蛋白片段-连接肽-NELL-1蛋白片段；
(2)THSD7A蛋白片段-连接肽-NELL-1蛋白片段-连接肽-PLA2R蛋白片段；
(3)NELL-1蛋白片段-连接肽-PLA2R蛋白片段-连接肽-THSD7A蛋白片段；
(4)NELL-1蛋白片段-连接肽-THSD7A蛋白片段-连接肽-PLA2R蛋白片段。
优选的，PLA2R蛋白的3段蛋白片段，相邻表位之间的连接肽Linker(GGGGSGGGS)n，其中，n为4或5。
优选的，PLA2R蛋白片段与THSD7A蛋白片段之间的连接肽Linker(GGGGSGGGS)n，其中，n为2、3或4。
优选的，THSD7A蛋白片段与NELL-1蛋白片段之间的连接肽Linker(GGGGSGGGS)n，其中，n为1或4；
优选的，PLA2R蛋白片段与NELL-1蛋白片段之间的连接肽Linker(GGGGSGGGS)n，其中，n为2或4。
作为一种优选方式，PLA2R蛋白的3段蛋白片段相邻之间的连接表现为CysR-FnⅡ-CTLD1-Linker-CTLD5-Linker-CTLD7，氨基酸序列为SEQ ID NO.4。
作为一种优选方式，所述PLA2R-THSD7A-NELL-1融合蛋白的氨基酸序列为SEQ IDNO.7-SEQ ID NO.10中的任意一种。
本发明还提供一种如上述的抗原PLA2R-THSD7A-NELL-1融合蛋白在制备膜性肾病免疫诊断试剂盒中的用途。
本发明还提供一种如上述的抗原PLA2R-THSD7A-NELL-1融合蛋白在制备免疫学诊断膜性肾病或在制备检测PLA2R、THSD7A与NELL-1中的至少一种的产品试剂中的用途。
本发明还提供一种膜性肾病免疫诊断试剂，采用上述的抗原PLA2R-THSD7A-NELL-1融合蛋白作为检测抗原。
本发明还提供一种膜性肾病免疫诊断试剂盒，采用上述的抗原PLA2R-THSD7A-NELL-1融合蛋白作为检测抗原。
本发明还提供一种与上述的PLA2R-THSD7A-NELL-1融合蛋白相关的生物材料，所述生物材料为如下任意一种，
(1)所述抗原PLA2R-THSD7A-NELL-1融合蛋白的核酸分子，抗原PLA2R-THSD7A-NELL-1融合蛋白的核酸分子的核苷酸序列为SEQ ID NO.11-SEQ ID NO.16中的任意一种；
(2)构建编码所述抗原PLA2R-THSD7A-NELL-1融合蛋白的重组质粒；
(3)表达盒，含有(1)中核酸分子；
(4)重组载体，含有(1)中核酸分子或(3)中表达盒；
(5)重组原核细胞，含有(1)中核酸分子、(3)中表达盒或(4)中重组载体，且在目的基因前加入KOZAK序列，C端加入Flag标签序列。
本发明提供一种抗原PLA2R-THSD7A-NELL-1融合蛋白，包括PLA2R蛋白片段、THSD7A蛋白片段和NELL-1蛋白片段，三者之间通过连接肽连接构成线性片段。该抗原避免了现有技术中用于膜性肾病的检测抗原存在空间位阻的问题，显著降低了阳性漏检率，准确性和特异性得到有效提高，同时能短时间一次性获得成本较低的抗原，且抗原的稳定性较好，在生产过程中批间差较小。该抗原和与其相关的生物材料均可以用于制备诊断模型肾炎或检测PLA2R、THSD7A、NELL-1产品，该产品的稳定性，准确性等性能也得到有效改善。
本发明提供的一种诊断膜性肾病的试剂或试剂盒，采用PLA2R-THSD7A-NELL-1融合蛋白抗，具有试剂灵敏度高，特异度好的特点。
具体实施方式
结合以下实施例对本发明作进一步说明。
本实施例提供一种抗原PLA2R-THSD7A-NELL-1融合蛋白，包括PLA2R蛋白片段、THSD7A蛋白片段和NELL-1蛋白片段，三者之间通过连接肽Linker(GGGGSGGGS)n连接构成线性片段，n为1-5的整数。
PLA2R蛋白片段含有氨基酸序列为SEQ ID NO.1-SEQ ID NO.3的3段蛋白片段，PLA2R蛋白的3段优势抗原肽片段，具有PLA2R蛋白的免疫显性表位。各相邻表位之间通过连接肽Linker(GGGGSGGGS)n进行连接，不同相邻表位之间的连接肽可以相同也可以不相同。
THSD7A蛋白片段的氨基酸序列为SEQ ID NO.5，其具有THSD7A蛋白N端优势的免疫显性表位。
NELL-1蛋白片段的氨基酸序列为SEQ ID NO.6，其具有NELL-1蛋白N端优势的免疫显性表位－TSPN结构域。
连接肽为(GGGGSGGGS)n，其中，n为1-5的整数，且PLA2R蛋白片段、THSD7A蛋白片段和NELL-1蛋白片段两两之间的连接肽相同或不同。
抗原PLA2R-THSD7A-NELL-1融合蛋白，连接方式可为如下任意一种：
(1)THSD7A蛋白片段-连接肽-PLA2R蛋白片段-连接肽-NELL-1蛋白片段；
(2)THSD7A蛋白片段-连接肽-NELL-1蛋白片段-连接肽-PLA2R蛋白片段；
(3)NELL-1蛋白片段-连接肽-PLA2R蛋白片段-连接肽-THSD7A蛋白片段；
(4)NELL-1蛋白片段-连接肽-THSD7A蛋白片段-连接肽-PLA2R蛋白片段。
该抗原避免了现有技术中用于膜性肾病的检测抗原存在空间位阻的问题，显著降低了阳性漏检率，准确性和特异性得到有效提高，同时能短时间一次性获得成本较低的抗原，且抗原的稳定性较好，在生产过程中批间差较小。该抗原和与其相关的生物材料均可以用于制备诊断模型肾炎或用于制备检测PLA2R、THSD7A、NELL-1中的至少一种的产品，该产品的稳定性，准确性等性能也得到有效改善。
需要说明的是，抗原PLA2R-THSD7A-NELL-1结构例如可以为PLA2R蛋白片段-Linker-THSD7A蛋白片段-Linker-NELL-1蛋白片段、THSD7A蛋白片段-Linker-NELL-1蛋白片段-Linker-PLA2R蛋白片段，或者，PLA2R蛋白片段-Linker-NELL-1蛋白片段-Linker-THSD7A蛋白片段等等，同时，可以理解的是，例如“PLA2R蛋白片段-Linker-THSD7A蛋白片段-Linker-NELL-1蛋白片段”中，“PLA2R蛋白片段-Linker-THSD7A蛋白片段”的“Linker”和“THSD7A蛋白片段-Linker-NELL-1蛋白片段”中的“Linker”可以是相同的，比如“PLA2R蛋白片段-Linker-THSD7A蛋白片段”的“Linker”为(GGGGSGGGS)3，“THSD7A蛋白片段-Linker-NELL-1蛋白片段”中的“Linker”也为(GGGGSGGGS)3；也可以是不同的，比如“PLA2R蛋白片段-Linker-THSD7A蛋白片段”的“Linker”为(GGGGSGGGS)4，“THSD7A蛋白片段-Linker-NELL-1蛋白片段”中的“Linker”也为(GGGGSGGGS)2。
可以作为一种实施方式，PLA2R蛋白的3段蛋白片段，相邻表位之间的连接肽Linker(GGGGSGGGS)n，其中，n为4或5。
可以作为另一种实施方式，PLA2R蛋白片段与THSD7A蛋白片段之间的连接肽Linker(GGGGSGGGS)n，其中，n为2、3或4。
可以作为另一种实施方式，THSD7A蛋白片段与NELL-1蛋白片段之间的连接肽Linker(GGGGSGGGS)n，其中，n为1或4。
可以作为另一种实施方式，PLA2R蛋白片段与NELL-1蛋白片段之间的连接肽Linker(GGGGSGGGS)n，其中，n为2或4。
可以作为另一种实施方式，PLA2R蛋白的3段蛋白片段相邻之间的连接表现为CysR-FnⅡ-CTLD1-Linker-CTLD5-Linker-CTLD7，氨基酸序列为SEQ ID NO.4。
可以作为另一种实施方式，PLA2R-THSD7A-NELL-1融合蛋白的氨基酸序列为SEQID NO.7-SEQ ID NO.10中的任意一种。
MLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKK(SEQ ID NO.1)。
PWLFYQDAEYLFHTFASEWLNFEFVCSWLHSDLLTIHSAHEQEFIHSKIKALSKYGASWWIGLQEERANDEFRWRD GTPVIYQNWDTGRERTVNNQSQRCGFISSITGLWGSEEC(SEQ ID NO.2)。
MPNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAICHV(SEQ ID NO.3)。
MLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSPWLFYQDAEYLFHTFASEWLNFEFVCSWLHSDLLTIHSAHEQEFIHSKIKALSKYGASWWIGLQEERANDEFRWRDGTPVIYQNWDTGRERTVNNQSQRCGFISSITGLWGSEECGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSMPNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAICHV(SEQID NO.4)。
MGLQARRWASGSRGAAGPRRGVLQLLPLPLPLPLLLLLLLRPGAGRAAAQGEAEAPTLYLWKTGPWGRCMGDECGPGGIQTRAVWCAHVEGWTTLHTNCKQAERPNNQQNCFKVCDWHKELYDWRLGPWNQCQPVISKSLEKPLECIKGEEGIQVREIACIQKDKDIPAEDIICEYFEPKPLLEQACLIPCQ(SEQ ID NO.5)。
MPMDLILVVWFCVCTARTVVGFGMDPDLQMDIVTELDLVNTTLGVAQVSGMHNASKAFLFQDIEREIHAAPHVSEKLIQLFRNKSEFTILATVQQKPSTSGVILSIRELEHSYFELESSGLRDEIRYHYIHNGKPRTEALPYRMADGQWHKVALSVSASHLLLHVDCNRIYERVIDPPDTNLPPGINLWLGQRNQKHGLFKGIIQDGKIIFMP(SEQ IDNO.6)。
MGLQARRWASGSRGAAGPRRGVLQLLPLPLPLPLLLLLLLRPGAGRAAAQGEAEAPTLYLWKTGPWGRCMGDECGPGGIQTRAVWCAHVEGWTTLHTNCKQAERPNNQQNCFKVCDWHKELYDWRLGPWNQCQPVISKSLEKPLECIKGEEGIQVREIACIQKDKDIPAEDIICEYFEPKPLLEQACLIPCQGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSMLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSWLFYQDAEYLFHTFASEWLNFEFVCSWLHSDLLTIHSAHEQEFIHSKIKALSKYGASWWIGLQEERANDEFRWRDGTPVIYQNWDTGRERTVNNQSQRCGFISSITGLWGSEECSVSMPSICGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSMPNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAICHVPGGGGSGGGSGGGGSGGGSMPMDLILVVWFCVCTARTVVGFGMDPDLQMDIVTELDLVNTTLGVAQVSGMHNASKAFLFQDIEREIHAAPHVSEKLIQLFRNKSEFTILATVQQKPSTSGVILSIRELEHSYFELESSGLRDEIRYHYIHNGKPRTEALPYRMADGQWHKVALSVSASHLLLHVDCNRIYERVIDPPDTNLPPGINLWLGQRNQKHGLFKGIIQDGKIIFMP(SEQ ID NO.7)。
MGLQARRWASGSRGAAGPRRGVLQLLPLPLPLPLLLLLLLRPGAGRAAAQGEAEAPTLYLWKTGPWGRCMGDECGPGGIQTRAVWCAHVEGWTTLHTNCKQAERPNNQQNCFKVCDWHKELYDWRLGPWNQCQPVISKSLEKPLECIKGEEGIQVREIACIQKDKDIPAEDIICEYFEPKPLLEQACLIPCQGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSMPMDLILVVWFCVCTARTVVGFGMDPDLQMDIVTELDLVNTTLGVAQVSGMHNASKAFLFQDIEREIHAAPHVSEKLIQLFRNKSEFTILATVQQKPSTSGVILSIRELEHSYFELESSGLRDEIRYHYIHNGKPRTEALPYRMADGQWHKVALSVSASHLLLHVDCNRIYERVIDPPDTNLPPGINLWLGQRNQKHGLFKGIIQDGKIIFMPGGGGSGGGSGGGGSGGGSMLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSWLFYQDAEYLFHTFASEWLNFEFVCSWLHSDLLTIHSAHEQEFIHSKIKALSKYGASWWIGLQEERANDEFRWRDGTPVIYQNWDTGRERTVNNQSQRCGFISSITGLWGSEECSVSMPSICGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSMPNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAICHVP(SEQ ID NO.8)。
MPMDLILVVWFCVCTARTVVGFGMDPDLQMDIVTELDLVNTTLGVAQVSGMHNASKAFLFQDIEREIHAAPHVSEKLIQLFRNKSEFTILATVQQKPSTSGVILSIRELEHSYFELESSGLRDEIRYHYIHNGKPRTEALPYRMADGQWHKVALSVSASHLLLHVDCNRIYERVIDPPDTNLPPGINLWLGQRNQKHGLFKGIIQDGKIIFMPGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSMLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSWLFYQDAEYLFHTFASEWLNFEFVCSWLHSDLLTIHSAHEQEFIHSKIKALSKYGASWWIGLQEERANDEFRWRDGTPVIYQNWDTGRERTVNNQSQRCGFISSITGLWGSEECSVSMPSICGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSMPNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAICHVPGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSRPGAGRAAAQGEAEAPTLYLWKTGPWGRCMGDECGPGGIQTRAVWCAHVEGWTTLHTNCKQAERPNNQQNCFKVCDWHKELYDWRLGPWNQCQPVISKSLEKPLECIKGEEGIQVREIACIQKDKDIPAEDIICEYFEPK(SEQ ID NO.9)。
MPMDLILVVWFCVCTARTVVGFGMDPDLQMDIVTELDLVNTTLGVAQVSGMHNASKAFLFQDIEREIHAAPHVSEKLIQLFRNKSEFTILATVQQKPSTSGVILSIRELEHSYFELESSGLRDEIRYHYIHNGKPRTEALPYRMADGQWHKVALSVSASHLLLHVDCNRIYERVIDPPDTNLPPGINLWLGQRNQKHGLFKGIIQDGKIIFMPGGGGSGGGSRPGAGRAAAQGEAEAPTLYLWKTGPWGRCMGDECGPGGIQTRAVWCAHVEGWTTLHTNCKQAERPNNQQNCFKVCDWHKELYDWRLGPWNQCQPVISKSLEKPLECIKGEEGIQVREIACIQKDKDIPAEDIICEYFEPKGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSMLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSWLFYQDAEYLFHTFASEWLNFEFVCSWLHSDLLTIHSAHEQEFIHSKIKALSKYGASWWIGLQEERANDEFRWRDGTPVIYQNWDTGRERTVNNQSQRCGFISSITGLWGSEECSVSMPSICGGGGSGGGSGGGGSGGGSGGGGSGGGSGGGGSGGGSMPNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAICHVP(SEQ ID NO.10)。
