刚地弓形虫RACK1蛋白与PKC蛋白结合作用的研究

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。

Study of the protein interaction between RACK1 and PKC of Toxoplasma gondii

Objective To detect the protein interaction between RACK1 and PKC of Toxoplasrna gondii. Methods The RACK1 gene was amplified with PCR using the cDNA sequence of Toxoplasma gondii as a template. The restriction endonucleases EcoR I and Xho I were used to digest the PCR products of RACK1 and PKC. Fragments were ligated and transformed into E. coil BL21 to construct the recombinant plasmid pGEX-4T 1-RACK1. IPTG （0.8 mmol/L） was used to induce the recombinant plasmid to express the protein RACK1. The protein products were identified and purified using SDS-PAGE and Ni IDA affinity chromatography. Western blotting detected binding between RACK1 and PKC. Results PCR amplified the target gene with an open reading frame of 966 bp . The prokaryotic expression vector pGEX-4T-1- RACK1 was successfully constructed, and the pGEX-4T 1 RACK1 expression vector was induced for 4 h--6 h after it was transformed into E. coli BL21. After expression of the target protein was induced, SDS-PAGE revealed it to be 36 kpt. Western blotting revealed protein interaction between RACK1 and PKC. Conclusion The prokaryotic expression vector pGEX-4T-1-RACK1 was constructed, and the expressed product RACK1 binds to PKC protein.