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目的构建葡萄球菌肠毒素A(SEA)和小鼠B71基因真核共表达载体。方法采用PCR和RTPCR方法分别克隆了带B71跨膜区的SEA(SEAB7tm)和小鼠B71基因,中间通过内部核糖体进入位点(Internalribosomeentrysite,IRES)序列的连接克隆至真核表达载体pcDNA3.1+。利用阳离子脂质体将重组质粒转染B16细胞,间接免疫荧光法检测B71和SEA分子在B16细胞膜表面的表达情况。结果测序结果与Genebank中公布的SEA、小鼠B71cDNA序列相符,双标记间接免疫荧光检测结果表明B71、SEA同时在转染的B16细胞膜上表达。结论成功构建了SEA和小鼠B71真核共表达载体,为进一步研究SEA和B71联合应用抗肿瘤免疫治疗及其免疫机理奠定了基础。
Objective To construct eukaryotic co-expression vector of staphylococcal enterotoxin A (SEA) and mouse B71 gene. Methods SEA (SEAB7tm) and mouse B71 genes with B71 transmembrane region were cloned by PCR and RTPCR respectively. The cDNA was cloned into the eukaryotic expression vector pcDNA3.1 by the internal ribosome entry site (IRES) +. The recombinant plasmids were transfected into B16 cells by cationic liposomes, and the expression of B71 and SEA on the surface of B16 cell membrane was detected by indirect immunofluorescence. Results The sequencing results were in accordance with the sequence of SEA and mouse B71 cDNAs published in Genebank. Indirect immunofluorescence assay showed that both B71 and SEA were expressed on the transfected B16 cell membrane simultaneously. Conclusion The eukaryotic expression vector of SEA and mouse B71 was constructed successfully, which laid the foundation for the further study on the antitumor immunotherapy and the immune mechanism of SEA and B71.