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目的构建空肠弯曲菌omp18基因的重组表达质粒、克隆表达rOMP18重组蛋白、对表达产物进行鉴定并评价其抗原性。方法 PCR扩增空肠弯曲菌的omp18基因片段,将其连接到表达载体pET32a(+)中并转化至大肠杆菌(E.co-liBL21(DE3)),诱导表达后SDS-PAGE分析其表达产物并纯化目的蛋白。应用空肠弯曲菌感染免疫血清,利用Western-blot方法分析其抗原性。结果与结论单、双酶鉴定结果显示已成功构建rOMP18的重组表达载体pET32a-omp18,IPTG诱导表达产物的相对分子质量与理论相符34kD(HIS16kD)。Western-blot检测结果表明重组蛋白rOMP18具有抗原性,为空肠弯曲菌感染的特异抗体的检测提供候选抗原。
Objective To construct the recombinant expression plasmid of omp18 gene of Campylobacter jejuni and clone and express the rOMP18 recombinant protein. The expressed product was identified and its antigenicity was evaluated. Methods The omp18 gene fragment of Campylobacter jejuni was amplified by PCR. The fragment was ligated into expression vector pET32a (+) and transformed into E. coli (E.co-liBL21 (DE3)). The expressed product was analyzed by SDS- Purify the target protein. The immune serum was infected with Campylobacter jejuni and its antigenicity was analyzed by Western-blot. RESULTS AND CONCLUSION Single and double enzyme identification results showed that the rOMP18 recombinant expression vector pET32a-omp18 was successfully constructed. The relative molecular mass of IPTG-induced expression product was 34kD (HIS16kD). The results of Western-blot showed that recombinant protein rOMP18 has antigenicity and provided candidate antigens for the detection of specific antibodies against Campylobacter jejuni.