变异链球菌葡聚糖结合蛋白D基因失活菌株的构建和鉴定

Construction of glucan-binding protein D gene inactivation mutant of Streptococcus mutans

目的利用基因打靶技术构建变异链球菌葡聚糖结合蛋白D基因(gbpD)失活株,用于葡聚糖结合蛋白D基因功能的研究。方法体外培养变异链球菌UA159菌株并以其基因组为模板,对gbpD基因内部序列进行PCR扩增,连接自杀载体pVA8912,分别用酶切及PCR鉴定;转化变异链球菌UA159株,用PCR及Westernblot鉴定。结果经鉴定PCR产物及插入片段大小与预期值相符,且为所需目的基因片段,成功构建了自杀质粒pVA8912-gbpD;经PCR鉴定及Westernblot鉴定,gb-pD基因失活株基因组中gbpD基因内部成功插入目的片段,且该菌株不表达GbpD蛋白。结论成功构建了用于变异链球菌gbpD基因打靶的自杀质粒和gbpD基因失活株,为该基因功能的研究奠定了基础。

Objective To construct an insertional inactivation mutant of Streptococus mutans glucanbinding protein D for the further functional study of the protein. Methods An intemal fragmont of gbpD coding sequence was PCR amplified from S. mutans UA159 chromosomal DNA, and cloned into suicide vector pVA8912 to obtain plasmid pVA8912/gbpD. Then the plasmid was transformed S. mutans UA159, Cells with the plasmid integrated into the chromosome were selected on erythromycin plate and were identified by PCR and Western blot. Results The internal fragment of gbpD was successfully cloned into pVA8912 and transformed into S. mutans UA159. The gbpD mutant was identified by PCR and Western blot. Conclusion The construction of the gbpD gene deficiency mutant strain of S. mutans is successful.