铜绿假单胞菌重组质粒pGEX-LasR的构建及在大肠埃希菌中的表达效率  

Construction of recombinant plasmid pGEX-LasR derived from Pseudomonas aeruginosa and its expression in Escherichia coli

作　　者：欧兴坤 李文桂[1] OuXingkun;Li Wengui(Institute of Infections and Parasitic Diseases,the First Affiliated Hospitalt Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院传染病寄生虫病研究所,400016

出　　处：《国际流行病学传染病学杂志》2021年第4期281-285,共5页International Journal of Epidemiology and Infectious Disease

基　　金：重庆市科委地方病重大专项基金(2008AB5055、2008AB5008、2008AB5054)。

摘　　要：目的:构建铜绿假单胞菌重组质粒pGEX-LasR,并在大肠埃希菌中研究其表达效率。方法:提取铜绿假单胞菌PA01株DNA,PCR扩增lasR基因,并将其与pGEX-1λT连接,构建重组质粒pGEX-LasR。将pGEX-LasR转入大肠埃希菌BL21(DE3)中,筛选阳性克隆,提取质粒进行PCR和双酶切鉴定,然后异丙基硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE和Western印迹试验分析和鉴定表达产物。结果:PCR扩增出717 bp的lasR基因;双酶切和PCR显示重组质粒构建成功;SDS-PAGE发现重组BL21(DE3)可表达相对分子质量约为53000的LasR重组蛋白,表达量约占菌体总量的21%。Western印迹试验显示,LasR重组蛋白可与铜绿假单胞菌感染的鼠血清发生特异性反应。结论:本研究成功构建了lasR基因的原核表达载体pGEX-LasR,该质粒可在大肠埃希菌BL21(DE3)中高效率表达具有免疫反应性的LasR重组蛋白。Objective To construct the recombinant plasmid pGEX-LasR of P.aeruginosa and observe its expression fficiency in Escherichia coli.Methods DNA of P.aeruginosa PA01 was extracted and lasR gene was amplfied by PCR.Then the PCR products were inserted into pCEX-1XT to construct recombinant plasmid pGEX-LasR.The obtained plasmid was transferred into E.coli BL21(DE3)to screen the positive clones.After identification with PCR and double enzyme digestion,the recombinant BL21(DE3)was induced using isopropyl-bpeta-D-thiogalactopyranoside(PTG),The expression products were analyzed by SDS-PAGE and western-blot.Results The amplified lasR gene was 717 bp.Double enzyme digestion and PCR proved that the recombinant plasmid pGEX-LasR was constructed scessfully.The results of SDS-PAGE and westem-blot indicated that the recombinant E.coli BL21(DE3)expressed a protein with a molecular weight of 53000,which contained about 21%of total somatic proteins and recognized the serum of mice infected with P.aeruginosa speifcally.Conclusions The recombinant plasmid pGEX-LasR is constructed sucessfully,and it can express the immune-specifie LasR protein eficiently in the recombinant E.coli BL21(DE3).

关 键 词：假单胞菌 铜绿 pGEX-LasR 大肠埃希菌 重组质粒 诱导表达 

分 类 号：Q78[生物学—分子生物学] R37[医药卫生—病原生物学]