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目的在大肠埃希菌中高效表达及纯化弓形虫GRA6抗原,为制作弓形虫感染基因工程诊断试剂盒奠定基础。方法将重组pGEX-GRA6表达载体转化大肠埃希菌BL21-Codon Plus (DE3)-RP菌株,在异丙基硫代-β-D半乳糖苷(IPTG)诱导下表达。超声破壁后,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物的表达形式,并对表达产物以硫酸铵沉淀、Sephedax G50脱盐和GST亲和层析柱进行目的蛋白的纯化。通过Western blotting检测其纯化的重组抗原的免疫反应性。结果GRA6以融合蛋白(GST-GRA6)的形式在大肠埃希菌中得到了高效表达。对表达产物可溶性分析表明,表达蛋白在上清液中和包涵体中均有表达。在上清液中表达的可溶性蛋白经纯化后蛋白纯度可达90%以上。Western blotting分析表明纯化蛋白能被弓形虫感染的人血清所识别。结论GRA6在大肠埃希菌中得到了高效表达,重组抗原经纯化后能特异性地被弓形虫感染者血清所识别。
Objective To express and purify Toxoplasma gondii GRA6 antigen efficiently in Escherichia coli and lay a foundation for the production of Toxoplasma gondii genetic engineering diagnostic kit. Methods The recombinant pGEX-GRA6 expression vector was transformed into Escherichia coli BL21-Codon Plus (DE3) -RP strain and expressed under the induction of isopropyl thio-β-D galactoside (IPTG). After sonication, the expressed product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the expressed product was precipitated with ammonium sulfate precipitation, Sephedax G50 desalting and GST affinity chromatography Purification of the protein of interest. Immunoreactivity of the purified recombinant antigen was tested by Western blotting. Results GRA6 was highly expressed in Escherichia coli as a fusion protein (GST-GRA6). Soluble expression of the expressed product showed that the expressed protein was expressed both in the supernatant and in the inclusion body. Purified protein expressed in the supernatant after purification of protein purity up to 90%. Western blotting analysis showed that the purified protein was recognized by human serum infected with Toxoplasma gondii. Conclusion GRA6 was highly expressed in Escherichia coli. The purified recombinant antigen was specifically recognized by the serum of Toxoplasma gondii infection.