金黄色葡萄球菌肠毒素A Asp227Ala基因的克隆及表达

Cloning and Expressing of Staphylococcal enterotoxin A Asp227ala gene

目的 :金黄色葡萄球菌肠毒素AAsp2 2 7ala基因的克隆及表达。方法 :利用错配PCR方法 ,从含有金黄色葡萄球菌肠毒素A(StaphylococcalenterotoxinA ,SEA)基因的质粒中扩增出约 72 0bp的DNA片段 ,将其克隆到表达载体 7ZTS中 ,并转化于JM10 9(DE3)。结果 :重组质粒的测序结果表明 ,它含有 70 2bp(不包括N端 72bp的信号肽编码区 ) ,其核苷酸序列与文献报道完全一致 ,推导的氨基酸序列显示 2 2 7位的天冬氨酸已突变为丙氨酸。结论 :该基因所表达的蛋白为可溶性蛋白 ,表达量占总蛋白 5 1.5 %。表达的蛋白与天然肠毒素A产生的抗体能发生凝集作用 ,具有与天然SEA类同的抗原活性。

Objective:cloning and expressing of Staphylococcal enterotoxin A Asp227ala gene.Methods:The 720 bp-long gene segment was amplified from a plasmid with Staphylococcal enterotoxins A (SEA) encoding gene by PCR using Taq DNA polymerase.The production of PCR was inserted to expression vector 7ZTS and transformed into E.coli.JM109 (DE3).Results:The sequencing of the recombined plasmid proved that Asp227of SEA gene was mutated into Ala227 of SEA.The nucleotide sequence is in accord with that reported before.It contains as 702 bp (the N-terminal signal peptide encoding region is not included).Conclusion:The recombined protein is a soluble protein and up to 51.5% of total protein.It can occur agglutination reaction with the antibody producted by natural Enterotoxin A and has the same antigen activity as natural SEA.