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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 construct a recombinant plasmid encoding ROP2 and major surface antigen 1 of RH strain Toxoplasma gondii RH strain, and to prepare purified and renatured fusion proteins for the rapid diagnosis of toxoplasmosis and for the preparation of protein vaccines. Methods The ROP2 and P30 gene fragments were amplified from the genomic DNA of Toxoplasma gondii by PCR and cloned into the pMD18-T vector respectively. The plasmids containing the exogenous gene were identified by PCR, double enzyme digestion and sequencing. The pMD-ROP2 The prokaryotic expression vector pET-30a (+) was constructed by constructing the prokaryotic expression vector pET-ROP2, and then the P30 gene fragment in pMD-P30 was subcloned into the prokaryotic expression vector pET-30a EcoR I digested pET-30a (+) vector was ligated with kanamycin LB screening, digestion and PCR identification. The positive recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and induced by IPTG. The expressed product was identified by SDS-PAGE. A large number of expressed fusion proteins were purified and refolded and analyzed by Western blot. Results The specific ROP2 and P30 gene fragments were amplified from the DNA of RH strain Toxoplasma gondii and the vectors pET-ROP2 and pET-P30 were successfully cloned. Conclusion The recombinant plasmids pET-ROP2 and pET-P30 were constructed successfully, and the purified and refolded Toxoplasma gondii ROP2 and P30 highly expressed products were obtained, which laid the foundation for the diagnosis and vaccine research of toxoplasmosis.