本实施例的抗原PLA2R-THSD7A-NELL-1融合蛋白在制备膜性肾病免疫诊断试剂盒中的用途。
本实施例的抗原PLA2R-THSD7A-NELL-1融合蛋白在制备免疫学诊断膜性肾病或在制备检测PLA2R、THSD7A与NELL-1中的至少一种的产品试剂中的用途。
作为另一实施方式，膜性肾病免疫诊断试剂或者试剂盒，采用抗原PLA2R-THSD7A-NELL-1融合蛋白作为检测抗原。该试剂或者试剂盒的检测原理可以是酶联免疫吸附测定(ELISA)、线性免疫印迹法(LIA)、化学发光免疫分析(CLIA)和免疫荧光法(IF)等。
作为另一实施方式，PLA2R-THSD7A-NELL-1融合蛋白相关的生物材料，生物材料为如下任意一种。
(1)编码抗原PLA2R-THSD7A-NELL-1融合蛋白的核酸分子，抗原PLA2R-THSD7A-NELL-1融合蛋白的核酸分子的核苷酸序列为SEQ ID NO.11-SEQ ID NO.16中的任意一种。
(2)构建编码所述抗原PLA2R-THSD7A-NELL-1融合蛋白的重组质粒。
(3)表达盒，含有(1)中核酸分子。
(4)重组载体，含有(1)中核酸分子或(3)中表达盒；重组真核细胞或重组原核细胞。
(5)重组原核细胞，含有(1)中核酸分子、(3)中表达盒或(4)中重组载体，且在目的基因前加入KOZAK序列，C端加入Flag标签序列。
该抗原PLA2R-THSD7A-NELL-1的制备方法，将编码抗原PLA2R-THSD7A-NELL-1的核酸分子导入宿主得到重组表达系统，重组表达系统表达得到抗原rPLA2R-THSD7A-NELL-1。其中，核酸分子的表达载体优选为pcDNA3.1(+/-)载体，宿主优选为人胚胎肾细胞293(HEK293)。
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本发明。
ATGCTGCTGTCGCCGTCGCTGCTGCTGCTGCTGCTGCTGGGGGCGCCGCGGGGCTGCGCCGAGGGTGTGGCGGCGGCGCTTACCCCCGAGCGGCTCCTGGAGTGGCAGGATAAAGGAATATTTGTTATCCAAAGTGAGAGTCTCAAGAAATGCATTCAAGCAGGTAAATCGGTTCTGACCCTGGAGAACTGCAAGCAAGCAAACAAGCACATGCTGTGGAAATGGGTTTCAAACCATGGCCTCTTTAACATAGGAGGCAGTGGTTGCCTGGGCCTGAATTTCTCCGCCCCAGAGCAGCCATTAAGCTTATATGAATGTGACTCCACCCTCGTTTCCTTACGGTGGCGCTGTAACAGGAAGATGATCACAGGCCCGCTGCAGTACTCTGTCCAGGTGGCGCATGACAACACAGTGGTGGCCTCACGGAAGTATATTCATAAGTGGATTTCTTATGGGTCAGGTGGTGGAGACATTTGTGAATATCTACACAAAGATTTGCATACAATCAAAGGGAACACCCACGGGATGCCGTGTATGTTTCCCTTCCAGTATAACCATCAGTGGCATCATGAATGTACCCGTGAAGGTCGGGAAGATGACTTACTGTGGTGTGCCACGACAAGCCGTTATGAAAGAGATGAAAAGTGGGGATTTTGCCCTGATCCCACCTCTGCAGAAGTAGGTTGTGATACTATTTGGGAGAAGGACCTCAATTCACACATTTGCTACCAGTTCAACCTGCTTTCATCTCTCTCTTGGAGTGAGGCACATTCTTCATGCCAGATGCAAGGAGGTACGCTGTTAAGTATTACAGATGAAACTGAAGAAAATTTCATAAGGGAGCACATGAGCAGTAAAACAGTGGAGGTGTGGATGGGCCTCAATCAGCTGGATGAACACGCTGGCTGGCAGTGGTCTGATGGAACGCCGCTCAACTATCTGAATTGGAGCCCAGAGGTAAATTTTGAGCCATTTGTTGAAGATCACTGTGGAACATTTAGTTCATTTATGCCAAGTGCCTGGAGGAGTCGGGATTGTGAGTCCACCTTGCCATATATATGTAAAAAA(SEQ ID NO.11)。
TGGCTCTTTTATCAGGATGCAGAATACCTTTTTCATACCTTTGCCTCAGAATGGTTGAACTTTGAGTTTGTCTGTAGCTGGCTGCACAGTGATCTTCTCACAATTCATTCTGCACATGAGCAAGAATTCATCCACAGCAAAATAAAAGCGCTATCAAAGTATGGTGCAAGTTGGTGGATTGGACTTCAAGAAGAAAGAGCCAATGATGAATTTCGCTGGAGAGATGGAACACCAGTGATATACCAGAACTGGGACACAGGAAGAGAAAGAACTGTGAATAATCAGAGCCAGAGATGTGGCTTTATTTCTTCTATAACAGGACTCTGGGGTAGTGAAGAGTGTTCAGTTTCTATGCCTAGTATCTGTAAGCGA(SEQ ID NO.12)。
ATGCCCAATACCTTAGAATATGGAAACAGAACTTACAAAATAATTAATGCAAATATGACTTGGTATGCAGCAATAAAAACCTGCCTGATGCACAAAGCACAACTGGTCAGCATCACAGACCAGTATCACCAGTCCTTCCTCACTGTTGTCCTCAACCGGCTAGGATATGCCCACTGGATTGGACTGTTCACCACAGATAATGGTCTTAATTTTGACTGGTCTGATGGCACCAAATCTTCTTTCACTTTTTGGAAAGATGAGGAGTCCTCCCTCCTTGGTGACTGCGTTTTTGCCGACAGCAACGGACGCTGGCATAGCACAGCCTGCGAGTCATTTCTGCAAGGTGCCATTTGTCATGTGCCA(SEQ IDNO.13)。
ATGCTGCTGTCGCCGTCGCTGCTGCTGCTGCTGCTGCTGGGGGCGCCGCGGGGCTGCGCCGAGGGTGTGGCGGCGGCGCTTACCCCCGAGCGGCTCCTGGAGTGGCAGGATAAAGGAATATTTGTTATCCAAAGTGAGAGTCTCAAGAAATGCATTCAAGCAGGTAAATCGGTTCTGACCCTGGAGAACTGCAAGCAAGCAAACAAGCACATGCTGTGGAAATGGGTTTCAAACCATGGCCTCTTTAACATAGGAGGCAGTGGTTGCCTGGGCCTGAATTTCTCCGCCCCAGAGCAGCCATTAAGCTTATATGAATGTGACTCCACCCTCGTTTCCTTACGGTGGCGCTGTAACAGGAAGATGATCACAGGCCCGCTGCAGTACTCTGTCCAGGTGGCGCATGACAACACAGTGGTGGCCTCACGGAAGTATATTCATAAGTGGATTTCTTATGGGTCAGGTGGTGGAGACATTTGTGAATATCTACACAAAGATTTGCATACAATCAAAGGGAACACCCACGGGATGCCGTGTATGTTTCCCTTCCAGTATAACCATCAGTGGCATCATGAATGTACCCGTGAAGGTCGGGAAGATGACTTACTGTGGTGTGCCACGACAAGCCGTTATGAAAGAGATGAAAAGTGGGGATTTTGCCCTGATCCCACCTCTGCAGAAGTAGGTTGTGATACTATTTGGGAGAAGGACCTCAATTCACACATTTGCTACCAGTTCAACCTGCTTTCATCTCTCTCTTGGAGTGAGGCACATTCTTCATGCCAGATGCAAGGAGGTACGCTGTTAAGTATTACAGATGAAACTGAAGAAAATTTCATAAGGGAGCACATGAGCAGTAAAACAGTGGAGGTGTGGATGGGCCTCAATCAGCTGGATGAACACGCTGGCTGGCAGTGGTCTGATGGAACGCCGCTCAACTATCTGAATTGGAGCCCAGAGGTAAATTTTGAGCCATTTGTTGAAGATCACTGTGGAACATTTAGTTCATTTATGCCAAGTGCCTGGAGGAGTCGGGATTGTGAGTCCACCTTGCCATATATATGTAAAAAAGGAGGAGGAGGTTCCGGTGGTGGAGGATCTGGTGGAGGGGGCAGTGGGGGTGGTGGTAGCGGAGGAGGAGGTTCCGGTGGTGGAGGATCTGGTGGAGGGGGCAGTGGGGGTGGTGGTAGCTGGCTCTTTTATCAGGATGCAGAATACCTTTTTCATACCTTTGCCTCAGAATGGTTGAACTTTGAGTTTGTCTGTAGCTGGCTGCACAGTGATCTTCTCACAATTCATTCTGCACATGAGCAAGAATTCATCCACAGCAAAATAAAAGCGCTATCAAAGTATGGTGCAAGTTGGTGGATTGGACTTCAAGAAGAAAGAGCCAATGATGAATTTCGCTGGAGAGATGGAACACCAGTGATATACCAGAACTGGGACACAGGAAGAGAAAGAACTGTGAATAATCAGAGCCAGAGATGTGGCTTTATTTCTTCTATAACAGGACTCTGGGGTAGTGAAGAGTGTTCAGTTTCTATGCCTAGTATCTGTAAGCGAGGAGGAGGAGGAAGTGGAGGAGGAGGATCAGGAGGTGGTGGATCAGGCGGCGGTGGATCAGGAGGAGGAGGAAGTGGAGGAGGAGGATCAGGAGGTGGTGGATCAGGCGGCGGTGGATCAGGAGGAGGAGGAAGTGGAGGAGGAGGATCAATGCCCAATACCTTAGAATATGGAAACAGAACTTACAAAATAATTAATGCAAATATGACTTGGTATGCAGCAATAAAAACCTGCCTGATGCACAAAGCACAACTGGTCAGCATCACAGACCAGTATCACCAGTCCTTCCTCACTGTTGTCCTCAACCGGCTAGGATATGCCCACTGGATTGGACTGTTCACCACAGATAATGGTCTTAATTTTGACTGGTCTGATGGCACCAAATCTTCTTTCACTTTTTGGAAAGATGAGGAGTCCTCCCTCCTTGGTGACTGCGTTTTTGCCGACAGCAACGGACGCTGGCATAGCACAGCCTGCGAGTCATTTCTGCAAGGTGCCATTTGTCATGTGCCA(SEQ ID NO.14)。
ATGGGGCTGCAAGCCAGGCGCTGGGCGTCCGGGAGCCGGGGCGCTGCGGGGCCGCGCCGGGGCGTCCTGCAGCTGCTGCCGCTGCCGCTGCCGCTGCCGCTGCTCCTGCTGCTGCTGCTACGCCCGGGCGCCGGCAGGGCTGCGGCGCAGGGCGAGGCGGAGGCGCCCACCCTCTATCTGTGGAAGACTGGTCCATGGGGCCGATGTATGGGAGATGAATGTGGTCCCGGAGGCATCCAAACGAGGGCTGTGTGGTGTGCTCATGTGGAGGGATGGACTACACTGCATACTAACTGTAAGCAGGCCGAGAGACCCAATAACCAGCAGAATTGTTTCAAAGTTTGCGATTGGCACAAAGAGTTGTACGACTGGAGACTGGGACCTTGGAATCAGTGTCAGCCCGTGATTTCAAAAAGCCTAGAGAAACCTCTTGAGTGCATTAAGGGGGAAGAAGGTATTCAGGTGAGGGAGATAGCGTGCATCCAGAAAGACAAAGACATTCCTGCGGAGGATATCATCTGTGAGTACTTTGAGCCCAAGCCTCTCCTGGAGCAGGCTTGCCTCATTCCTTGCCAG(SEQ ID NO.15)。
ATGCCGATGGATTTGATTTTAGTTGTGTGGTTCTGTGTGTGCACTGCCAGGACAGTGGTGGGCTTTGGGATGGACCCTGACCTTCAGATGGATATCGTCACCGAGCTTGACCTTGTGAACACCACCCTTGGAGTTGCTCAGGTGTCTGGAATGCACAATGCCAGCAAAGCATTTTTATTTCAAGACATAGAAAGAGAGATCCATGCAGCTCCTCATGTGAGTGAGAAATTAATTCAGCTGTTCCGGAACAAGAGTGAATTCACCATTTTGGCCACTGTACAGCAGAAGCCATCCACTTCAGGAGTGATACTGTCCATTCGAGAACTGGAGCACAGCTATTTTGAACTGGAGAGCAGTGGCCTGAGGGATGAGATTCGGTATCACTACATACACAATGGGAAGCCAAGGACAGAGGCACTTCCTTACCGCATGGCAGATGGACAATGGCACAAGGTTGCACTGTCAGTTAGCGCCTCTCATCTCCTGCTCCATGTCGACTGTAACAGGATTTATGAGCGTGTGATAGACCCTCCAGATACCAACCTTCCCCCAGGAATCAATTTATGGCTTGGCCAGCGCAACCAAAAGCATGGCTTATTCAAAGGGATCATCCAAGATGGGAAGATCATCTTTATGCCG(SEQ ID NO.16)
S1：本发明目的基因合成及重组载体的构建。
利用NCBI平台的GenBank数据库检索PLA2R的相关信息，结合对PLA2R多个抗原肽的筛选结果，按照表1所示，以PLA2R存在的3段抗原显性表位设计截短体。
表1
截短肽编号 优势表位 氨基酸残基序列
PLA2R-1(P1) CysR-FnⅡ-CTLD1 1-356
PLA2R-3(P2) CTLD5 816-939
PLA2R-4(P3) CTLD7 1116-1234
采用基因合成的方法，将编码SEQ ID NO.1的SEQ ID NO.10(PLA2R-1蛋白，简称P1)、编码SEQ ID NO.2的SEQ ID NO.11(PLA2R-2蛋白，简称P2)、编码SEQ ID NO.3的SEQ IDNO.12(PLA2R-3蛋白，简称P3)按照表2所示的结构通过连接肽连接到pcDNA3.1(+/-)载体上，构建成重组载体，并在目的基因前加入KOZAK序列。
表2
采用基因合成的方法，将编码SEQ ID NO.4的SEQ ID NO.14(PLA2R蛋白，简称P)、编码SEQ ID NO.5的SEQ ID NO.15(THSD7A蛋白，简称T)、编码SEQ ID NO.6的SEQ ID NO.16(NELL-1蛋白，简称N)按照如下表3所示的结构通过连接肽连接到pcDNA3.1(+/-)载体上，构建成重组载体，并在重组蛋白C端引入FLAG标签。
表3
P0T SD－SEQ ID NO.14－SEQ ID NO.15
P1T SD－SEQ ID NO.14－(GGGGSGGGS)1－SEQ ID NO.15
