大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体构建

摘要

A 126bp-length DNA fragment and a 2214bp-length DNA fragment from Actinobacillus pleuropneumoniae plasmid pKMA2425 were synthesized and then cloned into pGSI.The constructed recombinant plasmid was named pGSIA.Spectinomycin resistance gene amplified by PCR from pTKRED and linear pGSIA(126bp-length DNA fragment+pGSI+2214 bp-length DNA fragment) amplified by PCR from circular pGSIA were ligated together and then transformed into Actinobacillus pleuropneumoniae by electroporation.In the selection pressure of spectinomycin, correctly ligated recombinant plasmid was gained by PCR identification and named pGSIAS.Sequencing result of pGSIAS was as expected.In the selection pressure of spectinomycin, Actinobacillus pleuropneumoniae serotypes 1～10 containing pGSIAS growed well.In the selection pressure of ampicillin, Escherichia coli containing pGSIAS growed well.The results showed that the sequence of Escherichia coli-Actinobacillus pleuropneumoniae shuttle vector pGSIAS was correct.In addition, pGSIAS could replicate itself and express related genes on the vector in Actinobacillus pleuropneumoniae or Escherichia coli.%合成胸膜肺炎放线杆菌质粒pKMA2425上长度为126 bp及2214 bp的2个DNA片段,克隆到大肠埃希氏菌质粒pGSI中,获得重组质粒pGSIA.PCR扩增pTKRED上的大观霉素抗性基因及线性化的pGSIA(126 bp DNA片段-pGSI-2214 bp DNA片段),连接,电转化胸膜肺炎放线杆菌,在大观霉素的选择压力下筛选鉴定到连接正确的质粒,命名为pGSIAS.pGSIAS测序结果符合预期.在大观霉素的选择压力下,含有pGSIAS的胸膜肺炎放线杆菌血清1～10型菌株均生长良好;在氨苄青霉素的选择压力下,含有pGSIAS的大肠埃希氏菌生长良好.结果表明本研究构建的大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体pGSIAS序列正确,在胸膜肺炎放线杆菌及大肠埃希氏菌中均能复制并表达质粒上的相关基因.