【作者】 李迪； 谷鸿喜； 张凤民； 钟照华； 程志；

【Author】 LI Di,GU Hong-xi,ZHANG Feng-min,et al (Department of Microbiology, Harbin Medical University, Harbin 150086, China)

【机构】 哈尔滨医科大学微生物教研室；

【摘要】 目的构建重组原核表达质粒获得HPV18 L1活性蛋白,为进一步研制HPV18基因工程疫苗打下基础。方法以重组质粒pBB322-HPV18为模板,利用PCR方法扩增HPV18 L1DNA片段,将HPV18 L1DNA与 pUC19质粒重组构建pUC19-HPV18 L1重组质粒。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pQE32质粒做表达载体构建pQE32-HPV18 L1重组质粒,并用酶切电泳验证。结果 PCR扩增DNA片段约为1．7Kb,与预期结果相同。pUC19-HPV18 L1克隆重组质粒酶切后显示4．39Kb,酶切图谱与预期相同。测序验证插入片段全序列无改变。pQE32-HPV18 L1表达重组质粒酶切后显示其大小约5．16Kb,酶切图谱亦与预期相同。结论 pQE32-HPV18 L1表达重组质粒构建成功。更多还原

【Abstract】 Objective To construct a recombinant plasmid pQE32-HPV18 L1 for obtaining the HPV18 L1 protein expressed in E. coli and developing the genetically engineered vaccine. Methods PCR was used to amplify the HPV18 L1 gene from plasmid pBR322-HPV18 in which the HPV18 L1 was cloned. A new plasmid, pUC19- HPV18 L1, was then constructed by inserting the amplified HPV18 L1 gene into pUC19, a pro-karyotic expression vector. Restriction analysis and sequencing were used to confirm the structure of pUC19HPV18 L1.A new plasmid, pQE32- HPV18 L1, was then constructed by inserting the amplified HPV18 L1 gene into pQE32 a prokaryotic expression vector.Results A DNA fragment in size of 1.7Kb was amplified. Restriction analysis showed that the amplified gene was inserted into pUC19 correctly. Sequencing showed there was no mutation in both ends of the inserted L1 gene. Conclusion The plasmid pQE32-HPV18 L1 is constructed successfully.更多还原