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目的构建编码弓形虫RH株棒状体蛋白2(ROP2)和主要表面抗原1的重组表达质粒,纯化和复性的融合蛋白为弓形虫病快速诊断试剂盒及蛋白质疫苗的研制作准备. 方法用PCR技术从弓形虫基因组DNA中扩增出 ROP2和P30基因片段,分别克隆入pMD18-T载体,并对重组入外源基因的质粒通过PCR、双酶切和测序鉴定,将pMD-ROP2中 ROP2基因片段经EcoRⅠ和HindⅢ酶切、连接等反应,亚克隆入pET-30a(+)原核表达载体,构建pET-ROP2载体,然后再将pMD-P30中的P30基因片段与经同样NcoⅠ和EcoRⅠ酶切的pET-30a(+)载体连接,经含卡那霉素的LB平板筛选,酶切和PCR鉴定.阳性重组质粒转化到大肠埃希菌BL21(DE3)中,经IPTG诱导,表达产物用SDS-PAGE进行鉴定.大量的表达融合蛋白经纯化和复性后,用Western blot 分析. 结果从弓形虫RH株DNA中扩增出特异的ROP2和P30基因片段,成功克隆出pET-ROP2和pET-P30载体. 结论成功构建了pET-ROP2和pET-P30重组体,获得纯化和复性的弓形虫 ROP2 和P30的高效表达产物,为弓形虫病的诊断和疫苗研究奠定了基础.“,”Objective To obtain the functional fusion protein of rhoptry protein 2(ROP2) and major surface antigen 1 of Toxoplasma gondii in order to further prepare for the diagnosis and polyvalent vaccines of toxoplasmosis. Metthods The ROP2 and P30 genes from genomic DNA of T. gondii RH strain were amplified by PCR, and were inserted into pMD18-T cloning vector, respectively. ROP2 fragment was subcloned to pET-30a(+) digested by EcoRⅠand Hind Ⅲ to construct pET-ROP2, P30 fragment was subcloned to pET-30a(+) digested by NcoⅠand EcoRⅠto create pET-P30. The resulting recombinant plasmids were transformed into E. coli BL21 (DE3) and were induced with IPTG. Expression proteins identified by SDS-PAGE were further purified and refolded. Results The study showed that sizes of ROP2 and P30 genes were 1 212 and 684 bp with corresponding molecular weight of 50 ku and 30 ku. Conclusion The recombinant proteins could specifically react with sera and P30 monoclonal antibody against T. gondii.