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目的:研究人乳头瘤病毒(HPV)的主要衣壳蛋白L1和主要的转化蛋白E7的融合基因在原核细胞中的表达,为研制HPV16L1E7疫苗和诊断试剂盒提供依据。方法:采用聚合酶链反应(PCR)以重组质粒pUC19L1E7为模板扩增HPV16L1E7基因片段,克隆至载体pMD18-T中,并用限制性内切酶将HPV16L1E7基因切下,克隆至原核表达质粒pQE30中,经IPTG诱导表达,再使用SDS-PAGE和蛋白印迹检测其抗原性和表达水平。结果:HPV16L1E7融合蛋白能被抗HPV16L1抗体识别,分子量为66KD,经IPTG诱导4h后,表达的HPV16L1E7融合蛋白占菌体总蛋白量的25%以上。结论:成功构建的表达载体pOE30-L1E7可在原核表达细胞中高效表达,且表达出的HPV16L1E7融合蛋白具有反应活性,可与HPV16抗体发生反应。
Objective: To study the expression of fusion gene of human papillomavirus (HPV) major capsid protein L1 and major transforming protein E7 in prokaryotic cells, and to provide the basis for the development of HPV16L1E7 vaccine and diagnostic kit. Methods: HPV16 L1E7 gene fragment was amplified by polymerase chain reaction (PCR) from plasmid pUC19L1E7 and cloned into vector pMD18-T. The HPV16 L1E7 gene was excised by restriction endonuclease and cloned into prokaryotic expression plasmid pQE30. After induced by IPTG, the antigenicity and expression level were detected by SDS-PAGE and Western blotting. Results: HPV16 L1E7 fusion protein could be recognized by anti-HPV16 L1 antibody with a molecular weight of 66 kD. After induced by IPTG for 4 h, the expressed HPV16 L1 E7 fusion protein accounted for more than 25% of the total bacterial protein. CONCLUSION: The successfully constructed expression vector pOE30-L1E7 can be highly expressed in prokaryotic cells, and the expressed HPV16 L1E7 fusion protein is reactive and can react with HPV16 antibody.