轻型链球菌临床分离株psaA基因敲除株的构建及其毒力影响的初步研究

Construction of psaA gene-knockout clinical isolate of Streptocococcus mitis and a preliminary study on its virulence change

目的构建轻型链球菌(S.mitis)临床分离株S.mitis008肺炎链球菌表面蛋白A(psaA)基因敲除菌株S.mitis00801,并检测其生长及毒力影响,为进一步分析轻型链球菌属细菌间psaA基因水平转移的功能学意义奠定基础。方法采用长臂同源多聚酶链反应技术(LFH-PCR)构建含红霉素耐药基因(erm)片段及psaA基因上、下游同源序列的连接片段,并转化至S.mitis008中,在含红霉素的平板上筛选psaA基因敲除菌株,并采用PCR鉴定;进一步观察psaA基因敲除株S.mitis00801体外生长情况,并利用小鼠模型观察S.mitis008和S.mitis00801鼻咽部定植能力的差异。结果 PCR结果均证实轻型链球菌psaA基因敲除菌株S.mitis00801构建成功;S.mitis008和S.mitis00801体外生长情况比较无差异;在轻链球菌CBA/n小鼠鼻咽部定植模型中,psaA基因敲除菌株S.mitis00801定植能力较未敲除株S.mitis008明显减少,存在统计学差异(P<0.01)。结论 LFH-PCR技术构建psaA基因敲除菌株S.mitis00801方法切实可行,psaA基因的敲除对细菌的体外生长无影响,但降低了细菌在体内鼻咽部定植的能力。

Objective To understand the function significance of the events of frequently horizontal pneumococcal surface adhesin （psaA） gene transfer and recombination exist among the Streptococcus mitis （S.mitis） group strains through the construction of psaA gene-knockout clinical isolate S.mitis00801 of Streptocococcus mitis and the study on its virulence change.Methods Constructing a homologous sequences fragment of erm gene and psaA gene upstream and downstream through long flanking homology poly merase chain reaction （LFH-PCR） technology,and transforming into S.mitis008,psaA gene-knockout strain S.mitis00801 was screened out from plate containing erythromycin and was identified by PCR.And then the growth in vitro and nasopharyngeal colonization in vivo were compared between S.mitis008 and S.mitis00801.Result The results of PCR confirmed psaA gene was knockout successfully in the strain S.mitis00801.There was no difference in growth in vitro between S.mitis008 and S.mitis00801.In the model of nasopharyngeal colonization in CBA/n mice,it was significantly fewer S.mitis00801 than S.mitis008 in mice nasophary,and there was a statistically significant difference （P 〈 0.01）.Conclusion It is success and feasible using LFH-PCR technology to build psaA gene-knockout strain S.mitis00801.S.mitis00801 psaA gene knocked was no effect on the bacteria growth in vitro,in contrast to S.mitis008,but knocking psaA gene reduces the ability of the bacterial nasopharyngeal colonization in vivo.