【作者】 龚业莉；

【导师】 夏焕章；

【作者基本信息】 沈阳药科大学 ， 微生物与生化药学， 2009， 硕士

【摘要】 目的提高16型人乳头瘤病毒(HPV16)L1基因以杆状病毒为载体,在昆虫细胞中的表达水平,为研制预防性HPV疫苗奠定基础。方法根据昆虫细胞密码子偏性对野生型HPV16L1基因进行改造,全基因合成密码子优化型HPV16L1基因,利用Bac-to-Bac表达系统获得重组杆状病毒,感染昆虫细胞Sf9和High Five。Western blot鉴定表达产物；电镜下观察病毒样颗粒形成。利用ELISA法评价HPV16L1基因的优化效果,并探讨L1蛋白在两种昆虫细胞中表达的最佳条件。结果Sf9和High Five细胞的表达产物经Western blot检测,在相对分子质量56 kD处均出现HPV16L1的特异性条带；电镜下可见病毒样颗粒在两种昆虫细胞的核内形成；ELISA法结果显示密码子优化型HPV16L1基因的表达水平显著高于野生型(P<0.01)。High Five细胞表达的最佳条件为MOI=10,表达时相72 h,其L1蛋白表达量至少比Sf9细胞高3倍。结论密码子优化技术确实能够促进HPV16L1蛋白的高效表达,而High Five细胞表现出的显著优势尤其值得关注。更多还原

【Abstract】 Objective To enhance the expression level of human papillomavirus (HPV)16 L1 through Baculovirus Expression System, in order to lay a foundation of development of prophylactic HPV vaccines.Methods We optimized the codon usage of wild type HPV16L1 gene according to the codon bias of insect cells and the full length optimized HPV16L1 gene was obtained by nucleotide synthesis. In order to infect both Sf9 and High Five insect cells, the recombinant baculovirus was obtained using Baculovirus Expression System. The expressed product was identified by Western blot; the virus-like particles (VLPs) of HPV 16 was confirmed by electron microscopy; and ELISA was used to evaluate the effect of the optimized gene as well as to determine the optimal expression conditions in both Sf9 and High Five insect cells.Results The expressed protein, with a relative molecular mass of 56kD, showed specific reaction with HPV16L1 antiserum; The intranuclear VLPs of HPV16L1 were observed by electron microscopy; The result of ELISA indicated that the expression level of optimized HPV16L1 gene was significantly higher than the wild type; And the optimal expression condition for High Five cells was determined as MOI 10 and expression phase 72 hours, in which the L1 protein was expressed at least 3 times higher than that in Sf9 cells.Conclusion Codon optimization could indeed enhance the expression level of HPV16L1 gene in insect cells; The significant advantage of High Five was worth attention.更多还原