弓形虫蛋白激酶C受体1蛋白的表达与抗原性分析

Expression and antigenic analysis of the protein RACK1 of Toxoplasma gondii

目的构建弓形虫蛋白激酶C受体1基因的pET-30a(+)-RACK1重组质粒,表达、纯化RACK1蛋白。方法PCR扩增RACK1基因的cDNA序列,用SacI、NcoI限制性内切酶对RACK1基因的PCR产物及pET-30a(+)质粒进行双酶切,连接,转化大肠杆菌BL21,构建重组质粒。IPTG诱导表达,亲和层析柱纯化表达产物,SDS-PAGE和Western blotting对表达产物进行分析鉴定。结果 PCR扩增出966bp的RACK1完整基因序列,成功构建RACK1基因的pET-30a(+)-RACK1重组质粒,表达出约36kDa的RACK1蛋白,蛋白具有抗原性。结论成功构建弓形虫RACK1基因的pET-30a(+)-RACK1重组质粒,纯化出RACK1蛋白,为进一步进行RACK1蛋白在弓形虫入侵分子机制中的作用研究奠定基础。

The recombinant plasmid pET-30a （＋）-RACK1 for the RACK1 gene of Toxoplasma gondii was constructed in this study, and the RACK1 protein was expressed and purified. The cDNA sequence of RACK1 was amplificated by PCR, the products of RACK1 and pET-30a （＋） plasmid were double digested by the restriction endonuclease Sacl and Ncol, and then connected and transformed into E. coli BL21 to construct the recombinant plasmid. SDS-PAGE and Western blotting were used to analyze and identify the expression products after the recombinant plasmid was induced to express the protein RACK1 with IPTG, and the protein product was purified by affinity chromatography column. A 966 bp full gene sequence of RACK1 was amplificated by PCR, and pET-30a （＋）-RACK1 recombinant plasmid was constructed successfully. The expressed RACK1 protein was about 36 kD and showed antigenicity. It＇s considered that the recombinant plasmid pET-30a （＋）-RACK1 of RACKI was constructed successfully and the RACK1 protein was purified, which lays the foundation for the further study on the invasion molecular mechanism of Toxoplasma gondii.