CN112195154A - 一种基因改造表达改造型pla2r受体的细胞及其应用 - Google Patents
一种基因改造表达改造型pla2r受体的细胞及其应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域，具体地说涉及一种基因改造表达PLA2R受体的细胞及其应用。本发明目的在于提供一种基因改造的表达改造型PLA2受体的细胞。本发明另一个目的在于提供前一目的细胞的应用。本发明为了实现前述目的，本发明对人PLA2R基因进行了突变处理。为了更好的实现前述目的，本发明利用蛋白的糖基化修饰导致蛋白糖链结构发生改变，从而蛋白的功能性表位发生了变化，突变导致去糖基化修饰的PLA2R蛋白，通过检测去糖基化修饰PLA2R的CHO细胞，可见细胞表面存在大量表达的去糖基化的PLA2R，利用该细胞膜碎片进行包被，然后进行酶联检测，结果显示敏感性>97%，特异性100%。
Description
技术领域
本发明属于生物医药技术领域，具体地说涉及一种基因改造表达PLA2R受体的细胞及其应用。
背景技术
膜性肾小球肾炎（membranous glomerulonephritis），又称为膜性肾病（membranous nephropathy，MN），是导致成人肾病综合征的常见病因之一，约占原发性肾小球肾炎的10%。儿童发病率低，大多为继发性MN。本病光镜和电镜下病理特点为上皮下免疫复合物沉积及基底膜增厚。原发性MN的免疫复合物只分布于毛细血管襻而不分布在系膜区，一般无内皮、系膜或上皮细胞增生。继发性MN的免疫复合物除分布于毛细血管襻外，还可分布在系膜区。因此，电镜下见到系膜区有电子致密物沉积，往往提示继发性MN。成人肾病综合征者，特别是年龄在35岁以上，隐匿起病、病情发展缓慢者要考虑本病的可能性。确诊需靠肾活检病理诊断，早期病变光镜下肾小球可正常，易误诊为微小病变型肾病，此时电镜和免疫荧光检查尤其重要。活检会给患者带来巨大的痛苦，因为多种现在研究表明，抗PLA2R相关表位的抗体与原发性膜性肾小球肾炎具有良好的相关性，特异性与敏感度均可达到80%以上，为了更好的实现利用抗PLA2R相关表位的抗体检测达到减少临床患者痛苦，以及提高抗PLA2R相关表位的抗体检出的特异性与敏感度，发明人进行了以下内容的发明。
发明内容
本发明目的在于提供一种基因改造的表达改造型PLA2受体的细胞。
本发明另一个目的在于提供前一目的细胞的应用。
本发明为了实现前述目的，本发明对人PLA2R基因进行了突变处理，具体序列见序列1：PLA2R，具体突变部分序列2：突变糖基化区域，相对应于序列1：PLA2R中4191-4392。为了更好的实现前述目的，本发明利用蛋白的糖基化修饰导致蛋白糖链结构发生改变，从而蛋白的功能性表位发生了变化，突变导致去糖基化修饰的PLA2R蛋白，通过检测去糖基化修饰PLA2R的CHO细胞，可见细胞表面存在大量表达的去糖基化的PLA2R，利用该细胞膜碎片进行包被，然后进行酶联检测，结果显示敏感性>97%，特异性 100%。本发明实施例中采用CHO细胞株进行了验证，另外本发明也使用293细胞进行验证，同样可以实现本发明。
本发明另外建立了一种外分泌性sPLA2R基因突变型，具体序列见序列4：序列4：sPLA2R。经过检测，基因突变改造后的sPLA2R分泌量大量提高
本发明通过基因改造的表达PLA2受体的人人293/CHO细胞。表面表达有经过突变修饰的PLA2R受体的细胞，利用蛋白的糖基化修饰导致蛋白糖链结构发生改变，从而蛋白的功能性表位发生了变化，突变导致去糖基化修饰的PLA2R蛋白。申请保护293/CHO细胞表面表达这个蛋白而赋与这个细胞的新功能，就是表面表达有PLA2R受体的细胞。具体的，可以采用各种常规真核细胞表达载体，如用慢病毒lentivirus把PLA2R的表达框插入入到293/CHO细胞的基因组中，这样该受体就会表达在293/CHO细胞的表面。这个细胞可以用于检测，细胞破碎后包被固相载体，可以做包被抗原，用间接法ELISA测抗体。改造后的293/CHO细胞也可以与病人血清的抗体结合，然后利用二抗，流式细胞仪检测抗细胞表面面特异受体相应抗体。我们的发明包括一种被改造基因后的细胞以及该细胞用于后续的检测细胞表面特征性受体蛋白，以该蛋白作为靶点，检测针对该靶点的和配体或抗体。序列文件中，标注了糖基化位点。只要在基因合成的时候，把对应的天冬酰胺改成丙氨酸就可以去除糖基化。糖基化就是通过突变去除糖基化，或在某个位点增加一个糖基化。用突变的基因去除糖基化，这样就不再是原来的PLA2R。把PLA2A蛋白和片段进行基因改造和修饰，例如糖基化的PLa2R，我们用修饰过的PLA2R做检测比不修饰效果更好，检出率更高，特异性更好。
具体实施方式
实施例1：
