论文部分内容阅读
目的:重组表达弓形虫致密颗粒蛋白7(GRA7),分析其免疫活性。方法:采用聚合酶链反应(PCR)从刚地弓形虫基因组DNA中扩增GRA7基因,经克隆和测序分析后,亚克隆至表达载体pET-23a(+),转化大肠埃希菌BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达GRA7,用His-bindTM亲和层析柱纯化,免疫印迹和小鼠免疫分析其抗原性。结果:PCR扩增出大小约660bp的GRA7基因片段,并被亚克隆到原核表达载体pET-23a(+),构建了原核重组表达质粒pET-GRA7;表达纯化获得分子量约29,000的重组GRA7;重组GRA7能被弓形虫感染兔血清所识别,并诱导小鼠产生抗体滴度达1:12800。结论:获得了具有良好免疫学活性的重组抗原GRA7。
OBJECTIVE: To express Toxoplasma gondii granule protein 7 (GRA7) recombinantly and analyze its immunocompetence. METHODS: GRA7 gene was amplified from genomic DNA of Toxoplasma gondii by polymerase chain reaction (PCR). After cloning and sequencing analysis, GRA7 gene was subcloned into expression vector pET-23a (+) and transformed into E. coli BL21 GRA7 was induced by propyl-β-D-thiogalactopyranoside (IPTG), purified by His-bindTM affinity chromatography and immunoblotted and immunologically analyzed for its antigenicity. Results: The GRA7 gene was amplified by PCR and subcloned into the prokaryotic expression vector pET-23a (+). The prokaryotic recombinant plasmid pET-GRA7 was constructed. The GRA7 gene was expressed and purified to obtain recombinant GRA7 with a molecular weight of 29,000. GRA7 can be recognized by Toxoplasma gondii infected rabbit serum and induced antibody titer of 1: 12800 in mice. Conclusion: The recombinant antigen GRA7 with good immunological activity was obtained.