抗辐射菌lexA基因的克隆及在大肠杆菌中表达

MOLECULAR CLONING AND EXPRESSION OF lexA GENE FROM THE RADIORESISTANT BACTERIUM Deinococcus radiodurans

研究了旨在克隆抗辐射菌 Ｄｅｉｎｏｃｏｃｃｕｓ ｒｕｄｉｏｄｕｒａｎｓ的 ｌｅｘＡ基因并构建其表达载体，以便进一步研究ｌｅｘＡ基因的功能及其在辐射抗性中的作用。主要的实验步骤包括分离提取抗辐射菌基因组 ＤＮＡ，分离出 ｌｅｘＡ基因并测定其序列，克隆于质粒载体 ＰＵＣ１９，用电击穿孔法将重组的质粒导入大肠杆菌 ＪＭ１０９， ＳＤＳ－ＰＡＧＥ检测基因的表达。用定点突变技术，改变 ｌｅｘＡ基因核糖体结合位点（ＲＢＳ），以增强 ｌｅｘＡ基因的表达。结果表明， ｌｅｘＡ基因位于基因组 ＤＮＡ的 ＢｌｎＩ－ＡｓｃＩ片断中，由 ６３０个碱基对（ｂＰ）组成， 编码 ２１０个氨基酸（ａａ），理论上推定，分子量为 ２２．５ｋＤ，等电点为 ６．４。用 ｐＵＣ１９构建的 ｌｅｘＡ基因表达载体能在大肠杆菌 ＪＭ１０９中表达；但表达效率低，改变ｌｅｘＡ的核糖体结合位点ＲＢＳ可提高该基因表达水平；使进一步分离纯化 ｌｅｘＡ蛋白质以及深入研究该基因的作用成为可能。

In order to investigate the role of lexA gene in the radioresistance of Deinococcus radioduruns, the expressing vector of lexA gene was constructed. The genomic DNA was extracted from wild type Deinococcus radiodurans KD8301. The lcxA gene was isolated from the genomic DNA and sequenced. The Plasmid vector pUC19 was used to clone lexA gene and the electroporation was employed to transfer the recombinant plasmid into E. coli JM109. SDSPAGE was used to check the expression of lexA gene. The ribosome binding site (RBS) of lcxA gene was mutated by means of Site -Directed Mutagenesis technique for the optimum of gene expression. The results indicated that the lexA gene was located in the BlnI-AscI fragment of Deinocococus radiodurans genomic DNA, containing 630bp and coding 210 aa. The theoretically deduced molecular weight and the isoelectric point wele 2.5KDa and 6.4 respectively. IcxA geny inserted into pUC19 could be expressed in JM109, but at very low level. After optimizing the RBS of lexA gene, higher level of lcxA expression was obeserved. The results make it possible to isolate and purify lcxA protein, and further to investigate the function of lexA gene in Deinococcus radiodurans.