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

Abstract:

Abstract：[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°C in the presence of kan for three rounds. To induce excision of the plasmid vector sequence, the culture was shifted to 30°C 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°C. 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.