人乳头瘤病毒16型结构基因L1在昆虫细胞中的表达及其生物学活性

目的 研究与宫颈癌及人类其它多种组织癌症密切相关的人乳头瘤病毒16型（HPV16）的晚期基因L1的表达及其生物学活性。方法 采用PCR法从pBSSK-B/16L1扩增了来自中国妇女鲍温病组织标本的HPV16晚期基因L1，装入杆状病毒转移载体；在DH10Bac内通过转座子Tn7的介导，使携带有杆状病毒多角体蛋白基因启动子Ppolh的HPV16 L1基因整合入杆状病毒，形成重组杆状病毒；转染昆虫细胞Sf9进行表达；将感染72h的Sf9细胞包埋、切片、染色后，电镜观察。提取L1蛋白，免疫BALB／ c小鼠。结果 重组杆状病毒在Sf9细胞内高效表达出L1蛋白，经Western blot发现能与L1抗体特异地结合；薄层扫描显示所表达的L1蛋白占Sf9细胞总蛋白的比例高达31％。经透射电镜观察表明，在细胞核里有大量的重组杆状病毒形成；并且产生了大量的由HPV16 L1蛋白单体自组装的病毒样颗粒（virus-like particles,VLP)。小鼠免疫实验结果表明，所表达的HPV16 L1蛋白单体自组装的病毒样颗粒（virus-like particles,VLP)。小鼠免疫实验结果表明，所表达的HPV16 L1蛋白具有强免疫原性。结论 此研究为今后L1基因分子流行病学检查、L1蛋白的结构生物学研究、疫苗研制以及HPV相关基础性研究提供了有用的资料。

Expression of human papillomavirus type 16 L1 structure gene in Sf9 cells and its products assembling into virus-like particles

Objective To study the biological activities of the L1 geneproducts of human papillomavirus 16 (HPV16) which is believed to be related with cervical cancer and many of other human cancers. Methods The HPV16 L1 gene was amplified from pBSSK-B/16L1 (derived from Bowen's disease tissue of Chinese woman) by the PCR, and the L1 gene was recombined into the baculovirus transfer vector pFastbacⅠ; The recombinant baculovirus was generated by the action of Tn7 transposon in E. coli strain, DH10Bac, with the action of Tn7 transposon, the HPV16 L1 gene carrying the polyhedrin promoter, was integrated into the baculovrius genome DNA. The insect Sf9 cells were transfected with the recombinant baculovirus, Sf9 cells infected by the recombinant baculovirus of HPV16 L1 were emebeded, sectioned and stained, then examined by transmission electron microscopy (TEM). Results The L1 proteins were efficiently expressed in Sf9 cells, and the L1 antibody combined specially the proteins in Western blot, L1 proteins takes 31% of the Sf9 cell total proteins. TEM observation showed many recombinant baculoviruses and virus-like particles (VLP) which were formed by HPV16 L1 protein monomers in Sf9 cell nucleic. The BALB/c mice immunization results showed that the expressed HPV16 L1 proteins had good immunogenicity. Conclusion The results suggested that the results will be valuable for epidemiological investigation, the structural biological study of HPV16 L1 protein, development of the vaccine as well as other related researches HPV.