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目的构建弓形虫致密颗粒蛋白6(GRA6)基因重组质粒并在大肠埃希菌中进行表达。方法根据弓形虫GRA6基因序列设计并合成引物,用PCR方法从弓形虫基因组DNA中扩增GRA6基因片段,扩增产物经1.0%琼脂糖凝胶电泳鉴定后,再克隆到pGEX-4T载体中,重组质粒经限制性内切酶BamHI、EcoRI酶切鉴定并测序后,将重组质粒转化大肠埃希菌BL21后并用IPTG诱导表达,产物经SDS-PAGE分析并纯化,以Western blot分析其反应原性。结果以弓形虫DNA为模板扩增GRA6基因片段,经1%琼脂糖凝胶电泳鉴定扩增产物大小为693bp,与理论值相符;重组质粒经酶切鉴定构建成功,测序结果与GenBank上弓形虫RH株GRA6序列AF239283.1同源性100%;重组质粒转化菌经IPTG诱导后表达的GRA6融合蛋白分子质量单位约为52ku,与GST-GRA6的理论分子质量相符,该融合蛋白可被GST标签抗体识别。结论成功重组了弓形虫GRA6基因,表达蛋白具有反应原性,为弓形虫诊断抗原试剂盒的制备奠定了基础。
Objective To construct the recombinant plasmid of Toxoplasma gondii (GRT) 6 gene and express it in Escherichia coli. Methods According to the sequence of GRA6 gene of Toxoplasma gondii, primers were designed and synthesized. The GRA6 gene fragment was amplified from the genomic DNA of Toxoplasma gondii by PCR. The amplified product was identified by 1.0% agarose gel electrophoresis and cloned into pGEX-4T vector. The recombinant plasmid was identified by restriction enzyme BamHI, EcoRI and sequenced. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. The product was analyzed by SDS-PAGE and purified by Western blot, . Results The fragment of GRA6 gene was amplified by using Toxoplasma gondii DNA as a template. The amplified product was identified by 1% agarose gel electrophoresis. The amplified product was 693bp, which was consistent with the theoretical value. The recombinant plasmid was successfully constructed by restriction endonuclease analysis. RH strain GRA6 sequence homology of AF239283.1 100%; recombinant plasmid transformed bacteria induced by IPTG GRA6 fusion protein molecular mass unit is about 52ku, consistent with the theoretical molecular mass of GST-GRA6, the fusion protein can be GST tag Antibody recognition. Conclusion The GRA6 gene of Toxoplasma gondii was successfully recombined, and the expressed protein was reactive. It laid the foundation for the diagnosis of Toxoplasma gondii antigen kit.