人乳头瘤病毒11型L1基因原核表达系统的构建及鉴定

发布时间：2019-01-23 16:41

【摘要】：目的构建人乳头瘤病毒11型(HPV11)L1原核表达系统pBAD(B)鄄HPV11L1,表达HPVL1蛋白,为进一步制备基因工程疫苗奠定基础。方法PCR法从尖锐湿疣组织标本中扩增人乳头瘤病毒11型L1基因片段,克隆至pBluescript质粒,并测序。将其从克隆重组质粒中切下连入表达载体,建立pBAD(B)鄄HPV11L1原核表达重组质粒,在大肠杆菌宿主菌Top10中,经阿拉伯糖诱导,表达融合蛋白L1,经SDS鄄PAGE电泳和Westernblot进行鉴定。结果3株HPV11L1经测序发现变异一致,每株均有2个核苷酸变异,但未影响氨基酸变化。经SDS鄄PAGE鉴定L1蛋白相对分子质量为59×103,与预期值相同,Westernblot证明表达蛋白能与单克隆抗体反应,成功构建了pBAD(B)鄄HPV11L1原核表达系统。结论所构建的pBAD(B)鄄HPV11L1原核表达系统能够高效表达HPV11L1蛋白。
[Abstract]:Objective to construct a prokaryotic expression system of human papillomavirus type 11 (HPV11) L1 pBAD (B)-HPV11L1, to express HPVL1 protein. Methods Human papillomavirus type 11 L1 gene was amplified from condyloma acuminatum tissue by PCR and cloned into pBluescript plasmid and sequenced. The recombinant plasmid was digested from the cloned recombinant plasmid and inserted into the expression vector. PBAD (B) HPV11L1 prokaryotic expression plasmid was established. The fusion protein L1 was induced by arabinose in Escherichia coli Top10 and identified by SDS PAGE electrophoresis and Westernblot. Results the three HPV11L1 strains showed the same variation by sequencing. Each strain had 2 nucleotide variations, but had no effect on the change of amino acids. The relative molecular weight of L1 protein identified by SDS PAGE was 59 脳 103, which was the same as the expected value. Westernblot proved that the expressed protein could react with monoclonal antibody and successfully constructed the pBAD (B) HPV11L1 prokaryotic expression system. Conclusion the constructed pBAD (B)-HPV11L1 prokaryotic expression system can efficiently express HPV11L1 protein.
【作者单位】：哈尔滨医科大学微生物学教研室哈尔滨医科大学微生物学教研室第一临床医学院皮肤科哈尔滨医科大学微生物学教研室哈尔滨医科大学微生物学教研室哈尔滨医科大学微生物学教研室哈尔滨医科大学微生物学教研室
【基金】：黑龙江省科技攻关项目资助(GB02c111) 黑龙江省青年基金(QC03C04)
【分类号】：R346

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