The expression and preliminary evaluation of HPV6bL2△N360E7E6 fusion protein in E.coli for genital warts

Objective To express HPV6bL2△N360E7E6 fusion protein in E.coli and preliminarily evaluate its immune effect.Methods Three HPV6b gene fragments,which were L2(1-360 bp),E7 and E6,were fused by overlapping PCR,then were inserted into a prokaryotic expression vector and expressed in E.coli.C57BL/6 mice were immunized with purified fusion protein plus Al(OH)3 and/or CpG adjuvants through intramuscular route,the cellular and humoral immune responses were detected by IFN-γ ELISPOT and ELISA respectively.Results Protein plus CpG adjuvant could induce the strongest cellular immune response to E7 and E6,high antibody titer against L2 could be detected in all immunized groups but there were no significant difference among these groups.Conclutions HPV6bL2△N360E7E6 gene was successfully cloned into pQE30 vector and expressed in E.coli,the fusion protein was also purified and proved that could induce strong cellular and humoral immune responses with appropriate adjuvant in C57 BL/ 6 mice and could be used for future research.     

用于治疗尖锐湿疣的HPV6型L2△N360E7E6融合蛋白的原核表达及其免疫效果的初步评价

目的 原核表达人乳头瘤病毒（HPV）6型L2AN360E7E6融合蛋白并对其免疫效果进行初步评价.方法 用重叠PCR将HPV6b 12（1～360 bp）、E7、E6三个基因片段融合,原核表达HPV6bL2△N360E7E6融合蛋白,蛋白纯化后与Al（OH）3、CpG佐剂配伍肌内注射免疫C57BL/6小鼠,使用IFN-γ ELISPOT与ELISA分别对其细胞免疫和体液免疫效果进行评价.结果 蛋白＋CpG佐剂组与其他免疫组相比,针对E7与E6均有明显较强的细胞免疫反应;各免疫组均能检测到高滴度的抗L2的抗体,但各组之间无明显差异.结论 利用pQE30原核表达系统成功克隆、表达和纯化了HPV6bL2△N360E7E6融合蛋白,且该蛋白与合适佐剂配伍能在C57BL/6小鼠体内诱发强的细胞免疫和体液免疫反应,为该蛋白的后期研究奠定了基础.     

HPV31和52 L2融合蛋白的原核高效表达及免疫效果评价

目的 高效表达HPV31和52型L2融合蛋白疫苗,并评价其免疫效果.方法 根据GenBank上公布的HPV31、52型L2蛋白11-200位氨基酸序列,设计合成两个型别L2该区域对应的优化密码子基因融合序列,并将其克隆至原核表达载体中.利用原核表达系统表达HPV31和52 L2融合蛋白,纯化目的蛋白后免疫小鼠,用血清抗体检测及HPV假病毒体外中和试验评价融合蛋白疫苗的免疫效果.结果 HPV31和52 L2融合蛋白在原核表达体系中呈高效表达,表达量约占全菌20％.融合蛋白加铝佐剂免疫小鼠后,血清中能检测到高滴度的总抗体及一定水平的中和抗体和交叉中和抗体.结论 HPV31和52 L2融合蛋白能够刺激机体产生广谱中和抗体,为高危型广谱HPVL2蛋白疫苗研发提供了实验室依据.