P2T SD－SEQ ID NO.14－(GGGGSGGGS)2－SEQ ID NO.15
P3T SD－SEQ ID NO.14－(GGGGSGGGS)3－SEQ ID NO.15
P4T SD－SEQ ID NO.14－(GGGGSGGGS)4－SEQ ID NO.15
P5T SD－SEQ ID NO.14－(GGGGSGGGS)5－SEQ ID NO.15
T0N SD－SEQ ID NO.15－SEQ ID NO.16
T1N SD－SEQ ID NO.15－(GGGGSGGGS)1－SEQ ID NO.16
T2N SD－SEQ ID NO.15－(GGGGSGGGS)2－SEQ ID NO.16
T3N SD－SEQ ID NO.15－(GGGGSGGGS)3－SEQ ID NO.16
T4N SD－SEQ ID NO.15－(GGGGSGGGS)4－SEQ ID NO.16
T5N SD－SEQ ID NO.15－(GGGGSGGGS)5－SEQ ID NO.16
P0N SD－SEQ ID NO.14－SEQ ID NO.16
P1N SD－SEQ ID NO.14－(GGGGSGGGS)1－SEQ ID NO.16
P2N SD－SEQ ID NO.14－(GGGGSGGGS)2－SEQ ID NO.16
P3N SD－SEQ ID NO.14－(GGGGSGGGS)3－SEQ ID NO.16
P4N SD－SEQ ID NO.14－(GGGGSGGGS)4－SEQ ID NO.16
P5N SD－SEQ ID NO.14－(GGGGSGGGS)5－SEQ ID NO.16
。
S2：重组质粒转染HEK293T细胞。
将HEK293T细胞接种于6孔板，转染当天，检测细胞数及活力，细胞活力必须在95％以上。以30mL细胞转染体系计算，细胞数调整为2.5×106～3×106个/mL，总细胞数约为7.5×107个，总细胞培养体积调整为25.5mL。将30μL质粒用1.5mL Opti-MEMI无血清培养基稀释，并混匀，室温放置5min；将80μL转染试剂ExpiF ectamineTM 293用1.5mL Opti-MEMI无血清培养基稀释，并混匀，室温放置5min；将后面的液体加入前面的液体中，期间不断摇晃，最后振荡混匀，室温放置20min；最后将3mL转染混合液加入到待转染的细胞中。转染6h后，弃去原液，加含浓度为100mL/L胎牛血清的DMEM培养基。
S3：抗原的诱导表达和纯化和应用。
转染后16～18h，向转染细胞中加入150μL ExpiF ectamineTM 293TransfectionEnhancer 1以及1.5mL ExpiF ectamineTM 293Transfection Enhancer 2。培养4～5d(具体培养时间按照细胞数及活力进行调整)后收集细胞及上清。超声波裂解细胞，提取总蛋白。
使用ANTI-FLAG M2 Affinity Gel(Sigma)完成FLAG标签融合蛋白的分离纯化的整个操作过程须在2～8℃下进行以避免抗FLAG单抗M2失活：ANTI-FLAG M2 affinity gel置于冰上融化，混合均匀后吸取10μL于1.5mL干净离心管，在4℃下，8000×g离心30秒，用微量进样器小心吸去上清；向离心管加入1mL TBS(pH 7.4)，重悬混匀亲和凝胶，4℃下，8000×g离心30秒，用微量进样器吸去上清；重复清洗两步，以彻底清洗亲和凝胶；亲和凝胶中加入1mL细胞提取物，4℃，10rpm孵育过夜，4℃，8000×g离心30秒，微量进样器吸去上清；向亲和凝胶中加入1mL Buffer A，重悬混匀亲和凝胶，4℃下，8000×g离心30秒，用微量进样器吸去上清，重复操作清洗两次；向亲和凝胶中加入洗脱液，在冰上轻柔震荡1小时，4℃下，8000×g离心30秒，吸取上清转移到1.5mL干净离心管，上清即为分离纯化的FLAG标签融合蛋白；另外，各重组蛋白并留样20μL。
S4：抗原的应用。
将上述步骤留样的样品，进行SDS-PAGE检测，且所得蛋白命名为P1-n-P2、P1-n-P3、P2-n-P3、PnT、TnN、PnN等，其中n＝0、1、2、3、4、5。
S5：试验例1和例2－磁微粒包被及反应性测试。
将S1和S2制备的各蛋白包被在磁微粒载体上。
磁微粒包被物稀释缓冲液的配制：
编号试剂名称浓度如下表4，取磷酸二氢钾(KH2PO4)0.24g、磷酸氢二钠(Na2HPO4·12H2O)2.90g、NaCl 8.00g、KCl 0.20g，加入适量超纯水中，待完全溶解后，在25±1℃条件下调溶液pH至7.4±0.2，加入0.5mL的Proclin 300，混合均匀，加入超纯水定容至1000mL。
表4
化合物标记物稀释缓冲液的配制：
编号试剂名称浓度如下表5，取磷酸二氢钠(NaH2PO4)5.3g、磷酸氢二钠(Na2HPO4·12H2O)5.9g、NaCl 9.0g，加入适量超纯水中，待完全溶解后，在25±1℃条件下调溶液pH至6.3±0.1，加入0.5mL的Proclin 300，混合均匀，加入超纯水定容至1000mL。
表5
发光化合物标记的抗体标记物：经发光化合物标记的抗体标记物浓缩液用化合物标记物稀释缓冲液稀释至工作浓度0.5ug/mL。
S6反应流程。
磁珠包被：将所述融合抗原PLA2R-THSD7A-NELL-1与磁珠进行包被；
待检样本处理：以样本稀释液(康润生物科技产专属稀释液)40倍稀释样本；
检测抗体(带发光化合物标记的标记抗体)处理：用化合物标记物稀释缓冲液以1:5000的倍数稀释检测抗体，另外，以磁微粒包被物稀释缓冲液稀释磁微粒包被物，终浓度为0.5mg/mL。
系统反应条件：
试剂(或样本) 剂量 步骤
样本用量 10μL 0
试剂1用量 40μL 21
试剂2用量 40μL 12
磁珠用量 40μL 11
第一次孵育(抗原与样本反应)的时间为10min，然后洗涤3次，每次1min；第二次孵育的时间(检抗与样本结合)为5min，然后洗涤4次。
按照试验例1的方法将P1-0-P2，P1-1-P2，P1-2-P2，P1-3-P2，P1-4-P2，P1-5-P2，P2-0-P3，P2-1-P3，P2-2-P3，P2-3-P3，P2-4-P3，P2-5-P3，P1-0-P3，P1-1-P3，P1-2-P3，P1-3-P3，P1-4-P3，P1-5-P3的蛋白分别包被于磁微粒中，检测124例经肾脏活检明确的MN患者血清样本中的特异性抗体，比较特异性，实验结果如下表6所示。
表6
根据表6的结果，可以得出，P1-n-P2、P1-n-P3和P2-n-P3最优n值分别为4、4和5，即P1与P2之间的Linker数目为4，P1与P3之间的Linker数目为4，P2与P3之间的Linker数目为5。其PLA2R融合蛋白氨基酸序列和核酸序列分别为SEQ ID NO.4和SEQ ID NO.14。
按照S2的方法制备P0T，P1T，P2T，P3T，P4T，P5T，T0N，T1N，T2N，T3N，T4N，T5N，P0N，P1N，P2N，P3N，P4N，P5N，将所制备的蛋白分别包被于磁微粒中，检测124例经肾脏活检明确的MN患者血清样本中的特异性抗体，比较特异性，实验结果如下表7～9所示。
表7
包被抗原 检测出阳性数量/样本总数 特异性
P0T 84/124 67.74％
P1T 90/124 72.58％
P2T 90/124 72.58％
P3T 86/124 69.35％
P4T 98/124 79.03％
P5T 81/124 65.32％
根据表7的结果，可以得出，P-n-T最优n＝4，即P与T之间的Linker数目为4。
表8
包被抗原 检测出阳性数量/样本总数 特异性
T0N 14/124 11.29％
T1N 17/124 13.71％
T2N 15/124 12.10％
T3N 12/124 9.68％
T4N 16/124 12.90％
T5N 15/124 12.10％
根据表8的结果，可以得出，T-n-N最优n＝1，即T与N之间的Linker数目为1。
表9
包被抗原 检测出阳性数量/样本总数 特异性
P0N 95/124 76.61％
P1N 95/124 76.61％
P2N 101/124 81.45％
P3N 101/124 81.45％
P4N 104/124 83.87％
P5N 99/124 79.84％
根据表9的结果，可以得出，P-n-N最优n＝4，即P与N之间的Linker数目为4。
选择氨基酸序列为SEQ ID NO.4的PLA2R融合蛋白，简称P。将P、T、N通过连接肽Linker串联在一起，P与T之间连接的linker数目为4，T与N之间连接的linker数目为1，P与N之间连接的linker数目为4。P、T、N顺序可以互换，但是如果P与T相连，则linker数目为4，T与N相连，则linker数目为1，如果P与N相连，则linker数目为4，分别命名为P4T1N、P4N1T、N4P4T、N1T4P、T1N4P和T4P4N，按照试验例1的方法，制备抗原PLA2R-THSD7A-NELL-1融合蛋白。
按照试验例1的方法，运用各个抗原PLA2R-THSD7A-NELL-1融合蛋白进行抗原-微磁粒包被及性能测试，检测338例(124例临床结果为膜性肾病患者血清样本和214例健康献血者血清样本)样本(简称为测试样本)血清中的特异性抗体，比较灵敏度和特异性，实验结果如下表10所示。表10为抗原PLA2R-THSD7A-NELL-1融合蛋白灵敏度和特异性分析实验结果。
表10
根据表10的结果，可得，P4T1N对样品检测的特异性、灵敏度均较高，P4T1N简称为PLA2R-THSD7A-NELL-1(P4T1N)。
S7.灵敏度、特异性对比实验。
按照试验例1的方法，对PLA2R-THSD7A-NELL-1(P4T1N)进行抗原-微磁粒包被及性能测试，检测124例临床结果为膜性肾病患者血清样本中抗体的特异性和灵敏度，其中对照试剂盒：抗磷脂酶A2受体抗体IgG检测试剂盒(酶联免疫吸附法)(EA 1254-9601G：96人份/盒，EUROIMMUN)。结果如下表11所示。
表11
本试剂盒检测结果与临床结果符合率95.16％，通过对比结果显示本试剂盒检测的阳性率均高于单一检测项的试剂盒。较PLA2R单一检测项在灵敏度上由72.58％提升至95.16％。
临床把膜性肾病分为四期，I期为最轻的时期，比较好治疗，IV期为最严重的时期，难以治愈。从挑选50例有明确临床分期信息的MN血清样本进行试剂盒的特异性和灵敏度测试，其中对照试剂盒：抗磷脂酶A2受体抗体IgG检测试剂盒(酶联免疫吸附法)(EA 1254-9601G：96人份/盒，EUROIMMUN)。结果如下表12所示。
表12
根据表12的结果可以看出，本试剂盒提升了在轻度膜性肾病患者的检测灵敏度。
使用338例测试样本血清进行检测，使用市场上三款商品化试剂盒与本试剂盒分别检测，结果显示单独检测的阳性率分别为72.58％、4.84％、6.45％(表13)，本试剂盒的阳性率为95.16％，通过对比结果显示本试剂盒检测的阳性率均高于单一检测项的试剂盒。结果如下表13所示。
表13
根据表13的结果，可以看出，本试剂盒提升了在膜性肾病患者的检测灵敏度。
在测试样本中的24例临床结果为膜性肾病患者血清样本中，有15例弱阳样本，使用基于细胞的分析(cell-based assay，CBA)的方法学检测均为阳性，其检测抗体比例为1:10或1:64。以本试剂盒测试以上样本，其中对照试剂盒：抗磷脂酶A2受体抗体IgG检测试剂盒(酶联免疫吸附法)(EA 1254-9601G：96人份/盒，EUROIMMUN)。结果如下表14所示。
表14
可见，本试剂盒有更高的检测灵敏度。本发明的一种诊断膜性肾病的化学发光免疫分析试剂盒，在不降低试剂灵敏度或特异度条件下，减少试剂盒组分，减少了资源的消耗。试剂架仅包被PLA2R-THSD7A-NELL-1融合抗原的磁微粒溶液和具有化学发光化合物的标记物的标记的羊抗人IgG抗体溶液，提升了仪器空间利用率。整个反应过程全自动化操作，简便易行。提高了反应时间，25min内即可获得诊断结果，结合样本临床信息，诊断结果准确可靠。
需要说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
序列表
<110> 广州市康润生物科技有限公司
<120> 抗原PLA2R-THSD7A-NELL-1融合蛋白及其产品和用途
<130> GZZRH0504-22-1-1445
<140> 2022104571463
<141> 2022-04-28
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 356
<212> PRT
<213> 人(Homo sapiens)
<400> 1
Met Leu Leu Ser Pro Ser Leu Leu Leu Leu Leu Leu Leu Gly Ala Pro
1 5 10 15
Arg Gly Cys Ala Glu Gly Val Ala Ala Ala Leu Thr Pro Glu Arg Leu
20 25 30
Leu Glu Trp Gln Asp Lys Gly Ile Phe Val Ile Gln Ser Glu Ser Leu
35 40 45
Lys Lys Cys Ile Gln Ala Gly Lys Ser Val Leu Thr Leu Glu Asn Cys
50 55 60
Lys Gln Ala Asn Lys His Met Leu Trp Lys Trp Val Ser Asn His Gly
65 70 75 80
Leu Phe Asn Ile Gly Gly Ser Gly Cys Leu Gly Leu Asn Phe Ser Ala
85 90 95
Pro Glu Gln Pro Leu Ser Leu Tyr Glu Cys Asp Ser Thr Leu Val Ser
100 105 110
Leu Arg Trp Arg Cys Asn Arg Lys Met Ile Thr Gly Pro Leu Gln Tyr
115 120 125
Ser Val Gln Val Ala His Asp Asn Thr Val Val Ala Ser Arg Lys Tyr
130 135 140
Ile His Lys Trp Ile Ser Tyr Gly Ser Gly Gly Gly Asp Ile Cys Glu
145 150 155 160
Tyr Leu His Lys Asp Leu His Thr Ile Lys Gly Asn Thr His Gly Met
165 170 175
Pro Cys Met Phe Pro Phe Gln Tyr Asn His Gln Trp His His Glu Cys
180 185 190
Thr Arg Glu Gly Arg Glu Asp Asp Leu Leu Trp Cys Ala Thr Thr Ser
195 200 205
Arg Tyr Glu Arg Asp Glu Lys Trp Gly Phe Cys Pro Asp Pro Thr Ser
210 215 220
Ala Glu Val Gly Cys Asp Thr Ile Trp Glu Lys Asp Leu Asn Ser His
225 230 235 240
Ile Cys Tyr Gln Phe Asn Leu Leu Ser Ser Leu Ser Trp Ser Glu Ala
245 250 255
His Ser Ser Cys Gln Met Gln Gly Gly Thr Leu Leu Ser Ile Thr Asp
260 265 270
Glu Thr Glu Glu Asn Phe Ile Arg Glu His Met Ser Ser Lys Thr Val
275 280 285
Glu Val Trp Met Gly Leu Asn Gln Leu Asp Glu His Ala Gly Trp Gln
290 295 300
Trp Ser Asp Gly Thr Pro Leu Asn Tyr Leu Asn Trp Ser Pro Glu Val
305 310 315 320
Asn Phe Glu Pro Phe Val Glu Asp His Cys Gly Thr Phe Ser Ser Phe