委托基因合成公司合成附件基因序列3：DHFR的基因，并分别在5‘端和3’端添加“TCTAGAGCCACC”和”GTCGAT“，插入到载体pIRES (ClonTech)的酶切位点”Xba I”和“Sal I”之间，得到载体pIRES-DHFR。委托基因合成公司合成附件基因序列1：PLA2R的基因，并分别在5‘端和3’端添加CTCGAGGCCACC”和”ACGCGT“，插入到载体pIRES-DHFR载体XhoI/MluI中，得到载体pIRES-DHFR-PLA2R。从基因合成公司获得含有载体pIRES-DHFR-PLA2R甘油菌后，取100微升甘油菌接种10毫升的经过121℃高温灭菌20分钟的LB培养基（10g/L NaCl, 10g/L 蛋白胨，5g/L酵母抽提物，pH7.0）中，过夜培养后，采用生工生物工程（上海）股份有限公司的“SanPrep柱式质粒DNA小量抽提试剂盒”，按说明书抽提质粒，获得120μL DNA溶液，含量为1.20 μg/μL。
从液氮罐中，取出冻存的CHO-S细胞1支，37℃温育直至完全融化。1000 RPM离心10分钟后，将细胞接种于经过37℃预热的20 mL的CD-CHO 无血清培养基，并添加无菌过滤的谷氨酰胺溶液（200mM）至浓度为6mM。取200μL的细胞悬浮液，加入等体积的0.4% 台盼蓝溶液，血球计算板计数，活细胞密度为4.6×105 cells/mL，细胞活率为 95%。培养3天后 ，再次取200μL的细胞悬浮液，加入等体积的0.4% 台盼蓝溶液，血球计算板计数，活细胞密度为4×106 cells/mL，活率99%。
取2毫升细胞悬浮液接种于20 mL的CD-CHO 无血清培养基（含谷氨酰胺6mM）,继续培养2天，取200μL的细胞悬浮液，加入等体积的0.4% 台盼蓝溶液，血球计算板计数，活细胞密度为3×106 cells/mL，活率98%。
取3.3毫升的细胞悬浮液于15毫升的无菌离心管中，1000 RPM离心10分钟，在净化工作台内无菌操作，倒出培养上清，将细胞重新悬浮于800μL的CD-CHO培养基（含谷氨酰胺6mM）中，置于Bio-Rad 4mm厚的电冲击杯中，并加入17μL的pIRES-DHFR-PLA2R载体，轻轻混匀后，置于Bio-Rad 电转仪中电击转染（320 V, 电极1次，持续20mS）。然后将电转后的细胞转入含有20 mL的CD-CHO 无血清培养基（含谷氨酰胺6mM）中培养。
24小时后，取200μL的细胞悬浮液，加入等体积的0.4% 台盼蓝溶液，血球计算板计数，活细胞密度为4×105 cells/mL，活率90%。将细胞转移入2支15mL的无菌离心管，1000RPM离心10分钟，在净化工作台内无菌操作，倒出培养上清，将细胞重新悬浮于20 mL的CD-CHO培养基（含谷氨酰胺6mM）中,并加入G418至800 mg/L，继续培养。然后每过5天，取样计数细胞浓度，活率并进行换液。当细胞活率大于90%时，取细胞离心接种于20 mL的CD-CHO培养基（含谷氨酰胺6mM）中,接种密度为5×105 cells/mL，活率90%。将细胞转移入2支15mL的无菌离心管，1000 RPM离心10分钟105 cells/mL并加入MTX至50 nM，继续培养。此后每5天取样计数活细胞浓度和细胞活率，并换液。当细胞活率大于90%时，按照同样操作，进入MTX 浓度为500nM的细胞适应培养。当细胞活率超过90%时，对细胞进行冻存保种，细胞命名为CHO-S-PLA2R，并以抗人PLA2R单克隆抗体-FITC染色进行流式细胞分析，确认CHO-S-PLA2R细胞表面表达有人PLA2R蛋白。结果见附图2：CHO-S-PLA2R细胞流式细胞分析结果，对比附图1：CHO-S宿主细胞流式细胞分析结果。
实施例2：
委托基因合成公司合附件序列4：sPLA2R的基因，并分别在5‘端和3’端添加CTCGAGGCCACC”和”ACGCGT“，插入到载体pIRES-DHFR载体XhoI/MluI中，得到载体pIRES-DHFR-sPLA2R。从基因合成公司获得含有载体pIRES-DHFR-sPLA2R甘油菌后，取100微升甘油菌接种10毫升的经过121℃高温灭菌20分钟的LB培养基（10g/L NaCl, 10g/L 蛋白胨，5g/L酵母抽提物，pH7.0）中，过夜培养后，采用生工生物工程（上海）股份有限公司的“SanPrep柱式质粒DNA小量抽提试剂盒”，按说明书抽提质粒，获得120μLDNA溶液，含量为1.05 μg/μL。
从液氮罐中，取出冻存的CHO-S细胞1支，37℃温育直至完全融化。1000 RPM离心10分钟后，将细胞接种于经过37℃预热的20 mL的CD-CHO 无血清培养基，并添加无菌过滤的谷氨酰胺溶液（200mM）至浓度为6mM. 取200μL的细胞悬浮液，加入等体积的0.4% 台盼蓝溶液，血球计算板计数，活细胞密度为4.2×105 cells/mL，细胞活率为 96%。培养3天后 ，再次取200μL的细胞悬浮液，加入等体积的0.4% 台盼蓝溶液，血球计算板计数，活细胞密度为4×106 cells/mL，活率99%。
取2毫升细胞悬浮液接种于20 mL的CD-CHO 无血清培养基（含谷氨酰胺6mM）,继续培养2天，取200μL的细胞悬浮液，加入等体积的0.4% 台盼蓝溶液，血球计算板计数，活细胞密度为3×106 cells/mL，活率99%.
