周期型马来丝虫复合表位基因的构建及在真核细胞中的表达  

Construction of multi-epitope gene of periodic Brugia malayi and its expression in eukaryotic cells

作　　者：陆施娟[1] 蒋晗 夏瑜椰 方政[1] Lu Shijuan;Jiang Han;Xia Yuye;Fang Zheng(Medical Morphology Laboratory,Medical College,Nantong University,Nantong 226000,China)

机构地区：[1]南通大学医学院医学形态学实验室,南通226000

出　　处：《中华地方病学杂志》2022年第4期277-283,共7页Chinese Journal of Endemiology

摘　　要：目的构建周期型马来丝虫半胱氨酸蛋白酶抑制剂/3-磷酸甘油醛脱氢酶复合表位基因(CPI502/GAPDH720)真核表达质粒pcDNA3.1(+)-BmCPI502/BmGAPDH720,并观察其在人宫颈癌细胞(Hela细胞)中的蛋白表达。方法根据T细胞表位预测的基因序列设计引物,以质粒pGEM-T-CPI621为模版,利用反转录PCR(RT-PCR)扩增目的基因片段,将该片段克隆至原核表达质粒pET-28a(+)中,构建原核表达质粒pET28a(+)-BmCPI502。根据软件设计合适引物,RT-PCR分别扩增BmCPI502基因和BmGAPDH720基因,pcDNA3.1(+)、BmCPI502、BmGAPDH720分别进行双酶切后进行连接,构建复合表位基因真核表达质粒pcDNA3.1(+)-BmCPI502/BmGAPDH720。将复合表位基因重组质粒转染至Hela细胞后,进行RT-PCR验证,获得与预期相符目的条带;将表达产物利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行检测。结果获得了原核表达重组质粒pET28a(+)-BmCPI502,经酶切鉴定得到502 bp特异性片段,与预期值符合;成功构建了复合表位基因真核表达质粒pcDNA3.1(+)-BmCPI502/BmGAPDH720,酶切鉴定所产生的片段大小与预期符合;复合表位基因真核表达质粒pcDNA3.1(+)-BmCPI502/BmGAPDH720转染Hela细胞后得到稳定表达,SDS-PAGE鉴定分析显示重组蛋白相对分子质量(Mr×10^(3))约为50。结论成功构建了周期型马来丝虫复合表位基因真核表达质粒pcDNA3.1(+)-BmCPI502/BmGAPDH720,并在真核细胞中获得相应的重组蛋白,为进一步研究重组蛋白纯化和测定其生物学活性奠定了基础。Objective To construct a eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 of periodic Brugia malayi cysteine protease inhibitor/glyceraldehyde 3-phosphate dehydrogenase(CPI502/GAPDH720)multi-epitope gene and observe its protein expression in Hela cells.Methods Primers were designed according to the predicted gene sequences of T cell epitopes.The target gene fragment was amplified by reverse transcription(RT)-PCR using plasmid pGEM-T-CPI621 as template.The fragment was cloned into prokaryotic expression plasmid pET-28a(+)to construct prokaryotic expression plasmid pET28a(+)-BmCPI502.The BmCPI502 gene and BmGAPDH720 gene were amplified by RT-PCR,respectively.The gene fragments of pcDNA3.1(+),BmCPI502 and BmGAPDH720 were digested by double enzyme digestion and ligated with the target gene.The eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 was constructed.The recombinant plasmid was transfected into Hela cells and verified by RT-PCR to obtain the desired target bands.The expression product was detected by sodium dodecyl sulfate polyacrylamide gelelectrophoresis(SDS-PAGE).Results The recombinant plasmid pET28a(+)-BmCPI502 was obtained,and the 502 bp specific fragment was identified by enzyme digestion,which was in line with the expected value;the eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 of the multi-epitope gene was successfully constructed,and the fragment size was in line with the expected value.The eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 was transfected into Hela cells and the recombinant protein was stable expressed.SDS-PAGE analysis showed that the relative molecular weight(Mr×10^(3))of the recombinant protein was about 50.Conclusions The eukaryotic expression plasmid of pcDNA3.1(+)-BmCPI502/BmGAPDH720 of periodic Brugia malayi multi-epitope gene is successfully constructed,and the corresponding recombinant protein is obtained in eukaryotic cells.This study has laid a foundation for further study of the purification and biological activity of

关 键 词：周期型马来丝虫 半胱氨酸蛋白酶抑制剂 3-磷酸甘油醛脱氢酶 重组 表位基因 

分 类 号：Q782[生物学—分子生物学] R737.33[医药卫生—肿瘤]