利用昆虫-杆状病毒表达系统表达人乳头瘤病毒16/18/33/58亚型L1蛋白

目的:通过昆虫-杆状病毒表达系统获得人乳头瘤病毒(HPV)16/18/33/58亚型的主要衣壳蛋白L1。方法:克隆了HPV16/18/33/58亚型的L1蛋白基因,并采用密码子优化策略进行改造(记为HPV16/18/33/58亚型mL1),将优化基因片段插入pFastBac Dual载体获得重组载体,转化大肠杆菌DH10Bac感受态细胞后得到重组Bacmid,转染昆虫Sf9细胞,Western印迹和SDS-PAGE检测重组蛋白的表达。结果:获得了表达HPV16/18/33/58亚型mL1蛋白的重组杆状病毒;Western印迹和SDS-PAGE分析表明该重组杆状病毒感染昆虫Sf9细胞后表达mL1蛋白,且mL1蛋白主要分布在细胞中;优化了蛋白表达时间和感染复数,获得目的蛋白mL1的高效表达。结论:多个亚型HPV L1蛋白的克隆表达,为中国优势血清型疫苗的研制奠定了基础。

Production of HPV16/18/33/58 Major Capsid Protein L1 with Baculo virus Expression System

Objective: To obtain human papillomavirus(HPV) type 16/18/33/58 major capsid protein L1 with bac ulovirus expression system.Methods: The genes encoding the L1 proteins of HPV 16/18/33/58 serotype were cloned and modified by a variety of strategies to HPV16/18/33/58 mL1.The modified genes were inserted into the pFastBac Dual vector,and the recombinant bacmid was generated after the E.coli DH10Bac competent cell was transformed by the recombinant pFastBac Dual vector,recombinant baculovirus were transfected into insect cell Sf9.Results: Recombinant baculovirus expressing mL1 protein of HPV16/18/33/58 serotypes was obtained.Western blotting and SDS-PAGE analysis showed that the mL1 protein of the HPV strains was expressed when the Sf9 cells were infected by the recombinant baculovirus,and the mL1 proteins were found distributed mainly in the cell.The mL1 was highly expressed by optimized expression time and multiplicity of infection(MOI).Conclusion: Cloning and expression of L1 protein of multiple subtypes of HPV,laid the foundation for the Chinese indepen dent serotype vaccine.