325 330 335
Met Pro Ser Ala Trp Arg Ser Arg Asp Cys Glu Ser Thr Leu Pro Tyr
340 345 350
Ile Cys Lys Lys
355
<210> 2
<211> 115
<212> PRT
<213> 人(Homo sapiens)
<400> 2
Pro Trp Leu Phe Tyr Gln Asp Ala Glu Tyr Leu Phe His Thr Phe Ala
1 5 10 15
Ser Glu Trp Leu Asn Phe Glu Phe Val Cys Ser Trp Leu His Ser Asp
20 25 30
Leu Leu Thr Ile His Ser Ala His Glu Gln Glu Phe Ile His Ser Lys
35 40 45
Ile Lys Ala Leu Ser Lys Tyr Gly Ala Ser Trp Trp Ile Gly Leu Gln
50 55 60
Glu Glu Arg Ala Asn Asp Glu Phe Arg Trp Arg Asp Gly Thr Pro Val
65 70 75 80
Ile Tyr Gln Asn Trp Asp Thr Gly Arg Glu Arg Thr Val Asn Asn Gln
85 90 95
Ser Gln Arg Cys Gly Phe Ile Ser Ser Ile Thr Gly Leu Trp Gly Ser
100 105 110
Glu Glu Cys
115
<210> 3
<211> 120
<212> PRT
<213> 人(Homo sapiens)
<400> 3
Met Pro Asn Thr Leu Glu Tyr Gly Asn Arg Thr Tyr Lys Ile Ile Asn
1 5 10 15
Ala Asn Met Thr Trp Tyr Ala Ala Ile Lys Thr Cys Leu Met His Lys
20 25 30
Ala Gln Leu Val Ser Ile Thr Asp Gln Tyr His Gln Ser Phe Leu Thr
35 40 45
Val Val Leu Asn Arg Leu Gly Tyr Ala His Trp Ile Gly Leu Phe Thr
50 55 60
Thr Asp Asn Gly Leu Asn Phe Asp Trp Ser Asp Gly Thr Lys Ser Ser
65 70 75 80
Phe Thr Phe Trp Lys Asp Glu Glu Ser Ser Leu Leu Gly Asp Cys Val
85 90 95
Phe Ala Asp Ser Asn Gly Arg Trp His Ser Thr Ala Cys Glu Ser Phe
100 105 110
Leu Gln Gly Ala Ile Cys His Val
115 120
<210> 4
<211> 672
<212> PRT
<213> 人(Homo sapiens)
<400> 4
Met Leu Leu Ser Pro Ser Leu Leu Leu Leu Leu Leu Leu Gly Ala Pro
1 5 10 15
Arg Gly Cys Ala Glu Gly Val Ala Ala Ala Leu Thr Pro Glu Arg Leu
20 25 30
Leu Glu Trp Gln Asp Lys Gly Ile Phe Val Ile Gln Ser Glu Ser Leu
35 40 45
Lys Lys Cys Ile Gln Ala Gly Lys Ser Val Leu Thr Leu Glu Asn Cys
50 55 60
Lys Gln Ala Asn Lys His Met Leu Trp Lys Trp Val Ser Asn His Gly
65 70 75 80
Leu Phe Asn Ile Gly Gly Ser Gly Cys Leu Gly Leu Asn Phe Ser Ala
85 90 95
Pro Glu Gln Pro Leu Ser Leu Tyr Glu Cys Asp Ser Thr Leu Val Ser
100 105 110
Leu Arg Trp Arg Cys Asn Arg Lys Met Ile Thr Gly Pro Leu Gln Tyr
115 120 125
Ser Val Gln Val Ala His Asp Asn Thr Val Val Ala Ser Arg Lys Tyr
130 135 140
Ile His Lys Trp Ile Ser Tyr Gly Ser Gly Gly Gly Asp Ile Cys Glu
145 150 155 160
Tyr Leu His Lys Asp Leu His Thr Ile Lys Gly Asn Thr His Gly Met
165 170 175
Pro Cys Met Phe Pro Phe Gln Tyr Asn His Gln Trp His His Glu Cys
180 185 190
Thr Arg Glu Gly Arg Glu Asp Asp Leu Leu Trp Cys Ala Thr Thr Ser
195 200 205
Arg Tyr Glu Arg Asp Glu Lys Trp Gly Phe Cys Pro Asp Pro Thr Ser
210 215 220
Ala Glu Val Gly Cys Asp Thr Ile Trp Glu Lys Asp Leu Asn Ser His
225 230 235 240
Ile Cys Tyr Gln Phe Asn Leu Leu Ser Ser Leu Ser Trp Ser Glu Ala
245 250 255
His Ser Ser Cys Gln Met Gln Gly Gly Thr Leu Leu Ser Ile Thr Asp
260 265 270
Glu Thr Glu Glu Asn Phe Ile Arg Glu His Met Ser Ser Lys Thr Val
275 280 285
Glu Val Trp Met Gly Leu Asn Gln Leu Asp Glu His Ala Gly Trp Gln
290 295 300
Trp Ser Asp Gly Thr Pro Leu Asn Tyr Leu Asn Trp Ser Pro Glu Val
305 310 315 320
Asn Phe Glu Pro Phe Val Glu Asp His Cys Gly Thr Phe Ser Ser Phe
325 330 335
Met Pro Ser Ala Trp Arg Ser Arg Asp Cys Glu Ser Thr Leu Pro Tyr
340 345 350
Ile Cys Lys Lys Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
355 360 365
Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly
370 375 380
Gly Gly Gly Ser Gly Gly Gly Ser Pro Trp Leu Phe Tyr Gln Asp Ala
385 390 395 400
Glu Tyr Leu Phe His Thr Phe Ala Ser Glu Trp Leu Asn Phe Glu Phe
405 410 415
Val Cys Ser Trp Leu His Ser Asp Leu Leu Thr Ile His Ser Ala His
420 425 430
Glu Gln Glu Phe Ile His Ser Lys Ile Lys Ala Leu Ser Lys Tyr Gly
435 440 445
Ala Ser Trp Trp Ile Gly Leu Gln Glu Glu Arg Ala Asn Asp Glu Phe
450 455 460
Arg Trp Arg Asp Gly Thr Pro Val Ile Tyr Gln Asn Trp Asp Thr Gly
465 470 475 480
Arg Glu Arg Thr Val Asn Asn Gln Ser Gln Arg Cys Gly Phe Ile Ser
485 490 495
Ser Ile Thr Gly Leu Trp Gly Ser Glu Glu Cys Gly Gly Gly Gly Ser
500 505 510
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
515 520 525
Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Gly Gly Ser Met Pro Asn Thr Leu Glu Tyr Gly
545 550 555 560
Asn Arg Thr Tyr Lys Ile Ile Asn Ala Asn Met Thr Trp Tyr Ala Ala
565 570 575
Ile Lys Thr Cys Leu Met His Lys Ala Gln Leu Val Ser Ile Thr Asp
580 585 590
Gln Tyr His Gln Ser Phe Leu Thr Val Val Leu Asn Arg Leu Gly Tyr
595 600 605
Ala His Trp Ile Gly Leu Phe Thr Thr Asp Asn Gly Leu Asn Phe Asp
610 615 620
Trp Ser Asp Gly Thr Lys Ser Ser Phe Thr Phe Trp Lys Asp Glu Glu
625 630 635 640
Ser Ser Leu Leu Gly Asp Cys Val Phe Ala Asp Ser Asn Gly Arg Trp
645 650 655
His Ser Thr Ala Cys Glu Ser Phe Leu Gln Gly Ala Ile Cys His Val
660 665 670
<210> 5
<211> 192
<212> PRT
<213> 人(Homo sapiens)
<400> 5
Met Gly Leu Gln Ala Arg Arg Trp Ala Ser Gly Ser Arg Gly Ala Ala
1 5 10 15
Gly Pro Arg Arg Gly Val Leu Gln Leu Leu Pro Leu Pro Leu Pro Leu
20 25 30
Pro Leu Leu Leu Leu Leu Leu Leu Arg Pro Gly Ala Gly Arg Ala Ala
35 40 45
Ala Gln Gly Glu Ala Glu Ala Pro Thr Leu Tyr Leu Trp Lys Thr Gly
50 55 60
Pro Trp Gly Arg Cys Met Gly Asp Glu Cys Gly Pro Gly Gly Ile Gln
65 70 75 80
Thr Arg Ala Val Trp Cys Ala His Val Glu Gly Trp Thr Thr Leu His
85 90 95
Thr Asn Cys Lys Gln Ala Glu Arg Pro Asn Asn Gln Gln Asn Cys Phe
100 105 110
Lys Val Cys Asp Trp His Lys Glu Leu Tyr Asp Trp Arg Leu Gly Pro
115 120 125
Trp Asn Gln Cys Gln Pro Val Ile Ser Lys Ser Leu Glu Lys Pro Leu
130 135 140
Glu Cys Ile Lys Gly Glu Glu Gly Ile Gln Val Arg Glu Ile Ala Cys
145 150 155 160
Ile Gln Lys Asp Lys Asp Ile Pro Ala Glu Asp Ile Ile Cys Glu Tyr
165 170 175
Phe Glu Pro Lys Pro Leu Leu Glu Gln Ala Cys Leu Ile Pro Cys Gln
180 185 190
<210> 6
<211> 213
<212> PRT
<213> 人(Homo sapiens)
<400> 6
Met Pro Met Asp Leu Ile Leu Val Val Trp Phe Cys Val Cys Thr Ala
1 5 10 15
Arg Thr Val Val Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile
20 25 30
Val Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Ala Gln Val
35 40 45
Ser Gly Met His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Ile Glu
50 55 60
Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu
65 70 75 80
Phe Arg Asn Lys Ser Glu Phe Thr Ile Leu Ala Thr Val Gln Gln Lys
85 90 95
Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser
100 105 110
Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His
115 120 125
Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met
130 135 140
Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His
145 150 155 160
Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp
165 170 175
Pro Pro Asp Thr Asn Leu Pro Pro Gly Ile Asn Leu Trp Leu Gly Gln
180 185 190
Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys
195 200 205
Ile Ile Phe Met Pro
210
<210> 7
<211> 1130
<212> PRT
<213> 人(Homo sapiens)
<400> 7
Met Gly Leu Gln Ala Arg Arg Trp Ala Ser Gly Ser Arg Gly Ala Ala
1 5 10 15
Gly Pro Arg Arg Gly Val Leu Gln Leu Leu Pro Leu Pro Leu Pro Leu
20 25 30
Pro Leu Leu Leu Leu Leu Leu Leu Arg Pro Gly Ala Gly Arg Ala Ala
35 40 45
Ala Gln Gly Glu Ala Glu Ala Pro Thr Leu Tyr Leu Trp Lys Thr Gly
50 55 60
Pro Trp Gly Arg Cys Met Gly Asp Glu Cys Gly Pro Gly Gly Ile Gln
65 70 75 80
Thr Arg Ala Val Trp Cys Ala His Val Glu Gly Trp Thr Thr Leu His
85 90 95
Thr Asn Cys Lys Gln Ala Glu Arg Pro Asn Asn Gln Gln Asn Cys Phe
