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目的获得编码弓形虫RH株棒状体蛋白2和主要表面抗原1重组复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pMD18T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中的ROP2基因片段经EcoRⅠ和HindⅢ酶切,连接等反应,亚克隆入pET30a(+)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BglⅡandEcoRⅠ酶切的pET ROP2载体连接,构建pET ROP2P30载体,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氏菌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Westernblot分析。结果从弓形虫RH株基因组DNA中扩增出特异的ROP2和P30基因片段,成功构建成pET ROP2和pET ROP2P30载体,成功表达了弓形虫棒状体蛋白2和弓形虫棒状体蛋白与主要表面抗原1的融合复合蛋白,表达出的蛋白经纯化复性后具有免疫反应性。结论ROP2和P30基因重组后,在原核表达载体中表达出的蛋白经纯化复性后具有活性,获得纯化和复性的弓形虫ROP2和ROP2P30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 prepare a rapid diagnostic kit for Toxoplasma gondii and a protein vaccine for the preparation of recombinant Toxoplasma gondii RH strain 2 and major surface antigen 1 recombinant protein. Methods The ROP2 and P30 gene fragments were amplified from the genomic DNA of Toxoplasma gondii by PCR and cloned into the pMD18T vector respectively. The plasmids that had been inserted into the exogenous gene were identified by PCR, restriction enzyme digestion and sequencing. The ROP2 gene fragment was digested with EcoRⅠ and Hind Ⅲ, ligated and then subcloned into pET30a (+) prokaryotic expression vector to construct pET ROP2 vector. The P30 gene fragment in pMD P30 was ligated with pET ROP2 vector digested with BglII and EcoRⅠ, Construction of pET ROP2P30 vector, screening by kanamycin-containing LB plates, digestion and PCR identification. The positive recombinant plasmid was transformed into E. 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 genomic DNA of Toxoplasma gondii RH strain and successfully constructed into pET ROP2 and pET ROP2P30 vector and successfully expressed Toxoplasma gondii protein 2 and Toxoplasma gondii major surface antigen 1 Fusion protein, the expressed protein is immunoreactive after purification and refolding. CONCLUSION: The recombinant protein of ROP2 and P30 is prokaryotic expressed in prokaryotic expression vector. After purification and renaturation, the purified and renatured Toxoplasma gondii ROP2 and ROP2P30 are highly expressed, which can be used in the diagnosis and vaccine of toxoplasmosis Foundation.