取3.3毫升的细胞悬浮液于15毫升的无菌离心管中，1000 RPM离心10分钟，在净化工作台内无菌操作，倒出培养上清，将细胞重新悬浮于800μL的CD-CHO培养基（含谷氨酰胺6mM）中，置于Bio-Rad 4mm厚的电冲击杯中，并加入20μL的pIRES-DHFR-sPLA2R载体，轻轻混匀后，置于Bio-Rad 电转仪中电击转染（320 V, 电极1次，持续20mS）。然后将电转后的细胞转入含有20 mL的CD-CHO 无血清培养基（含谷氨酰胺6mM）中培养。
24小时后，取200μL的细胞悬浮液，加入等体积的0.4% 台盼蓝溶液，血球计算板计数，活细胞密度为4.3×105 cells/mL，活率91%。将细胞转移入2支15mL的无菌离心管，1000RPM离心10分钟，在净化工作台内无菌操作，倒出培养上清，将细胞重新悬浮于20 mL的CD-CHO培养基（含谷氨酰胺6mM）中,并加入G418至800 mg/L， 继续培养。然后每过5天，取样计数细胞浓度，活率并进行换液。当细胞活率大于90%时，取细胞离心接种于20 mL的CD-CHO培养基（含谷氨酰胺6mM）中,接种密度为5×105 cells/mL并加入MTX至50 nM，继续培养。此后每5天取样计数活细胞浓度和细胞活率，并换液。当细胞活率大于90%时，按照同样操作，进入MTX 浓度为500 nM的细胞适应培养。当细胞活率大于90%时，按照同样操作，进入MTX 浓度为1000 nM的细胞适应培养。当细胞活率超过90%时，对细胞进行冻存保种，细胞命名为CHO-S-sPLA2R，并以鼠抗人PLA2R单克隆抗体包板，兔抗HIS标签多抗-HRP进行ELISA试验，以镍柱亲和层析获得的纯化是sPLA2R作为参考品，对细胞批培养的样品进行分析检测。再分批培养中，sPLA2R 的浓度可以达到50 mg/L. 结果见附图3：sPLA2R表达量，附图4电泳结果：。
实施例3：
将实施例1中的CHO-S-PLA2R经过7天培养后离心去上清，将细胞沉淀使用无菌水吹起，破膜，差速离心获取细胞膜碎片，将细胞膜碎片20ug/ml包被酶联板，使用95例原发性MN患者血清以及100例体检正常人血清，将样本稀释后加入100ul/孔，加入HRP标记羊抗人IgG100ul/孔，后加入TMB显色液100ul/孔，以正常人血清检测OD均值加上 3倍标准差作为临界值，进行检测结果患者中检出阳性93例，正常人中无一例检出阳性。
实施例4：
将实施例2中的sPLA2R 5ug/ml包被酶联板，使用95例原发性MN患者血清以及100例体检正常人血清，将样本稀释后加入100ul/孔，加入HRP标记羊抗人IgG 100ul/孔，后加入TMB显色液100ul/孔，以正常人血清检测OD均值加上 3倍标准差作为临界值，进行检测结果患者中检出阳性93例，正常人中无一例检出阳性。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，本领域技术人利用可预期的替代方案进行实施亦属于本发明内容。
序列表
<110> 江苏汇智生物科技有限公司
<120> 一种基因改造表达改造型PLA2R受体的细胞及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 5
<211> 4392
<212> DNA
<213> 人工序列(未知)
<400> 5
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 gtaaaaaata tctaaaccac 1080
attgatcatg aaatagttga aaaagatgcg tggaaatatt atgctaccca ctgtgagcct 1140
ggctggaatc cctacaatcg taattgctac aaacttcaga aagaagaaaa gacctggcat 1200
gaggctctgc gttcttgtca ggctgataac agtgcattaa tagacataac ctcattagca 1260
gaggtggagt ttcttgtaac cctccttgga gatgaaaatg catcagaaac atggattggt 1320
ttgagcagca ataaaattcc agtttccttt gaatggtcta atgactcttc agtcatcttt 1380
actaattggc acacacttga gccccacatt tttccaaata gaagccagct gtgtgtctca 1440
gcagagcagt ctgagggaca ctggaaagtc aaaaattgtg aagaaagact tttttacatt 1500
tgtaaaaaag caggccatgt cctctctgat gctgaatcag gatgtcaaga gggatgggag 1560
agacatggtg gattctgtta caaaattgac acagtccttc gaagctttga ccaagcttcc 1620
agcggttatt actgtcctcc tgcacttgta accattacaa acaggtttga acaggctttt 1680
attaccagtt tgatcagtag tgtggtaaaa atgaaggaca gttatttttg gatagctctt 1740
