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本研究旨在克隆微小隐孢子虫黏附相关蛋白基因CP21开放阅读框,构建其原核表达质粒,并对获得的融合蛋白进行鉴定。从含CP21基因的噬菌体中提取模板DNA,PCR扩增基因片段,构建pET-28a-CP21原核表达质粒,转化至埃希氏大肠杆菌BL21(DE3)中,IPTG诱导表达,表达产物经SDS-PAGE和Western blotting分析鉴定。用原核表达蛋白免疫小鼠,制备抗重组蛋白多抗,对蛋白进行定位。结果表明成功扩增出CP21基因,并构建原核表达质粒,转化至大肠杆菌中表达出相对分子质量为25 ku的融合蛋白,Western blotting证明表达的CP21融合蛋白能与抗C.parvum多克隆抗体产生特异性反应。免疫荧光结果表明:CP21基因在子孢子和卵囊期均表达,且表达产物位于子孢子表膜和卵囊壁表面。CP21是一种与侵入机制有关的黏附相关蛋白。研究结果为微小隐孢子虫的免疫学诊断及疫苗研制奠定了基础。
The purpose of this study was to clone the open reading frame of Cryptosporidium parvum adhesion-related protein gene CP21, construct its prokaryotic expression plasmid, and identify the fusion protein obtained. The template DNA was extracted from the phage containing CP21 gene and the gene fragment was amplified by PCR. The prokaryotic expression plasmid pET-28a-CP21 was constructed and transformed into Escherichia coli BL21 (DE3). The expression product was induced by IPTG. And Western blotting analysis identified. Mice were immunized with the prokaryotic expression protein to prepare anti-recombinant polyclonal antibodies for protein localization. The results showed that the CP21 gene was successfully amplified and the prokaryotic expression plasmid was constructed. The fusion protein was expressed in Escherichia coli with a molecular weight of 25 ku. Western blotting showed that the expressed CP21 fusion protein was able to react with the anti-C.parvum polyclonal antibody Specific reaction. Immunofluorescence results showed that CP21 gene was expressed in both sporozoites and oocysts, and the expression product was located on the surface of sporozoites and oocysts. CP21 is an adhesion-related protein involved in invasion mechanisms. The results laid the foundation for the immunological diagnosis and vaccine development of Cryptosporidium parvum.