一种构建马链球菌兽疫亚种血红素受体基因缺失突变株的方法

Construction of in-frame deletion streptococcal hemoprotein receptor gene mutant in streptococcus equi subsp. zooepidemicus

【目的】探讨一种构建马链球菌兽疫亚种基因缺失突变株的方法。【方法】PCR扩增目的基因,用pJR700温度敏感载体系统,构建目的基因载体;反向PCR扩增目的基因缺失载体片段,连接,产生目的基因缺失载体;电转化缺失重组质粒导入感受态细胞,先在37℃卡拉霉素(kan)培养基中连续培养,然后在30℃不含kan液体培养基中传代,挑取抗生素敏感菌落,PCR扩增检测抗生素敏感菌染色体上目的基因片段和链黑霉素抗性实验确认血红素受体基因缺失。【结果】获得不含抗生素基因的马链球菌兽疫亚种血红素受体基因缺失突变株。【结论】用pJR700温度敏感载体系统,构建马链球菌兽疫亚种基因缺失突变株是可行的。

[Objective]To construct the in-frame deletion gene mutants in streptococcus equi subsp. zooepidemicusby （ S. zooepidemicus） by pJR700 plasmid,a thermosensitive delivery vector system. [Method]With PCR we amplified a 4017-bp DNA fragment of streptococcal hemoprotein receptor gene from chromatosome DNA. This DNA fragment was subcloned into the pJR700 plasmid to create pXL28. Using pXL28 as the template we got the DNA fragment deleted 1831 bp in streptococcal hemoprotein receptor gene by inverse PCR amplification and ligated the in-frame deletion DNA fragment by T4DNase to create pXL30. Then pXL30 was transformed into S. Zooepidemicus by electroperation. The kanamycin（kan）-resistant clones were selected at 37℃ in the presence of kan for three rounds. To induce excision of the plasmid vector sequence,the culture was shifted to 30℃ and grown without antibiotics for four rounds. Colonies with kan sensitivity,which indicated that the plasmid had been excised,were selected by plating on THY agar at 37℃. S. Zooepidemicus mutants were identified through PCR with primers homologous to the flanking regions and the streptonigrin sensitive test. [Result]The in-frame deletion streptococcal hemoprotein receptor mutants were successfully built in S. Zooepidemicus. [Conclusion]The thermosensitive delivery vector system of pJR700 could be utilized to construct the in-frame deletion gene mutant strains of S. Zooepidemicus.