caggaccaaa atgatacggg agaatacact tggaagccag tagggcagaa acccgagccg 1800
gtgcagtaca cacactggaa cacacaccag ccgcgctaca gtggtggctg tgttgccatg 1860
cgaggaaggc atccacttgg tcgctgggaa gtgaagcact gtcggcactt taaggcaatg 1920
tccttgtgca agcagccagt tgaaaatcag gaaaaagcag agtatgaaga gagatggccc 1980
tttcacccct gctatttgga ctgggagtca gagcctggtc tggccagttg cttcaaggta 2040
tttcatagtg aaaaagttct gatgaaaaga acatggagag aagctgaagc attttgcgaa 2100
gaatttggag ctcatcttgc aagctttgcc catattgagg aagagaattt tgtgaatgag 2160
ctcttacatt caaaatttaa ttggacagaa gaaaggcagt tctggattgg atttaataaa 2220
agaaacccac tgaatgccgg ctcatgggag tggtctgata gaactcctgt tgtctcttcg 2280
tttttagaca acacttattt tggagaagat gcaagaaact gtgctgttta taaggcaaac 2340
aaaacattgc tgcccttaca ctgtggttcc aaacgtgaat ggatatgcaa aatcccaaga 2400
gatgtgaaac ccaagattcc gttctggtac cagtacgatg taccctggct cttttatcag 2460
gatgcagaat acctttttca tacctttgcc tcagaatggt tgaactttga gtttgtctgt 2520
agctggctgc acagtgatct tctcacaatt cattctgcac atgagcaaga attcatccac 2580
agcaaaataa aagcgctatc aaagtatggt gcaagttggt ggattggact tcaagaagaa 2640
agagccaatg atgaatttcg ctggagagat ggaacaccag tgatatacca gaactgggac 2700
acaggaagag aaagaactgt gaataatcag agccagagat gtggctttat ttcttctata 2760
acaggactct ggggtagtga agagtgttca gtttctatgc ctagtatctg taagcgaaaa 2820
aaggtttggc tcatagagaa aaagaaagat acaccaaaac aacatggaac gtgtcccaaa 2880
ggatggctat attttaacta taagtgcctt ctgctgaata tccccaaaga cccaagcagt 2940
tggaagaact ggacgcatgc tcaacatttc tgtgctgaag aaggggggac cctggtcgcc 3000
attgaaagtg aggtggagca agctttcatt actatgaatc tttttggcca gaccaccagt 3060
gtgtggatag gtttacaaaa tgatgattat gaaacatggc taaatggaaa gcctgtggta 3120
tattctaact ggtctccatt tgatataata aatattccaa gtcacaatac cactgaagtt 3180
cagaaacaca ttcctctctg tgccttactc tcaagtaatc ctaattttca tttcactgga 3240
aaatggtatt ttgaagactg tggaaaggaa ggctatgggt ttgtttgtga aaaaatgcaa 3300
gatacttctg gacacggtgt aaatacatct gatatgtatc caatgcccaa taccttagaa 3360
tatggaaaca gaacttacaa aataattaat gcaaatatga cttggtatgc agcaataaaa 3420
acctgcctga tgcacaaagc acaactggtc agcatcacag accagtatca ccagtccttc 3480
ctcactgttg tcctcaaccg gctaggatat gcccactgga ttggactgtt caccacagat 3540
