表达破伤风毒素C片段的重组鼠伤寒沙门菌的构建与鉴定

为构建表达破伤风毒素C片段基因的减毒鼠伤寒沙门菌,将编码破伤风毒素C片段的基因插入到asd 的组成型表达载体pYA3333,转化asd基因突变的E.coliX6212,获得重组质粒pYA3333-TetC,再将重组质粒通过电穿孔法转入宿主菌,构建成表达破伤风毒素C片段基因的平衡致死的鼠伤寒沙门菌X4550(pYA3333-TetC)。免疫印迹试验证实,该重组菌表达的蛋白与预期的目的蛋白一致。重组菌X4550(pYA3333-TetC)的稳定性试验表明,在体外培养100代后,随机挑选的重组菌落全部都能生长,重组菌X4550(pYA3333-TetC)的生长曲线测定表明,X4550(pYA3333-TetC)和X4550(pYA3333)的生长状态基本一致。动物试验证明,该重组菌是安全的,破伤风毒素攻击后能提供良好的保护作用。

Construction and identification of recombinant Salmonella typhimurium strain expressing tetanus toxin fragment C

To construct a recombinant attenuated Salmonella typhimurium strain which can express tetanus toxin fragment C,the gene fragment encoding tetanus toxin fragment C was cloned into the expression vector pYA3333 containing asd gene,and a new recombinant plasmid pYA3333-TetC was generated.The recombinant plasmid pYA3333-TetC was transformed into an attenuated Salmonella(typhimurium) strain X4550 by electroporation,yielding the live vaccine strain X4550(pYA3333-TetC),which was a balanced-lethal recombinant.Stability and growth curve of the recombinant strain was determined.Results showed that the recombinant X4550(pYA3333-TetC) maintained good stability after 100 passages in vitro and the growth status of X4550(pYA3333-TetC) was the same as that of X4550(pYA3333).The recombinant strain X4550(pYA3333-TetC) was safe and could provide significant protection against a lethal challenge with tetanus toxin in immunized mice.