100 105 110
Lys Val Cys Asp Trp His Lys Glu Leu Tyr Asp Trp Arg Leu Gly Pro
115 120 125
Trp Asn Gln Cys Gln Pro Val Ile Ser Lys Ser Leu Glu Lys Pro Leu
130 135 140
Glu Cys Ile Lys Gly Glu Glu Gly Ile Gln Val Arg Glu Ile Ala Cys
145 150 155 160
Ile Gln Lys Asp Lys Asp Ile Pro Ala Glu Asp Ile Ile Cys Glu Tyr
165 170 175
Phe Glu Pro Lys Pro Leu Leu Glu Gln Ala Cys Leu Ile Pro Cys Gln
180 185 190
Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Ser Met Leu Leu Ser Pro Ser Leu Leu Leu Leu Leu Leu
225 230 235 240
Leu Gly Ala Pro Arg Gly Cys Ala Glu Gly Val Ala Ala Ala Leu Thr
245 250 255
Pro Glu Arg Leu Leu Glu Trp Gln Asp Lys Gly Ile Phe Val Ile Gln
260 265 270
Ser Glu Ser Leu Lys Lys Cys Ile Gln Ala Gly Lys Ser Val Leu Thr
275 280 285
Leu Glu Asn Cys Lys Gln Ala Asn Lys His Met Leu Trp Lys Trp Val
290 295 300
Ser Asn His Gly Leu Phe Asn Ile Gly Gly Ser Gly Cys Leu Gly Leu
305 310 315 320
Asn Phe Ser Ala Pro Glu Gln Pro Leu Ser Leu Tyr Glu Cys Asp Ser
325 330 335
Thr Leu Val Ser Leu Arg Trp Arg Cys Asn Arg Lys Met Ile Thr Gly
340 345 350
Pro Leu Gln Tyr Ser Val Gln Val Ala His Asp Asn Thr Val Val Ala
355 360 365
Ser Arg Lys Tyr Ile His Lys Trp Ile Ser Tyr Gly Ser Gly Gly Gly
370 375 380
Asp Ile Cys Glu Tyr Leu His Lys Asp Leu His Thr Ile Lys Gly Asn
385 390 395 400
Thr His Gly Met Pro Cys Met Phe Pro Phe Gln Tyr Asn His Gln Trp
405 410 415
His His Glu Cys Thr Arg Glu Gly Arg Glu Asp Asp Leu Leu Trp Cys
420 425 430
Ala Thr Thr Ser Arg Tyr Glu Arg Asp Glu Lys Trp Gly Phe Cys Pro
435 440 445
Asp Pro Thr Ser Ala Glu Val Gly Cys Asp Thr Ile Trp Glu Lys Asp
450 455 460
Leu Asn Ser His Ile Cys Tyr Gln Phe Asn Leu Leu Ser Ser Leu Ser
465 470 475 480
Trp Ser Glu Ala His Ser Ser Cys Gln Met Gln Gly Gly Thr Leu Leu
485 490 495
Ser Ile Thr Asp Glu Thr Glu Glu Asn Phe Ile Arg Glu His Met Ser
500 505 510
Ser Lys Thr Val Glu Val Trp Met Gly Leu Asn Gln Leu Asp Glu His
515 520 525
Ala Gly Trp Gln Trp Ser Asp Gly Thr Pro Leu Asn Tyr Leu Asn Trp
530 535 540
Ser Pro Glu Val Asn Phe Glu Pro Phe Val Glu Asp His Cys Gly Thr
545 550 555 560
Phe Ser Ser Phe Met Pro Ser Ala Trp Arg Ser Arg Asp Cys Glu Ser
565 570 575
Thr Leu Pro Tyr Ile Cys Lys Lys Gly Gly Gly Gly Ser Gly Gly Gly
580 585 590
Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
595 600 605
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Trp Leu Phe Tyr
610 615 620
Gln Asp Ala Glu Tyr Leu Phe His Thr Phe Ala Ser Glu Trp Leu Asn
625 630 635 640
Phe Glu Phe Val Cys Ser Trp Leu His Ser Asp Leu Leu Thr Ile His
645 650 655
Ser Ala His Glu Gln Glu Phe Ile His Ser Lys Ile Lys Ala Leu Ser
660 665 670
Lys Tyr Gly Ala Ser Trp Trp Ile Gly Leu Gln Glu Glu Arg Ala Asn
675 680 685
Asp Glu Phe Arg Trp Arg Asp Gly Thr Pro Val Ile Tyr Gln Asn Trp
690 695 700
Asp Thr Gly Arg Glu Arg Thr Val Asn Asn Gln Ser Gln Arg Cys Gly
705 710 715 720
Phe Ile Ser Ser Ile Thr Gly Leu Trp Gly Ser Glu Glu Cys Ser Val
725 730 735
Ser Met Pro Ser Ile Cys Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly
740 745 750
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
755 760 765
Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Met Pro Asn Thr Leu Glu
770 775 780
Tyr Gly Asn Arg Thr Tyr Lys Ile Ile Asn Ala Asn Met Thr Trp Tyr
785 790 795 800
Ala Ala Ile Lys Thr Cys Leu Met His Lys Ala Gln Leu Val Ser Ile
805 810 815
Thr Asp Gln Tyr His Gln Ser Phe Leu Thr Val Val Leu Asn Arg Leu
820 825 830
Gly Tyr Ala His Trp Ile Gly Leu Phe Thr Thr Asp Asn Gly Leu Asn
835 840 845
Phe Asp Trp Ser Asp Gly Thr Lys Ser Ser Phe Thr Phe Trp Lys Asp
850 855 860
Glu Glu Ser Ser Leu Leu Gly Asp Cys Val Phe Ala Asp Ser Asn Gly
865 870 875 880
Arg Trp His Ser Thr Ala Cys Glu Ser Phe Leu Gln Gly Ala Ile Cys
885 890 895
His Val Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly
900 905 910
Ser Gly Gly Gly Ser Met Pro Met Asp Leu Ile Leu Val Val Trp Phe
915 920 925
Cys Val Cys Thr Ala Arg Thr Val Val Gly Phe Gly Met Asp Pro Asp
930 935 940
Leu Gln Met Asp Ile Val Thr Glu Leu Asp Leu Val Asn Thr Thr Leu
945 950 955 960
Gly Val Ala Gln Val Ser Gly Met His Asn Ala Ser Lys Ala Phe Leu
965 970 975
Phe Gln Asp Ile Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu
980 985 990
Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Ile Leu Ala
995 1000 1005
Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg
1010 1015 1020
Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp
1025 1030 1035 1040
Glu Ile Arg Tyr His Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala
1045 1050 1055
Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser
1060 1065 1070
Val Ser Ala Ser His Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr
1075 1080 1085
Glu Arg Val Ile Asp Pro Pro Asp Thr Asn Leu Pro Pro Gly Ile Asn
1090 1095 1100
Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile
1105 1110 1115 1120
Ile Gln Asp Gly Lys Ile Ile Phe Met Pro
1125 1130
<210> 8
<211> 1130
<212> PRT
<213> 人(Homo sapiens)
<400> 8
Met Gly Leu Gln Ala Arg Arg Trp Ala Ser Gly Ser Arg Gly Ala Ala
1 5 10 15
Gly Pro Arg Arg Gly Val Leu Gln Leu Leu Pro Leu Pro Leu Pro Leu
20 25 30
Pro Leu Leu Leu Leu Leu Leu Leu Arg Pro Gly Ala Gly Arg Ala Ala
35 40 45
Ala Gln Gly Glu Ala Glu Ala Pro Thr Leu Tyr Leu Trp Lys Thr Gly
50 55 60
Pro Trp Gly Arg Cys Met Gly Asp Glu Cys Gly Pro Gly Gly Ile Gln
65 70 75 80
Thr Arg Ala Val Trp Cys Ala His Val Glu Gly Trp Thr Thr Leu His
85 90 95
Thr Asn Cys Lys Gln Ala Glu Arg Pro Asn Asn Gln Gln Asn Cys Phe
100 105 110
Lys Val Cys Asp Trp His Lys Glu Leu Tyr Asp Trp Arg Leu Gly Pro
115 120 125
Trp Asn Gln Cys Gln Pro Val Ile Ser Lys Ser Leu Glu Lys Pro Leu
130 135 140
Glu Cys Ile Lys Gly Glu Glu Gly Ile Gln Val Arg Glu Ile Ala Cys
145 150 155 160
Ile Gln Lys Asp Lys Asp Ile Pro Ala Glu Asp Ile Ile Cys Glu Tyr
165 170 175
Phe Glu Pro Lys Pro Leu Leu Glu Gln Ala Cys Leu Ile Pro Cys Gln
180 185 190
Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Ser Met Pro Met Asp Leu Ile Leu Val Val Trp Phe Cys
225 230 235 240
Val Cys Thr Ala Arg Thr Val Val Gly Phe Gly Met Asp Pro Asp Leu
245 250 255
Gln Met Asp Ile Val Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly
260 265 270
Val Ala Gln Val Ser Gly Met His Asn Ala Ser Lys Ala Phe Leu Phe
275 280 285
Gln Asp Ile Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys
290 295 300
Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Ile Leu Ala Thr
305 310 315 320
Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu
325 330 335
Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu
340 345 350
Ile Arg Tyr His Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala Leu
355 360 365
Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val
370 375 380
Ser Ala Ser His Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu
385 390 395 400
Arg Val Ile Asp Pro Pro Asp Thr Asn Leu Pro Pro Gly Ile Asn Leu
405 410 415
Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile
420 425 430
Gln Asp Gly Lys Ile Ile Phe Met Pro Gly Gly Gly Gly Ser Gly Gly
435 440 445
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Met Leu Leu Ser Pro
450 455 460