aatggtctta attttgactg gtctgatggc accaaatctt ctttcacttt ttggaaagat 3600
gaggagtcct ccctccttgg tgactgcgtt tttgccgaca gcaacggacg ctggcatagc 3660
acagcctgcg agtcatttct gcaaggtgcc atttgtcatg tgccacctga aacaagacaa 3720
tctgaacacc cagagttgtg ctcagaaaca tctattccct ggataaaatt taaaagtaat 3780
tgctacagtt tttctacagt cctagacagt atgagttttg aggctgctca tgaattttgc 3840
aaaaaggaag gttctaatct tttaacaatc aaggatgagg ctgaaaatgc atttctccta 3900
gaagagctgt ttgcttttgg ttcttctgtc cagatggttt ggttgaatgc tcaatttgat 3960
ggtaacaatg aaaccataaa gtggtttgat ggaactccca cagaccagtc aaactggggc 4020
attcggaagc cagacacaga ctacttcaag ccccatcatt gtgttgcctt gaggatccct 4080
gaaggattat ggcagctatc cccgtgtcaa gaaaaaaaag gctttatatg taaaatggag 4140
gcagatattc acactgcaga ggcgctgcca gaaaaaggac caagtcacag catcattcct 4200
cttgcggttg tactgacact gatagtcatt gtggccattt gcacactttc cttctgcata 4260
tacaagcata acggtggctt cttcaggaga cttgcagggt ttcggaatcc ttactatcct 4320
gcaaccaact ttagtacagt atatttagaa gaaaatattc tcatttctga tcttgagaag 4380
agtgaccaat aa 4392
<210> 6
<211> 201
<212> DNA
<213> 人工序列(未知)
<400> 6
atcattcctc ttgcggttgt actgacactg atagtcattg tggccatttg cacactttcc 60
ttctgcatat acaagcataa cggtggcttc ttcaggagac ttgcagggtt tcggaatcct 120
tactatcctg caaccaactt tagtacagta tatttagaag aaaatattct catttctgat 180
cttgagaaga gtgaccaata a 201
<210> 7
<211> 564
<212> DNA
<213> 人工序列(未知)
<400> 7
atggttcgac cgctgaactg catcgtcgcc gtgtcccaga atatgggcat cggcaagaac 60
ggagaccttc cctggccaat gctcaggaac gaattcaagt acttccaaag aatgaccacc 120
acctcctcag tggaaggtaa acagaacctg gtgattatgg gccggaaaac ctggttctcc 180
attcctgaga agaatcgacc tttaaaggac agaattaata tagttctcag tagagagctc 240
aaggaaccac cacaaggagc tcattttctt gccaaaagtc tggacgatgc cttaaaactt 300
attgaacaac cagagttagc agataaagtg gacatggttt ggatagttgg aggcagttcc 360
gtttacaagg aagccatgaa tcagccaggc catctcagac tctttgtgac aaggatcatg 420
caggaatttg aaagtgacac gttcttccca gaaattgatt tggagaaata taaacttctc 480
ccagagtacc caggggtcct ttctgaagtc caggaggaaa aaggcatcaa gtataaattt 540
gaagtctatg agaagaaagg ctaa 564
<210> 8
<211> 4218
<212> DNA
<213> 人工序列(未知)
<400> 8
atgctgctgt cgccgtcgct gctgctgctg ctgctgctgg gggcgccgcg gggctgcgcc 60
gagggtgtgg cggcggcgct tacccccgag cggctcctgg agtggcagga taaaggaata 120
