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目的:从人乳头瘤病毒(HPV)阳性的宫颈癌组织中克隆HPV18型主要衣壳蛋白L1基因全长,构建真核表达载体及验证目的蛋白的表达。方法:根据HPV18-L1基因全长设计一对特异引物,用PCR方法从宫颈癌组织DNA中获得L1基因,构建pMD18T重组质粒扩增L1基因,以pVAX1为真核表达载体构建重组表达质粒,转染入地鼠肾细胞BHK,采用免疫细胞化学方法检测HPV18-L1蛋白的表达。结果:从长春地区宫颈癌临床标本克隆到的HPV18-L1基因序列与Genebank报道序列高度同源,其真核表达产物能够与特异性抗体发生特异性结合。结论:成功构建pVAX1-HPV18-L1重组真核表达质粒,为研制地区特异性HPV预防性疫苗提供基础实验参考。
OBJECTIVE: To clone the full-length L1 gene of HPV18 major capsid protein from human papillomavirus (HPV) -positive cervical cancer tissue, construct eukaryotic expression vector and verify the expression of the target protein. Methods: According to the full length of HPV18-L1 gene, a pair of specific primers were designed. The L1 gene was obtained from cervical cancer tissue DNA by PCR. The pMD18T recombinant plasmid was constructed to amplify L1 gene. The recombinant plasmid was constructed by using pVAX1 as eukaryotic expression vector. Hamster kidney cells infected with BHK, the use of immunocytochemical detection of HPV18-L1 protein expression. Results: The sequence of HPV18-L1 cloned from clinical specimens of cervical cancer in Changchun area was highly homologous to the reported sequence of Genebank. The eukaryotic expression product could specifically bind to specific antibodies. CONCLUSION: The eukaryotic expression plasmid pVAX1-HPV18-L1 was successfully constructed and provided the basis for the development of a regional specific HPV vaccine.