重组超抗原葡萄球菌肠毒素A基因的克隆及表达载体的构建

Construction of expression vector of superantigen staphylococcal enterotoxin A gene

目的 克隆金黄色葡萄球菌肠毒素 A(SEA)全长基因并构建其表达载体。方法 以金黄色葡萄球菌的基因组 DNA为模板 ,用两条针对 SEA全长基因特异的引物 ,通过 PCR方法克隆 SEA全长基因 ,PCR产物与p GEM- T载体连接 ,经测序证实后进行亚克隆 ,构建其表达载体 p ET- 2 8b- SEA,再次测序验证。结果 克隆了 SEA全长基因 ,编码 2 5 7个氨基酸残基 ,经测序证实与 Genbank中收录的 SEA基因序列完全一致 ;并构建了 SEA的表达载体。结论 成功克隆了 SEA全长基因并构建了其表达载体 ,为进一步研究

Objective To clone the total sequence of staphylococcal enterotoxin A (SEA) gene and construct its expression vector. Methods A pair of primers special to the total sequence of SEA gene were synthesized by Sangon Company (Shanghai); SEA gene was cloned by PCR techniques, PCR product was ligated with the pGEM-T vector and confirmed by DNA sequencing; then, the target gene was subcloned into the expression vector of pET-28b and verified again by sequencing. Results The SEA gene was cloned and it consisted of 771 base pairs, encoded 257 amino acid residues: the targeting vector pET-28b-SEA was constructed. Conclusions The success of cloning and expression of SEA gene lays a good foundation for the further studies on the antitumor effects of the SEA fusion protein in immunotherapy.