Ser Leu Leu Leu Leu Leu Leu Leu Gly Ala Pro Arg Gly Cys Ala Glu
465 470 475 480
Gly Val Ala Ala Ala Leu Thr Pro Glu Arg Leu Leu Glu Trp Gln Asp
485 490 495
Lys Gly Ile Phe Val Ile Gln Ser Glu Ser Leu Lys Lys Cys Ile Gln
500 505 510
Ala Gly Lys Ser Val Leu Thr Leu Glu Asn Cys Lys Gln Ala Asn Lys
515 520 525
His Met Leu Trp Lys Trp Val Ser Asn His Gly Leu Phe Asn Ile Gly
530 535 540
Gly Ser Gly Cys Leu Gly Leu Asn Phe Ser Ala Pro Glu Gln Pro Leu
545 550 555 560
Ser Leu Tyr Glu Cys Asp Ser Thr Leu Val Ser Leu Arg Trp Arg Cys
565 570 575
Asn Arg Lys Met Ile Thr Gly Pro Leu Gln Tyr Ser Val Gln Val Ala
580 585 590
His Asp Asn Thr Val Val Ala Ser Arg Lys Tyr Ile His Lys Trp Ile
595 600 605
Ser Tyr Gly Ser Gly Gly Gly Asp Ile Cys Glu Tyr Leu His Lys Asp
610 615 620
Leu His Thr Ile Lys Gly Asn Thr His Gly Met Pro Cys Met Phe Pro
625 630 635 640
Phe Gln Tyr Asn His Gln Trp His His Glu Cys Thr Arg Glu Gly Arg
645 650 655
Glu Asp Asp Leu Leu Trp Cys Ala Thr Thr Ser Arg Tyr Glu Arg Asp
660 665 670
Glu Lys Trp Gly Phe Cys Pro Asp Pro Thr Ser Ala Glu Val Gly Cys
675 680 685
Asp Thr Ile Trp Glu Lys Asp Leu Asn Ser His Ile Cys Tyr Gln Phe
690 695 700
Asn Leu Leu Ser Ser Leu Ser Trp Ser Glu Ala His Ser Ser Cys Gln
705 710 715 720
Met Gln Gly Gly Thr Leu Leu Ser Ile Thr Asp Glu Thr Glu Glu Asn
725 730 735
Phe Ile Arg Glu His Met Ser Ser Lys Thr Val Glu Val Trp Met Gly
740 745 750
Leu Asn Gln Leu Asp Glu His Ala Gly Trp Gln Trp Ser Asp Gly Thr
755 760 765
Pro Leu Asn Tyr Leu Asn Trp Ser Pro Glu Val Asn Phe Glu Pro Phe
770 775 780
Val Glu Asp His Cys Gly Thr Phe Ser Ser Phe Met Pro Ser Ala Trp
785 790 795 800
Arg Ser Arg Asp Cys Glu Ser Thr Leu Pro Tyr Ile Cys Lys Lys Gly
805 810 815
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
820 825 830
Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
835 840 845
Gly Gly Ser Trp Leu Phe Tyr Gln Asp Ala Glu Tyr Leu Phe His Thr
850 855 860
Phe Ala Ser Glu Trp Leu Asn Phe Glu Phe Val Cys Ser Trp Leu His
865 870 875 880
Ser Asp Leu Leu Thr Ile His Ser Ala His Glu Gln Glu Phe Ile His
885 890 895
Ser Lys Ile Lys Ala Leu Ser Lys Tyr Gly Ala Ser Trp Trp Ile Gly
900 905 910
Leu Gln Glu Glu Arg Ala Asn Asp Glu Phe Arg Trp Arg Asp Gly Thr
915 920 925
Pro Val Ile Tyr Gln Asn Trp Asp Thr Gly Arg Glu Arg Thr Val Asn
930 935 940
Asn Gln Ser Gln Arg Cys Gly Phe Ile Ser Ser Ile Thr Gly Leu Trp
945 950 955 960
Gly Ser Glu Glu Cys Ser Val Ser Met Pro Ser Ile Cys Gly Gly Gly
965 970 975
Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly
980 985 990
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
995 1000 1005
Ser Met Pro Asn Thr Leu Glu Tyr Gly Asn Arg Thr Tyr Lys Ile Ile
1010 1015 1020
Asn Ala Asn Met Thr Trp Tyr Ala Ala Ile Lys Thr Cys Leu Met His
1025 1030 1035 1040
Lys Ala Gln Leu Val Ser Ile Thr Asp Gln Tyr His Gln Ser Phe Leu
1045 1050 1055
Thr Val Val Leu Asn Arg Leu Gly Tyr Ala His Trp Ile Gly Leu Phe
1060 1065 1070
Thr Thr Asp Asn Gly Leu Asn Phe Asp Trp Ser Asp Gly Thr Lys Ser
1075 1080 1085
Ser Phe Thr Phe Trp Lys Asp Glu Glu Ser Ser Leu Leu Gly Asp Cys
1090 1095 1100
Val Phe Ala Asp Ser Asn Gly Arg Trp His Ser Thr Ala Cys Glu Ser
1105 1110 1115 1120
Phe Leu Gln Gly Ala Ile Cys His Val Pro
1125 1130
<210> 9
<211> 1096
<212> PRT
<213> 人(Homo sapiens)
<400> 9
Met Pro Met Asp Leu Ile Leu Val Val Trp Phe Cys Val Cys Thr Ala
1 5 10 15
Arg Thr Val Val Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile
20 25 30
Val Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Ala Gln Val
35 40 45
Ser Gly Met His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Ile Glu
50 55 60
Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu
65 70 75 80
Phe Arg Asn Lys Ser Glu Phe Thr Ile Leu Ala Thr Val Gln Gln Lys
85 90 95
Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser
100 105 110
Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His
115 120 125
Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met
130 135 140
Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His
145 150 155 160
Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp
165 170 175
Pro Pro Asp Thr Asn Leu Pro Pro Gly Ile Asn Leu Trp Leu Gly Gln
180 185 190
Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys
195 200 205
Ile Ile Phe Met Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
225 230 235 240
Gly Gly Gly Gly Ser Gly Gly Gly Ser Met Leu Leu Ser Pro Ser Leu
245 250 255
Leu Leu Leu Leu Leu Leu Gly Ala Pro Arg Gly Cys Ala Glu Gly Val
260 265 270
Ala Ala Ala Leu Thr Pro Glu Arg Leu Leu Glu Trp Gln Asp Lys Gly
275 280 285
Ile Phe Val Ile Gln Ser Glu Ser Leu Lys Lys Cys Ile Gln Ala Gly
290 295 300
Lys Ser Val Leu Thr Leu Glu Asn Cys Lys Gln Ala Asn Lys His Met
305 310 315 320
Leu Trp Lys Trp Val Ser Asn His Gly Leu Phe Asn Ile Gly Gly Ser
325 330 335
Gly Cys Leu Gly Leu Asn Phe Ser Ala Pro Glu Gln Pro Leu Ser Leu
340 345 350
Tyr Glu Cys Asp Ser Thr Leu Val Ser Leu Arg Trp Arg Cys Asn Arg
355 360 365
Lys Met Ile Thr Gly Pro Leu Gln Tyr Ser Val Gln Val Ala His Asp
370 375 380
Asn Thr Val Val Ala Ser Arg Lys Tyr Ile His Lys Trp Ile Ser Tyr
385 390 395 400
Gly Ser Gly Gly Gly Asp Ile Cys Glu Tyr Leu His Lys Asp Leu His
405 410 415
Thr Ile Lys Gly Asn Thr His Gly Met Pro Cys Met Phe Pro Phe Gln
420 425 430
Tyr Asn His Gln Trp His His Glu Cys Thr Arg Glu Gly Arg Glu Asp
435 440 445
Asp Leu Leu Trp Cys Ala Thr Thr Ser Arg Tyr Glu Arg Asp Glu Lys
450 455 460
Trp Gly Phe Cys Pro Asp Pro Thr Ser Ala Glu Val Gly Cys Asp Thr
465 470 475 480
Ile Trp Glu Lys Asp Leu Asn Ser His Ile Cys Tyr Gln Phe Asn Leu
485 490 495
Leu Ser Ser Leu Ser Trp Ser Glu Ala His Ser Ser Cys Gln Met Gln
500 505 510
Gly Gly Thr Leu Leu Ser Ile Thr Asp Glu Thr Glu Glu Asn Phe Ile
515 520 525
Arg Glu His Met Ser Ser Lys Thr Val Glu Val Trp Met Gly Leu Asn
530 535 540
Gln Leu Asp Glu His Ala Gly Trp Gln Trp Ser Asp Gly Thr Pro Leu
545 550 555 560
Asn Tyr Leu Asn Trp Ser Pro Glu Val Asn Phe Glu Pro Phe Val Glu
565 570 575
Asp His Cys Gly Thr Phe Ser Ser Phe Met Pro Ser Ala Trp Arg Ser
580 585 590
Arg Asp Cys Glu Ser Thr Leu Pro Tyr Ile Cys Lys Lys Gly Gly Gly
595 600 605
Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly
610 615 620
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
625 630 635 640
Ser Trp Leu Phe Tyr Gln Asp Ala Glu Tyr Leu Phe His Thr Phe Ala
645 650 655
Ser Glu Trp Leu Asn Phe Glu Phe Val Cys Ser Trp Leu His Ser Asp
660 665 670
Leu Leu Thr Ile His Ser Ala His Glu Gln Glu Phe Ile His Ser Lys
675 680 685
Ile Lys Ala Leu Ser Lys Tyr Gly Ala Ser Trp Trp Ile Gly Leu Gln
690 695 700
Glu Glu Arg Ala Asn Asp Glu Phe Arg Trp Arg Asp Gly Thr Pro Val
705 710 715 720
Ile Tyr Gln Asn Trp Asp Thr Gly Arg Glu Arg Thr Val Asn Asn Gln
725 730 735
Ser Gln Arg Cys Gly Phe Ile Ser Ser Ile Thr Gly Leu Trp Gly Ser
740 745 750
Glu Glu Cys Ser Val Ser Met Pro Ser Ile Cys Gly Gly Gly Gly Ser
755 760 765
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
770 775 780
Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Met
785 790 795 800
Pro Asn Thr Leu Glu Tyr Gly Asn Arg Thr Tyr Lys Ile Ile Asn Ala
805 810 815
Asn Met Thr Trp Tyr Ala Ala Ile Lys Thr Cys Leu Met His Lys Ala
