【摘要】 目的构建微小隐孢子虫表面抗原CP23基因表达载体并获得重组表达蛋白。方法用微小隐孢子虫卵囊感染免疫抑制BALB/c小鼠,用不连续蔗糖密度梯度离心法纯化微小隐孢子虫卵囊,用酚-氯仿抽提法提取微小隐孢子虫基因组DNA,PCR扩增CP23基因片段,然后克隆至TA载体;通过酶切、测序鉴定后,将CP23基因片段用限制性内切酶切下克隆至pET-30a(+)载体,构建pET-30a-23质粒,转化大肠埃希菌BL21(DE3),IPTG诱导表达,表达蛋白用SDS-PAGE和Western blot分析鉴定。结果扩增出约340bp的微小隐孢子虫CP23基因片段并成功构建pET-30a-23质粒,表达出分子质量单位为27ku的融合蛋白,Western blot显示该蛋白能被抗微小隐孢子虫血清识别。结论成功构建pET-30a-23质粒,表达产物具有免疫反应性,为微小隐孢子虫的免疫学诊断及疫苗研制打下了基础。更多还原

【Abstract】 Objective To construct a recombinant plasmid containing CP23 gene of Cryptosporidium parvum and obtain recombinant protein. Methods Genomic DNA of C. parvum was extracted from oocysts of C. parvum. CP23 gene fragment was amplified from genomic DNA of C. parvum by PCR. The purified PCR fragment was ligated into the pMD18-T Simple Vector, then subcloned into the pET-30a(+). The recombinant plasmid was transformed into E. coli DH5α. The positive clones were screened and identified by PCR from bacteria directly and digested via double enzymes following with a sequencing of recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21 (DE3); the expression of protein was induced by IPTG. The expression product was identified by SDS-PAGE and Western blot, which was preceded with rabbit anti-C. parvum serum produced by our laboratory. Results The CP23 gene fragment was amplified correctly as the size of gene was about 342 bp; including an open reading fragment encoding 113 amino acids residues. The molecular weight of the whole expression protein was 27 ku on SDS-PAGE in accordance with the result of prestained protein marker. The expressed protein was further identified by Western blot performed with rabbit anti-C. parvum serum. Conclusion The recombinant plasmid is successfully constructed. The protein of CP23 gene of C. parvum is over-expressed and recognized by anti-serum. Our work would lay solid foundations for the diagnosis of cryptosporidiosis as well as study of vaccines.更多还原