SEA和B7-1基因真核共表达载体的构建及在B16细胞的表达

目的构建葡萄球菌肠毒素A（SEA）和小鼠B7-1基因真核共表达载体。方法采用PCR和RT-PCR方法分别克隆了带B7-1跨膜区的SEA（SEA-B7tm）和小鼠B71基因，中间通过内部核糖体进入位点（Internal ribosome entry site，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B7-1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B7-1 cDNA序列相符，双标记间接免疫荧光检测结果表明B7-1、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B7-1真核共表达载体，为进一步研究SEA和B7-1联合应用抗肿瘤免疫治疗及其免疫机理奠定了基础。

Construction of the eukaryotic coexpression vector of SEA and murine B7-1 and its expression in B16 cell line

Objective To construct the eukaryotic coexpression vector of SEA and murine B7-1. Methods The SEA with B7-1 transmembrane region (SEA -B7tm) and the murine B7-1 gene were cloned by PCR and RT-PCR, respectively. SEA-B7tm and B7-1 were linked by IRES sequence and cloned into eukaryotic expression vector pcDNA3.1 . The recombinant plasmid was transfected into B16 cell line by lipofectamine reagent. The expressions of B7-1 and SEA were observed by double-labeling indirect immunofluorescent stain. Results The cloned SEA-B7tm and the murine B7-1 gene were identical to those reported in GeneBank. The B7-1 and the SEA were simultaneously expressed on the membrane of B16. Conclusion The eukaryotic coexpression vector of SEA and murine B7-1 is constructed successfully. The results give a basis for coapplication of B7 and SEA in tumor treatment and research of its immunological mechanisms.