人乳头瘤病毒重组质粒的构建及蛋白表达
Construction of recombinant plasmid pQE32-HPV18L1 and protein expression
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摘要:目的 构建重组原核表达质粒获得人乳头瘤病毒18型L1片段(HPV18L1)活性蛋白, 为进一步研制人乳头瘤病毒18型(HPV18)基因工程疫苗打下基础。方法 以重组质粒(pBR322-HPV18)为模板, 利用PCR方法扩增HPV18L1DNA片段, 将HPV18L1DNA与质粒(pUC19)重组构建重组质粒(pUC19-HPV18L1)。用酶切电泳验证重组结果的正确性; 并通过测序检查质粒重组后序列有无变化。再利用质粒(pQE32)做表达载体构建重组质粒(pQE32-HPV18L1), 并用酶切电泳验证。将重组质粒pQE32-HPV18L1转入工程菌(M15), 用酶切电泳验证重组工程菌(pQE32-HPV18L1/M15)正确性。利用SDS-聚丙酰胺凝胶电泳(SDS-PAGE)检测HPV18L1目的蛋白。采用不同浓度异丙基-β-D-半乳糖苷(IPTG)、不同诱导时间来优化HPV18L1目的蛋白表达条件。利用Western印迹检测蛋白质(Westernblot)鉴定HPV18L1目的蛋白的特异性。结果 PCR扩增DNA片段约为17Kb, 与预期结果相同。克隆重组质粒pUC19-HPV18L1酶切后显示的酶切图谱与预期相同, 而且测序验证插入片段全序列无改变。表达重组质粒pQE32-HPV18L1酶切图谱亦与预期相同。蛋白表达条件优化结果为1mmol/LIPTG诱导4h后, 获得HPV18L1最佳蛋白表达量: SDS-PAGE显示, 约63KD处可见目的蛋白带Abstract:Objective To construct a recombinant prokaryotic expression vector for obtaining the HPV18L1 protein and develop the genetic engineering vaccine.Methods The L1 gene of HPV18 was amplified by PCR from DBR322-HPV18 and cloned into pUC19.The sequence of cloned HPV18L1 was confirmed by restriction analysis and DNA sequencing.A HPV18L1 prokaryotic expression plasmid, pQE32-HPV18L1, was then constructed by subclone.pQE32-HPV18L1 tranformed E.coli M15 was induced by IPTG.The expression of L1 protein was analyzed by SDS-PAGE and western blot.Results The amplified DNA fragment was in size of 1.7 Kb as expected.Restriction analysis showed that the amplified gene was inserted in pUC19 correctly.Sequence showed there was no mutation in both ends of the cloned L1.Restriction analysis showed that the recombinant pQE32-HPV18L1 right.A63KD protein could be seen in M15 induced by IPTG with SDS-PAGE and was positive while reacting with HPV18L1 antibody by western blot.Conclusion HPV18L1 was successfully cloned and expressed in this study.
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