820 825 830
Gln Leu Val Ser Ile Thr Asp Gln Tyr His Gln Ser Phe Leu Thr Val
835 840 845
Val Leu Asn Arg Leu Gly Tyr Ala His Trp Ile Gly Leu Phe Thr Thr
850 855 860
Asp Asn Gly Leu Asn Phe Asp Trp Ser Asp Gly Thr Lys Ser Ser Phe
865 870 875 880
Thr Phe Trp Lys Asp Glu Glu Ser Ser Leu Leu Gly Asp Cys Val Phe
885 890 895
Ala Asp Ser Asn Gly Arg Trp His Ser Thr Ala Cys Glu Ser Phe Leu
900 905 910
Gln Gly Ala Ile Cys His Val Pro Gly Gly Gly Gly Ser Gly Gly Gly
915 920 925
Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
930 935 940
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Arg Pro Gly Ala
945 950 955 960
Gly Arg Ala Ala Ala Gln Gly Glu Ala Glu Ala Pro Thr Leu Tyr Leu
965 970 975
Trp Lys Thr Gly Pro Trp Gly Arg Cys Met Gly Asp Glu Cys Gly Pro
980 985 990
Gly Gly Ile Gln Thr Arg Ala Val Trp Cys Ala His Val Glu Gly Trp
995 1000 1005
Thr Thr Leu His Thr Asn Cys Lys Gln Ala Glu Arg Pro Asn Asn Gln
1010 1015 1020
Gln Asn Cys Phe Lys Val Cys Asp Trp His Lys Glu Leu Tyr Asp Trp
1025 1030 1035 1040
Arg Leu Gly Pro Trp Asn Gln Cys Gln Pro Val Ile Ser Lys Ser Leu
1045 1050 1055
Glu Lys Pro Leu Glu Cys Ile Lys Gly Glu Glu Gly Ile Gln Val Arg
1060 1065 1070
Glu Ile Ala Cys Ile Gln Lys Asp Lys Asp Ile Pro Ala Glu Asp Ile
1075 1080 1085
Ile Cys Glu Tyr Phe Glu Pro Lys
1090 1095
<210> 10
<211> 1069
<212> PRT
<213> 人(Homo sapiens)
<400> 10
Met Pro Met Asp Leu Ile Leu Val Val Trp Phe Cys Val Cys Thr Ala
1 5 10 15
Arg Thr Val Val Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile
20 25 30
Val Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Ala Gln Val
35 40 45
Ser Gly Met His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Ile Glu
50 55 60
Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu
65 70 75 80
Phe Arg Asn Lys Ser Glu Phe Thr Ile Leu Ala Thr Val Gln Gln Lys
85 90 95
Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser
100 105 110
Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His
115 120 125
Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met
130 135 140
Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His
145 150 155 160
Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp
165 170 175
Pro Pro Asp Thr Asn Leu Pro Pro Gly Ile Asn Leu Trp Leu Gly Gln
180 185 190
Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys
195 200 205
Ile Ile Phe Met Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Arg Pro
210 215 220
Gly Ala Gly Arg Ala Ala Ala Gln Gly Glu Ala Glu Ala Pro Thr Leu
225 230 235 240
Tyr Leu Trp Lys Thr Gly Pro Trp Gly Arg Cys Met Gly Asp Glu Cys
245 250 255
Gly Pro Gly Gly Ile Gln Thr Arg Ala Val Trp Cys Ala His Val Glu
260 265 270
Gly Trp Thr Thr Leu His Thr Asn Cys Lys Gln Ala Glu Arg Pro Asn
275 280 285
Asn Gln Gln Asn Cys Phe Lys Val Cys Asp Trp His Lys Glu Leu Tyr
290 295 300
Asp Trp Arg Leu Gly Pro Trp Asn Gln Cys Gln Pro Val Ile Ser Lys
305 310 315 320
Ser Leu Glu Lys Pro Leu Glu Cys Ile Lys Gly Glu Glu Gly Ile Gln
325 330 335
Val Arg Glu Ile Ala Cys Ile Gln Lys Asp Lys Asp Ile Pro Ala Glu
340 345 350
Asp Ile Ile Cys Glu Tyr Phe Glu Pro Lys Gly Gly Gly Gly Ser Gly
355 360 365
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly
370 375 380
Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Met Leu
385 390 395 400
Leu Ser Pro Ser Leu Leu Leu Leu Leu Leu Leu Gly Ala Pro Arg Gly
405 410 415
Cys Ala Glu Gly Val Ala Ala Ala Leu Thr Pro Glu Arg Leu Leu Glu
420 425 430
Trp Gln Asp Lys Gly Ile Phe Val Ile Gln Ser Glu Ser Leu Lys Lys
435 440 445
Cys Ile Gln Ala Gly Lys Ser Val Leu Thr Leu Glu Asn Cys Lys Gln
450 455 460
Ala Asn Lys His Met Leu Trp Lys Trp Val Ser Asn His Gly Leu Phe
465 470 475 480
Asn Ile Gly Gly Ser Gly Cys Leu Gly Leu Asn Phe Ser Ala Pro Glu
485 490 495
Gln Pro Leu Ser Leu Tyr Glu Cys Asp Ser Thr Leu Val Ser Leu Arg
500 505 510
Trp Arg Cys Asn Arg Lys Met Ile Thr Gly Pro Leu Gln Tyr Ser Val
515 520 525
Gln Val Ala His Asp Asn Thr Val Val Ala Ser Arg Lys Tyr Ile His
530 535 540
Lys Trp Ile Ser Tyr Gly Ser Gly Gly Gly Asp Ile Cys Glu Tyr Leu
545 550 555 560
His Lys Asp Leu His Thr Ile Lys Gly Asn Thr His Gly Met Pro Cys
565 570 575
Met Phe Pro Phe Gln Tyr Asn His Gln Trp His His Glu Cys Thr Arg
580 585 590
Glu Gly Arg Glu Asp Asp Leu Leu Trp Cys Ala Thr Thr Ser Arg Tyr
595 600 605
Glu Arg Asp Glu Lys Trp Gly Phe Cys Pro Asp Pro Thr Ser Ala Glu
610 615 620
Val Gly Cys Asp Thr Ile Trp Glu Lys Asp Leu Asn Ser His Ile Cys
625 630 635 640
Tyr Gln Phe Asn Leu Leu Ser Ser Leu Ser Trp Ser Glu Ala His Ser
645 650 655
Ser Cys Gln Met Gln Gly Gly Thr Leu Leu Ser Ile Thr Asp Glu Thr
660 665 670
Glu Glu Asn Phe Ile Arg Glu His Met Ser Ser Lys Thr Val Glu Val
675 680 685
Trp Met Gly Leu Asn Gln Leu Asp Glu His Ala Gly Trp Gln Trp Ser
690 695 700
Asp Gly Thr Pro Leu Asn Tyr Leu Asn Trp Ser Pro Glu Val Asn Phe
705 710 715 720
Glu Pro Phe Val Glu Asp His Cys Gly Thr Phe Ser Ser Phe Met Pro
725 730 735
Ser Ala Trp Arg Ser Arg Asp Cys Glu Ser Thr Leu Pro Tyr Ile Cys
740 745 750
Lys Lys Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
755 760 765
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
770 775 780
Gly Ser Gly Gly Gly Ser Trp Leu Phe Tyr Gln Asp Ala Glu Tyr Leu
785 790 795 800
Phe His Thr Phe Ala Ser Glu Trp Leu Asn Phe Glu Phe Val Cys Ser
805 810 815
Trp Leu His Ser Asp Leu Leu Thr Ile His Ser Ala His Glu Gln Glu
820 825 830
Phe Ile His Ser Lys Ile Lys Ala Leu Ser Lys Tyr Gly Ala Ser Trp
835 840 845
Trp Ile Gly Leu Gln Glu Glu Arg Ala Asn Asp Glu Phe Arg Trp Arg
850 855 860
Asp Gly Thr Pro Val Ile Tyr Gln Asn Trp Asp Thr Gly Arg Glu Arg
865 870 875 880
Thr Val Asn Asn Gln Ser Gln Arg Cys Gly Phe Ile Ser Ser Ile Thr
885 890 895
Gly Leu Trp Gly Ser Glu Glu Cys Ser Val Ser Met Pro Ser Ile Cys
900 905 910
Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
915 920 925
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
930 935 940
Gly Gly Gly Ser Met Pro Asn Thr Leu Glu Tyr Gly Asn Arg Thr Tyr
945 950 955 960
Lys Ile Ile Asn Ala Asn Met Thr Trp Tyr Ala Ala Ile Lys Thr Cys
965 970 975
Leu Met His Lys Ala Gln Leu Val Ser Ile Thr Asp Gln Tyr His Gln
980 985 990
Ser Phe Leu Thr Val Val Leu Asn Arg Leu Gly Tyr Ala His Trp Ile
995 1000 1005
Gly Leu Phe Thr Thr Asp Asn Gly Leu Asn Phe Asp Trp Ser Asp Gly
1010 1015 1020
Thr Lys Ser Ser Phe Thr Phe Trp Lys Asp Glu Glu Ser Ser Leu Leu
1025 1030 1035 1040
Gly Asp Cys Val Phe Ala Asp Ser Asn Gly Arg Trp His Ser Thr Ala
1045 1050 1055
Cys Glu Ser Phe Leu Gln Gly Ala Ile Cys His Val Pro
1060 1065
<210> 11
<211> 1068
<212> DNA
<213> 人(Homo sapiens)
<400> 11
atgctgctgt cgccgtcgct gctgctgctg ctgctgctgg gggcgccgcg gggctgcgcc 60
gagggtgtgg cggcggcgct tacccccgag cggctcctgg agtggcagga taaaggaata 120
tttgttatcc aaagtgagag tctcaagaaa tgcattcaag caggtaaatc ggttctgacc 180
ctggagaact gcaagcaagc aaacaagcac atgctgtgga aatgggtttc aaaccatggc 240
ctctttaaca taggaggcag tggttgcctg ggcctgaatt tctccgcccc agagcagcca 300
ttaagcttat atgaatgtga ctccaccctc gtttccttac ggtggcgctg taacaggaag 360
atgatcacag gcccgctgca gtactctgtc caggtggcgc atgacaacac agtggtggcc 420
tcacggaagt atattcataa gtggatttct tatgggtcag gtggtggaga catttgtgaa 480