tttgttatcc aaagtgagag tctcaagaaa tgcattcaag ccggtaaatc 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 gtaaaaaata tctaaaccac 1080
attgatcatg aaatagttga aaaagatgcg tggaaatatt atgctaccca ctgtgagcct 1140
ggctggaatc cctacaatcg taattgctac aaacttcaga aagaagaaaa gacctggcat 1200
gaggctctgc gttcttgtca ggctgataac agtgcattaa tagacataac ctcattagca 1260
gaggtggagt ttcttgtaac cctccttgga gatgaaaatg catcagaaac atggattggt 1320
ttgagcagca ataaaattcc agtttccttt gaatggtcta atgactcttc agtcatcttt 1380
actaattggc acacacttga gccccacatt tttccaaata gaagccagct gtgtgtctca 1440
gcagagcagt ctgagggaca ctggaaagtc aaaaattgtg aagaaagact tttttacatt 1500
tgtaaaaaag caggccatgt cctctctgat gctgaatcag gatgtcaaga gggatgggag 1560
agacatggtg gattctgtta caaaattgac acagtccttc gaagctttga ccaagcttcc 1620
agcggttatt actgtcctcc tgcacttgta accattacaa acaggtttga acaggctttt 1680
attaccagtt tgatcagtag tgtggtaaaa atgaaggaca gttatttttg gatagctctt 1740
caggaccaaa atgatacggg agaatacact tggaagccag tagggcagaa acccgagccg 1800
gtgcagtaca cacactggaa cacacaccag ccgcgctaca gtggtggctg tgttgccatg 1860
cgaggaaggc atccacttgg tcgctgggaa gtgaagcact gtcggcactt taaggcaatg 1920
tccttgtgca agcagccagt tgaaaatcag gaaaaagcag agtatgaaga gagatggccc 1980
tttcacccct gctatttgga ctgggagtca gagcctggtc tggccagttg cttcaaggta 2040
tttcatagtg aaaaagttct gatgaaaaga acatggagag aagctgaagc attttgcgaa 2100
gaatttggag ctcatcttgc aagctttgcc catattgagg aagagaattt tgtgaatgag 2160
ctcttacatt caaaatttaa ttggacagaa gaaaggcagt tctggattgg atttaataaa 2220
agaaacccac tgaatgccgg ctcatgggag tggtctgata gaactcctgt tgtctcttcg 2280
tttttagaca acacttattt tggagaagat gcaagaaact gtgctgttta taaggcaaac 2340
aaaacattgc tgcccttaca ctgtggttcc aaacgtgaat ggatatgcaa aatcccaaga 2400
gatgtgaaac ccaagattcc gttctggtac cagtacgatg taccctggct cttttatcag 2460
gatgcagaat acctttttca tacctttgcc tcagaatggt tgaactttga gtttgtctgt 2520
agctggctgc acagtgatct tctcacaatt cattctgcac atgagcaaga gttcatccac 2580
agcaaaataa aagcgctatc aaagtatggt gcaagttggt ggattggact tcaagaagaa 2640
agagccaatg atgaatttcg ctggagagat ggaacaccag tgatatacca gaactgggac 2700
acaggaagag aaagaactgt gaataatcag agccagagat gtggctttat ttcttctata 2760
acaggactct ggggtagtga agagtgttca gtttctatgc ctagtatctg taagcgaaaa 2820
