弓形虫GRA6可溶性蛋白在大肠埃希菌中的表达及纯化

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.

Expression and purification of soluble fusion protein of Toxoplasma gondii GRA6 in E. coli

Objective To high express and purify toxoplasma gondii antigen GRA6 in E.coli which can be used to develop the genetic engineering diagnostic reagents.Methods The recombinant plasmid of pGEX-GRA6 was transformed to a bacterium BL21-Codon Plus(DE3)-RP and the recom- binant product was expressed under the inducement of isopropyl-beta-D-thiogalactosidase(IPTG). Cells were lysed by multiple rounds of sonication.The expression product was analyzed by using SDS- PAGE.Furthermore,it was purified by sedimentation of ammonium sulphate,desalting using Sephe- dax GS0 and affinity chromatography on glutathione-sepharose system.The immunogenicity of recom binant antigen purified was tested with Western blot.Results GRA6 was highly expressed in E.coli as fusion protein consisting of glutathione S-transferase and GRA6(GST-GRA6).The solubility anal- ysis of expression product indicated that this recombinant protein could be expressed in both superna- tant and inclusion bodies.The soluble protein of GRA6 in supernatant yield the final preparation at greater than 90% after purification.The recombinant protein purified could be recognized by human toxoplasmosis-infective sera with Western blotting analysis.Conclusions The soluble protein of GRA6 was highly expressed in E.coli and the recombinant antigen could be recognized by human tox- oplasmosis-infective serum after purification.The recombinant antigen can be used for devoloping kit to diagnose the acute and chronic infection of toxoplasma gondii.