摘要
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 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.
出处
《中国病原生物学杂志》
CSCD
北大核心
2014年第3期252-254,共3页
Journal of Pathogen Biology
基金
南阳市科技攻关计划项目(No.2011GG018)
