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目的:克隆幽门螺杆菌HpureB基因,并构建其核酸疫苗。方法:以HpNCTC11637株基因组DNA为模板,用PCR扩增ureB基因,并亚克隆至pMD18-T载体中。将目的基因经SalI、BglⅡ酶切纯化后插入pTCAE中,转化E.coliDH5α。经SalI、XhoI酶切并测序鉴定的阳性重组质粒命名为pT-ureB。以电穿孔法将pT-ureB转染CHO细胞,用Wes-tern blot检测UreB蛋白的表达。结果:克隆重组后得到pT-ureB。将pT-ureB以电穿孔法转染CHO细胞后,取其培养上清进行Western blot检测,在UreB的相对分子质量(Mr)为62000处出现特异性条带。结论:成功地构建了HpureB核酸疫苗。体外转染CHO细胞后,经Western blot检测证实有UreB蛋白的表达,为进一步的相关研究奠定了基础。
Objective: To clone Helicobacter pylori HpureB gene and construct its nucleic acid vaccine. Methods: ureB gene was amplified by PCR using genomic DNA of HpNCTC11637 as a template and subcloned into pMD18-T vector. The target gene was digested with SalI and BglII and inserted into pTCAE to transform E. coli DH5α. After SalI, XhoI digested and sequenced positive recombinant plasmid named pT-ureB. CHO cells were transfected with pT-ureB by electroporation, and the expression of UreB protein was detected by Wes-blot. Results: pT-ureB was obtained after cloning and recombination. After transfection of pT-ureB into CHO cells by electroporation, the culture supernatants were harvested for Western blot analysis. A specific band appeared at a molecular weight of 62,000 (Mr) of UreB. Conclusion: HpureB DNA vaccine was successfully constructed. After transfection of CHO cells in vitro, the expression of UreB protein was confirmed by Western blot, which laid the foundation for further related research.