tatctacaca aagatttgca tacaatcaaa gggaacaccc acgggatgcc gtgtatgttt 540
cccttccagt ataaccatca gtggcatcat gaatgtaccc gtgaaggtcg ggaagatgac 600
ttactgtggt gtgccacgac aagccgttat gaaagagatg aaaagtgggg attttgccct 660
gatcccacct ctgcagaagt aggttgtgat actatttggg agaaggacct caattcacac 720
atttgctacc agttcaacct gctttcatct ctctcttgga gtgaggcaca ttcttcatgc 780
cagatgcaag gaggtacgct gttaagtatt acagatgaaa ctgaagaaaa tttcataagg 840
gagcacatga gcagtaaaac agtggaggtg tggatgggcc tcaatcagct ggatgaacac 900
gctggctggc agtggtctga tggaacgccg ctcaactatc tgaattggag cccagaggta 960
aattttgagc catttgttga agatcactgt ggaacattta gttcatttat gccaagtgcc 1020
tggaggagtc gggattgtga gtccaccttg ccatatatat gtaaaaaa 1068
<210> 12
<211> 372
<212> DNA
<213> 人(Homo sapiens)
<400> 12
tggctctttt atcaggatgc agaatacctt tttcatacct ttgcctcaga atggttgaac 60
tttgagtttg tctgtagctg gctgcacagt gatcttctca caattcattc tgcacatgag 120
caagaattca tccacagcaa aataaaagcg ctatcaaagt atggtgcaag ttggtggatt 180
ggacttcaag aagaaagagc caatgatgaa tttcgctgga gagatggaac accagtgata 240
taccagaact gggacacagg aagagaaaga actgtgaata atcagagcca gagatgtggc 300
tttatttctt ctataacagg actctggggt agtgaagagt gttcagtttc tatgcctagt 360
atctgtaagc ga 372
<210> 13
<211> 363
<212> DNA
<213> 人(Homo sapiens)
<400> 13
atgcccaata ccttagaata tggaaacaga acttacaaaa taattaatgc aaatatgact 60
tggtatgcag caataaaaac ctgcctgatg cacaaagcac aactggtcag catcacagac 120
cagtatcacc agtccttcct cactgttgtc ctcaaccggc taggatatgc ccactggatt 180
ggactgttca ccacagataa tggtcttaat tttgactggt ctgatggcac caaatcttct 240
ttcacttttt ggaaagatga ggagtcctcc ctccttggtg actgcgtttt tgccgacagc 300
aacggacgct ggcatagcac agcctgcgag tcatttctgc aaggtgccat ttgtcatgtg 360
cca 363
<210> 14
<211> 2073
<212> DNA
<213> 人(Homo sapiens)
<400> 14
atgctgctgt cgccgtcgct gctgctgctg ctgctgctgg gggcgccgcg gggctgcgcc 60
gagggtgtgg cggcggcgct tacccccgag cggctcctgg agtggcagga taaaggaata 120
tttgttatcc aaagtgagag tctcaagaaa tgcattcaag caggtaaatc ggttctgacc 180
ctggagaact gcaagcaagc aaacaagcac atgctgtgga aatgggtttc aaaccatggc 240
ctctttaaca taggaggcag tggttgcctg ggcctgaatt tctccgcccc agagcagcca 300
ttaagcttat atgaatgtga ctccaccctc gtttccttac ggtggcgctg taacaggaag 360
atgatcacag gcccgctgca gtactctgtc caggtggcgc atgacaacac agtggtggcc 420
tcacggaagt atattcataa gtggatttct tatgggtcag gtggtggaga catttgtgaa 480
tatctacaca aagatttgca tacaatcaaa gggaacaccc acgggatgcc gtgtatgttt 540
cccttccagt ataaccatca gtggcatcat gaatgtaccc gtgaaggtcg ggaagatgac 600
ttactgtggt gtgccacgac aagccgttat gaaagagatg aaaagtgggg attttgccct 660
gatcccacct ctgcagaagt aggttgtgat actatttggg agaaggacct caattcacac 720
atttgctacc agttcaacct gctttcatct ctctcttgga gtgaggcaca ttcttcatgc 780
cagatgcaag gaggtacgct gttaagtatt acagatgaaa ctgaagaaaa tttcataagg 840
gagcacatga gcagtaaaac agtggaggtg tggatgggcc tcaatcagct ggatgaacac 900
gctggctggc agtggtctga tggaacgccg ctcaactatc tgaattggag cccagaggta 960
aattttgagc catttgttga agatcactgt ggaacattta gttcatttat gccaagtgcc 1020
tggaggagtc gggattgtga gtccaccttg ccatatatat gtaaaaaagg aggaggaggt 1080
tccggtggtg gaggatctgg tggagggggc agtgggggtg gtggtagcgg aggaggaggt 1140
tccggtggtg gaggatctgg tggagggggc agtgggggtg gtggtagctg gctcttttat 1200
caggatgcag aatacctttt tcataccttt gcctcagaat ggttgaactt tgagtttgtc 1260
tgtagctggc tgcacagtga tcttctcaca attcattctg cacatgagca agaattcatc 1320
cacagcaaaa taaaagcgct atcaaagtat ggtgcaagtt ggtggattgg acttcaagaa 1380
gaaagagcca atgatgaatt tcgctggaga gatggaacac cagtgatata ccagaactgg 1440
gacacaggaa gagaaagaac tgtgaataat cagagccaga gatgtggctt tatttcttct 1500
ataacaggac tctggggtag tgaagagtgt tcagtttcta tgcctagtat ctgtaagcga 1560
ggaggaggag gaagtggagg aggaggatca ggaggtggtg gatcaggcgg cggtggatca 1620
ggaggaggag gaagtggagg aggaggatca ggaggtggtg gatcaggcgg cggtggatca 1680
ggaggaggag gaagtggagg aggaggatca atgcccaata ccttagaata tggaaacaga 1740
acttacaaaa taattaatgc aaatatgact tggtatgcag caataaaaac ctgcctgatg 1800
cacaaagcac aactggtcag catcacagac cagtatcacc agtccttcct cactgttgtc 1860
ctcaaccggc taggatatgc ccactggatt ggactgttca ccacagataa tggtcttaat 1920
tttgactggt ctgatggcac caaatcttct ttcacttttt ggaaagatga ggagtcctcc 1980
ctccttggtg actgcgtttt tgccgacagc aacggacgct ggcatagcac agcctgcgag 2040
tcatttctgc aaggtgccat ttgtcatgtg cca 2073
<210> 15
<211> 576
<212> DNA
<213> 人(Homo sapiens)
<400> 15
atggggctgc aagccaggcg ctgggcgtcc gggagccggg gcgctgcggg gccgcgccgg 60
ggcgtcctgc agctgctgcc gctgccgctg ccgctgccgc tgctcctgct gctgctgcta 120
cgcccgggcg ccggcagggc tgcggcgcag ggcgaggcgg aggcgcccac cctctatctg 180
tggaagactg gtccatgggg ccgatgtatg ggagatgaat gtggtcccgg aggcatccaa 240
acgagggctg tgtggtgtgc tcatgtggag ggatggacta cactgcatac taactgtaag 300
caggccgaga gacccaataa ccagcagaat tgtttcaaag tttgcgattg gcacaaagag 360
ttgtacgact ggagactggg accttggaat cagtgtcagc ccgtgatttc aaaaagccta 420
gagaaacctc ttgagtgcat taagggggaa gaaggtattc aggtgaggga gatagcgtgc 480
atccagaaag acaaagacat tcctgcggag gatatcatct gtgagtactt tgagcccaag 540
cctctcctgg agcaggcttg cctcattcct tgccag 576
<210> 16
<211> 639
<212> DNA
<213> 人(Homo sapiens)
<400> 16
atgccgatgg atttgatttt agttgtgtgg ttctgtgtgt gcactgccag gacagtggtg 60
ggctttggga tggaccctga ccttcagatg gatatcgtca ccgagcttga ccttgtgaac 120
accacccttg gagttgctca ggtgtctgga atgcacaatg ccagcaaagc atttttattt 180
caagacatag aaagagagat ccatgcagct cctcatgtga gtgagaaatt aattcagctg 240
ttccggaaca agagtgaatt caccattttg gccactgtac agcagaagcc atccacttca 300
ggagtgatac tgtccattcg agaactggag cacagctatt ttgaactgga gagcagtggc 360
ctgagggatg agattcggta tcactacata cacaatggga agccaaggac agaggcactt 420
ccttaccgca tggcagatgg acaatggcac aaggttgcac tgtcagttag cgcctctcat 480
ctcctgctcc atgtcgactg taacaggatt tatgagcgtg tgatagaccc tccagatacc 540
aaccttcccc caggaatcaa tttatggctt ggccagcgca accaaaagca tggcttattc 600
aaagggatca tccaagatgg gaagatcatc tttatgccg 639

Claims (7)

1.抗原PLA2R-THSD7A-NELL-1融合蛋白，其特征在于：由PLA2R蛋白片段、THSD7A蛋白片段和NELL-1蛋白片段通过连接肽Linker（GGGGSGGGS）n连接组成，n为1-5的整数；
PLA2R蛋白片段由氨基酸序列为SEQ ID NO.1 - SEQ ID NO.3的3段蛋白片段通过连接肽Linker（GGGGSGGGS）n依次连接组成；
THSD7A蛋白片段的氨基酸序列为SEQ ID NO.5；
NELL-1蛋白片段的氨基酸序列为SEQ ID NO.6；
连接方式为如下任意一种，
（1）THSD7A蛋白片段-连接肽-PLA2R蛋白片段-连接肽-NELL-1蛋白片段；
（2）THSD7A蛋白片段-连接肽-NELL-1蛋白片段-连接肽-PLA2R蛋白片段；
（3）NELL-1蛋白片段-连接肽-PLA2R蛋白片段-连接肽-THSD7A蛋白片段；
（4）NELL-1蛋白片段-连接肽-THSD7A蛋白片段-连接肽-PLA2R蛋白片段；
PLA2R蛋白的3段蛋白片段，相邻表位之间的连接肽Linker（GGGGSGGGS）n，其中，n为4或5；
PLA2R蛋白片段与THSD7A蛋白片段之间的连接肽Linker（GGGGSGGGS）n，其中，n为2、3或4；
THSD7A蛋白片段与NELL-1蛋白片段之间的连接肽Linker（GGGGSGGGS）n，其中，n为1或4；
PLA2R蛋白片段与NELL-1蛋白片段之间的连接肽Linker（GGGGSGGGS）n，其中，n为2或4。
2.根据权利要求1所述的抗原PLA2R-THSD7A-NELL-1融合蛋白，其特征在于：PLA2R蛋白的3段蛋白片段相邻之间的连接表现为CysR-FnⅡ-CTLD1-Linker-CTLD5-Linker-CTLD7，氨基酸序列为SEQ ID NO.4。
3.如权利要求1-2任意一项所述的抗原PLA2R-THSD7A-NELL-1融合蛋白在制备膜性肾病免疫诊断试剂盒中的用途。
4.如权利要求1-2任意一项所述的抗原PLA2R-THSD7A-NELL-1融合蛋白在制备免疫学诊断膜性肾病或在制备检测PLA2R、THSD7A与NELL-1中的至少一种的试剂中的用途。
5.一种膜性肾病免疫诊断试剂，其特征在于：采用如权利要求1-2任意一项所述的抗原PLA2R-THSD7A-NELL-1融合蛋白作为检测抗原。
6.一种膜性肾病免疫诊断试剂盒，其特征在于：采用如权利要求1-2任意一项所述的抗原PLA2R-THSD7A-NELL-1融合蛋白作为检测抗原。
7.与如权利要求1-2任意一项所述的PLA2R-THSD7A-NELL-1融合蛋白相关的生物材料，其特征在于：所述生物材料为构建编码所述抗原PLA2R-THSD7A-NELL-1融合蛋白的重组质粒。
CN202210457146.3A 2022-04-28 2022-04-28 抗原pla2r-thsd7a-nell-1融合蛋白及其产品和用途 Active CN114891118B (zh)

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