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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HPV16L1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Westernblot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HPV16L1蛋白单体自组装的病毒样颗粒 (virus likeparticles,VLP)。小鼠免疫实验结果表明 ,所表达的HPV16L1蛋白具有强免疫原性。结论 此研究为今后L1基因分子流行病学调查、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
Objective To study the expression and biological activity of the late gene L1 of human papillomavirus type 16 (HPV16), which is closely related to cervical cancer and other kinds of human cancers. Methods The HPV16 late L1 gene was amplified from pBSSK B / 16L1 by PCR from Bowen’s disease in Chinese women and inserted into the baculovirus transfer vector. In DH10Bac, The HPV16 L1 gene of Ppolh was inserted into baculovirus to form a recombinant baculovirus. Sf9 cells were transfected with insect cells for expression. Sf9 cells infected for 72 h were stained, sectioned and stained for electron microscopy. L1 protein was extracted and BALB / c mice were immunized. Results Recombinant baculovirus could efficiently express L1 protein in Sf9 cells and could be specifically bound to L1 antibody by Western blotting. The results of TLC showed that the expressed L1 protein accounted for 31% of the total protein in Sf9 cells. Transmission electron microscopy showed that a large amount of recombinant baculovirus was formed in the nucleus and a large number of virus-like particles (VLPs) were self-assembled by HPV16 L1 protein monomer. Mouse immunization results show that the expressed HPV16L1 protein has strong immunogenicity. Conclusions This study provides useful information for the molecular epidemiology of L1 gene, the structural biology of L1 protein, the development of vaccines and the basic research on HPV in the future