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目的表达和纯化弓形虫P30(SAG1)蛋白,为弓形虫病快速诊断试剂盒及蛋白质疫苗的研制奠定基础。方法PCR法从弓形虫基因组DNA中扩增P30基因片段,P30产物克隆到表达质粒pET-30a(+)构建重组载体,将其转化到DH5α中。经PCR扩增和质粒酶切及基因测序鉴定后,阳性重组质粒转化到大肠埃氏菌BL21(DE3)中,经IPTG诱导,表达产物用SDS-PAGE和Western blot进行鉴定。大量的诱导表达产物用SNBC3S NTA Resin方法纯化并进行复性。结果扩增的P30基因片段为750bp,重组表达融合蛋白量单位为30ku,与理论值相符。结论成功构建重组体,获得纯化和复性的弓形虫主要表面抗原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 express and purify Toxoplasma gondii P30 (SAG1) protein and lay a foundation for the rapid diagnosis of toxoplasmosis and the development of protein vaccine. Methods P30 gene fragment was amplified from genomic DNA of Toxoplasma gondii by PCR. P30 product was cloned into expression vector pET-30a (+) to construct a recombinant vector and transformed into DH5α. After PCR amplification, plasmid digestion and sequencing, the positive recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. The expressed product was identified by SDS-PAGE and Western blot. A large number of induced expression products were purified by SNBC3S NTA Resin method and refolded. Results The amplified P30 gene fragment was 750bp, and the unit of recombinant fusion protein was 30ku, which was consistent with the theoretical value. Conclusion The recombinant plasmids were successfully constructed and the purified and refolded Toxoplasma gondii p30 gene was highly expressed, which laid the foundation for the diagnosis and vaccine research of toxoplasmosis.