分枝杆菌表达质粒的快速构建及ClpC2基因功能初步研究

Fast construction of mycobacterium expression plasmid and identification of the functions of ClpC2 gene

目的通过一种不受到序列限制、酶切连接反应一步进行的分子克隆方法,构建分枝杆菌Clp C2重组表达质粒,检测其在耻垢分枝杆菌中的蛋白表达并对其功能进行初步探讨。方法利用识别甲基化位点的内切酶Fsp EⅠ进行一步法酶切、连接反应,构建重组质粒PMV261(+)-Clp C2。将重组质粒转入耻垢分枝杆菌,利用SDS-PAGE和Western blot检测Clp C2基因在耻垢分枝杆菌中的表达以及氧化应激、酸应激对其功能进行初步探讨。结果利用Fsp EⅠ快速构建了分枝杆菌表达质粒Clp C2,将其成功电转至耻垢分枝杆菌中,SDS-PAGEA和Western blot检测其蛋白的表达,His单抗和兔多抗均能检测到目的条带,Mr大小27 570。氧化应激及酸应激实验表明Clp C2重组耻垢分枝杆菌与空载耻垢分枝杆菌在对抗应激方面无明显差异。结论成功构建了一种不受序列限制的分子克隆方法以及分枝杆菌Clp C2重组表达质粒,Western blot表明其具有一定的免疫原性,氧化应激及酸应激表明Clp C2重组耻垢分枝杆菌对抗应激方面无明显改变,为进一步深入研究Clp C2基因的功能奠定了基础。

This study was performed to construct a mycobacterium recombinant plasmid of PMV261（＋）-ClpC2 through a one step and sequence independent method for molecular clone and preliminary identification of its functions. The restriction enzyme FspE I which was only recognize the methylation base CmC was utilized to establish a one step and sequence independent method for molecular clone and construction of the mycobacterium recombinant PMV261（＋）-ClpC2 plasmid. Then the recombinant PMV261（＋）-ClpC2 plasmid was transformed into Mycobacterium smegmatis by the electrotransformation method. The expression of recombinant protein rClpC2 was detected by Western blotting, and its functions were preliminary investigated by oxidative stress test and acid stress test. The mycobacterium recombinant PMV261（＋）-ClpC2 plasmid was constructed in a shorter time using the restriction enzyme FspE I ; the expressed recombinant protein rClpC2 about 27.57 kD was identified by SDS-PAGE and Western blotting. The expression vector for ClpC2 gene of M. smegmatis was constructed successfully, and the rClpC2 protein was expressed. Oxidative stress test and acid stress test revealed no significant difference between the rClpC2 M. smegmatis and PMV261（＋）-M. smegmatis. Taken together, the result has laid a foundation for further study on the gene function of CIpC2.