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。

abstractsObjective: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 the recombinant protein.