铜绿假单胞菌外排泵MexA蛋白原核表达纯化

目的 构建MexA原核表达载体,并实现MexA原核表达、纯化.方法 从多重耐药的铜绿假单胞菌抽提DNA,经PCR扩增出mexA1基因与PUC18克隆载体连接,PCR、酶切及测序鉴定,再经PCR扩增出mexA基因,酶切纯化后克隆到原核表达载体PQE30,构建重组表达载体PQE30/mexA,PCR及酶切鉴定,IPTG诱导表达,SDS-PAGE、Western blot检测有无蛋白的表达.结果 已获得长约1.5kb PCR产物,酶切结果显示所构建的重组质粒已成功地克隆了mexA基因,序列分析结果与PA01mexA序列相同.SDS-PAGE检测表达产物,在相对分子量40kDa处有表达条带,诱导表达之菌体,超声破碎后,目的蛋白主要以包涵体形式存在.结论 获得mexA基因,并在E.coliM15中成功表达.融合蛋白纯化后作免疫原,为制备多克隆抗体打下基础.

Purification of MexA recombinant protein for antibody preparation in Pseudomonas aeruginosa

Objective Aimed to prepare purification MexA recombinant protein for antibody preparation. Methods Pseudomonas aeruginosa was cultured and then mexA1 temple DNA was extracted from the cultured germs.PCR was used to amplify the mexA1 gene from Pseudomonas aeruginosa DNA.PCR products were directly ligated into PUCk-18 vector,restriction enzymes cleavage analysis and sequencing,then PCR was used to amplify the mexA gene from PUC18/ mexA1 vector.After being digested with BamHⅠ+hindⅢ and purified,the mexA gene was inserted into the compatible site of prokaryotic expression vector PQE30,then the constructed recombinant plasmid was introduced into competent E.coliM15.The recombinant plasmid PQE30/mexA was transferred into an expression strain E.coliM15.The host bacterial harboring the expression plasmid were induced by IPTG and the product was identified by SDS-PAGE and Western-blot,then purified. Results The size of amplified mexA gene was about 1.5kp.The correct recombinant plasmid PQE30/mexA by restriction enzymes clearage analysis,DNA sequence of the cloned gene was the same as the published sequence.The fusion protein from the transformations was approximate size of 40 kDa in SDS-PAGE analysis.Most of the expressed recombinant proteins were in inclusion bodies form. Conclusion obtained mexA gene of Pseudomonas aeruginosa and expressed it successfully in E.coliM15.The purified fusion protein with the 6×his-binding protein of E.coliM15 is a suitable candidated of immunodiagnostic antigen.It will facilitate antibody preparation.