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目的研究刚地弓形虫RACK1蛋白与PKC蛋白之间的结合特性。方法采用PCR方法扩增弓形虫RACK1基因,双酶切后与pGEX-4T-1连接,构建原核表达载体pGEX-4T-1-RACK1,转化入BL21大肠埃希菌中,用0.8mmol/L IPTG诱导表达,表达产物用SDS-PAGE检测并进行Ni-IDA亲和层析纯化,采用Western blot分析RACK1蛋白与PKC蛋白的结合作用。结果 PCR扩增出966bp的RACK1基因开放读码框,成功构建pGEX-4T-1-RACK1原核表达载体,转化BL21后用IPTG诱导4h~6h,SDS-PAGE检测到约36ku的表达产物,Western blot检测RACK1蛋白能与PKC蛋白结合。结论成功构建了pGEX-4T-1原核表达载体,表达产物RACK1蛋白能与PKC蛋白结合。
Objective To study the binding characteristics between RACK1 protein and PKC protein of Toxoplasma gondii. Methods Toxoplasma gondii RACK1 gene was amplified by PCR and ligated with pGEX-4T-1 after double digestion. The prokaryotic expression vector pGEX-4T-1-RACK1 was constructed and transformed into E.coli BL21. The expressed product was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography. The binding of RACK1 protein and PKC protein was analyzed by Western blot. Results The open reading frame of 966bp RACK1 gene was amplified by PCR. The prokaryotic expression vector pGEX-4T-1-RACK1 was successfully constructed. The recombinant plasmid was transformed into BL21 and induced with IPTG for 4h to 6h. The expression product of about 36ku was detected by SDS-PAGE. Western blot Detection of RACK1 protein binds to PKC protein. Conclusion The prokaryotic expression vector pGEX-4T-1 was successfully constructed. The expression product RACK1 protein could bind to PKC protein.