aaggtttggc tcatagagaa aaagaaagat acaccaaaac aacatggaac gtgtcccaaa 2880
ggatggctat attttaacta taagtgcctt ctgctgaata tccccaaaga cccaagcagt 2940
tggaagaact ggacgcatgc tcaacatttc tgtgctgaag aaggggggac cctggtcgcc 3000
attgaaagtg aggtggagca agctttcatt actatgaatc tttttggcca gaccaccagt 3060
gtgtggatag gtttacaaaa tgatgattat gaaacatggc taaatggaaa gcctgtggta 3120
tattctaact ggtctccatt tgatataata aatattccaa gtcacaatac cactgaagtt 3180
cagaaacaca ttcctctctg tgccttactc tcaagtaatc ctaattttca tttcactgga 3240
aaatggtatt ttgaagactg tggaaaggaa ggctatgggt ttgtttgtga aaaaatgcaa 3300
gatacttctg gacacggtgt aaatacatct gatatgtatc caatgcccaa taccttagaa 3360
tatggaaaca gaacttacaa aataattaat gcaaatatga cttggtatgc agcaataaaa 3420
acctgcctga tgcacaaagc acaactggtc agcatcacag accagtatca ccagtccttc 3480
ctcactgttg tcctcaaccg gctaggatat gcccactgga ttggactgtt caccacagat 3540
aatggtctta attttgactg gtctgatggc accaaatctt ctttcacttt ttggaaagat 3600
gaggagtcct ccctccttgg tgactgcgtt tttgccgaca gcaacggacg ctggcatagc 3660
acagcctgcg agtcatttct gcaaggtgcc atttgtcatg tgccacctga aacaagacaa 3720
tctgaacacc cagagttgtg ctcagaaaca tctattccct ggataaaatt taaaagtaat 3780
tgctacagtt tttctacagt cctagacagt atgagttttg aggctgctca tgaattttgc 3840
aaaaaggaag gttctaatct tttaacaatc aaggatgagg ctgaaaatgc atttctccta 3900
gaagagctgt ttgcttttgg ttcttctgtc cagatggttt ggttgaatgc tcaatttgat 3960
ggtaacaatg aaaccataaa gtggtttgat ggaactccca cagaccagtc aaactggggc 4020
attcggaagc cagacacaga ctacttcaag ccccatcatt gtgttgcctt gaggatccct 4080
gaaggattat ggcagctatc cccgtgtcaa gaaaaaaaag gctttatatg taaaatggag 4140
gcagatattc acactgcaga ggcgctgcca gaaaaaggac caagtcacag ccaccaccat 4200
caccatcacc atcactaa 4218
Claims (9)
1.一种基因改造的表达改造型PLA2受体的细胞，其特征在于：含有经过糖基化修饰PLA2R受体基因。
2.根据权利要求1所述的细胞，其特征在于：所述的经过突变修饰PLA2R受体基因为将PLA2R基因序列中4191-4392把对应的天冬酰胺改成丙氨酸。
3.根据权利要求1或2所述的细胞，其特征在于：所述的突变修饰具体为序列：1。
4.根据权利要求1或2所述的细胞，其特征在于：所述的突变修饰具体为序列：4。
5.根据权利要求1或2所述的细胞，其特征在于：所述的细胞为CHO细胞或293细胞。
6.一种基因改造的表达改造型PLA2受体的细胞应用，其特征在于：使用权利1、2、3、5任意之一所述细胞膜或膜碎片进行抗PLA2受体抗体检测或使用权利4述细胞所分泌的蛋白进行抗PLA2受体抗体检测。
7.根据权利要求6所述的应用，其特征在于：将所述的细胞膜或膜碎片包被于固相载体，利用免疫原理进行检测。
8.根据权利要求6所述的应用，其特征在于：将所述的细胞膜或膜碎片用于免疫沉淀检测。
9.根据权利要求7所述的应用，其特征在于：所述的固相载体是酶标板、磁珠微球、微孔膜材料、免疫胶乳微球。
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