用PCR体外定点突变技术诱导霍乱毒素A亚基突变体的构建

目的： 有效去除霍乱毒素Ａ 亚基（ ＣＴ Ａ） 的毒性作用而保存其佐剂性能。方法： 采用ＰＣＲ 体外定点突变技术（ＰＣＲ ＳＤＭ） ，设计两对引物，引入一个突变位点，通过重叠延伸法两次ＰＣＲ 扩增，使ＣＴ Ａ 编码基因第６３ 位密码子由ＣＴＣ 突变为ＴＴＴ，亦将扩增片断克隆入ＰＵＣ１９ 载体。结果： ＤＮＡ 测序结果表明在预期位点已发生突变，用ＰＣＲ 诱导成功ＣＴ Ａ 突变体。结论： ＰＣＲ 诱导突变准确、简便，为深入研究ＣＴ 免疫佐剂的作用打下了基础

Construction of a mutant A subunit of cholera toxin elicited by polymerase chain reaction for site directed mutagenesis

Objective: To construct a mutant of cholera toxin (CT),lacking diarrheagenicity but retain adjuvanticity.Methods: We used the polymerase chain reaction (PCR) for the site directed mutagenesis.Two sets of primers(P 1,P 2,P 3,P 4)were designed according to the gene sequence of cholera toxin A subunit (CT A),and mismatches were introduced into P 2 and P 3 for serine (TCT) to phenylalanine (TTT) substitution at position 63.Mutagenesis was performed in a two step PCR,the amplified fragments from the second PCR which contain the mutation site were subcloned into the pUC19 vector and verified by sequencing analysis.Results: The sequencing analysis showed that the mutation site was correct,and the mutant of CT A subunit was constructed respectively.Conclusion: A mutant CT A has been constructed and identified,it is a promising candidate for further studies.