密码子优化型HPV16L1基因在两种昆虫细胞中的表达研究
发布时间：2018-11-09 18:01
【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(P0.01)。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。
[Abstract]:Objective to improve the expression level of L1 gene of human papillomavirus type 16 (HPV16) in insect cells using baculovirus as vector, so as to lay a foundation for the development of prophylactic HPV vaccine. Methods based on the codon bias of insect cells, the wild type HPV16L1 gene was modified, the codon optimized HPV16L1 gene was synthesized, and the recombinant baculovirus was obtained by using the Bac-to-Bac expression system. The expression products were identified by Sf9 and High Five.Western blot in infected insect cells. Virus-like particles were observed under electron microscope. ELISA method was used to evaluate the optimal effect of HPV16L1 gene expression and the optimal conditions for L1 protein expression in two insect cells were discussed. Results the expression products of Sf9 and High Five cells were detected by Western blot, and the specific bands of HPV16L1 were found at the relative molecular weight of 56 kD, and the virus like particles were formed in the nuclei of the two insect cells under electron microscope. The results of ELISA showed that the expression level of codon optimized HPV16L1 gene was significantly higher than that of wild type (P0.01). High Five cells expressed at 72 h after MOI=10, expression, and the expression of L1 protein was at least 3 times higher than that of Sf9 cells. Conclusion the codon optimization technique can promote the high expression of HPV16L1 protein, and the significant advantage of High Five cells is worthy of attention.
【学位授予单位】：沈阳药科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1;Q78
[Abstract]:Objective to improve the expression level of L1 gene of human papillomavirus type 16 (HPV16) in insect cells using baculovirus as vector, so as to lay a foundation for the development of prophylactic HPV vaccine. Methods based on the codon bias of insect cells, the wild type HPV16L1 gene was modified, the codon optimized HPV16L1 gene was synthesized, and the recombinant baculovirus was obtained by using the Bac-to-Bac expression system. The expression products were identified by Sf9 and High Five.Western blot in infected insect cells. Virus-like particles were observed under electron microscope. ELISA method was used to evaluate the optimal effect of HPV16L1 gene expression and the optimal conditions for L1 protein expression in two insect cells were discussed. Results the expression products of Sf9 and High Five cells were detected by Western blot, and the specific bands of HPV16L1 were found at the relative molecular weight of 56 kD, and the virus like particles were formed in the nuclei of the two insect cells under electron microscope. The results of ELISA showed that the expression level of codon optimized HPV16L1 gene was significantly higher than that of wild type (P0.01). High Five cells expressed at 72 h after MOI=10, expression, and the expression of L1 protein was at least 3 times higher than that of Sf9 cells. Conclusion the codon optimization technique can promote the high expression of HPV16L1 protein, and the significant advantage of High Five cells is worthy of attention.
【学位授予单位】：沈阳药科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1;Q78
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1 任占平,黄健辉,石U，
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