人乳头瘤病毒16型L2E7融合蛋白的优化表达、制备及其对肿瘤生长的抑制作用

Optimized expression, preparation of human papillomavirus 16 L2E7 fusion protein and its inhibitory effect on tumor growth in mice

摘要:

为提高人乳头瘤病毒 (HPV) 16型治疗性融合蛋白疫苗HPV16 L2E7在大肠杆菌中的表达量，根据大肠杆菌偏爱密码子，对HPV16 l2e7基因进行密码子优化，优化后的基因分别插入到pGEX-5X-1、pQE30和pET41a表达载体中，转化JM109、JM109 (DE3) 和BL21 (DE3) 表达菌，筛选出高表达菌株pET41a-HPV16sl2e7/BL21 (DE3)，目的蛋白从占全菌蛋白的10%以下提高到约28%，优化接种量、IPTG浓度、诱导温度和诱导时间，获得最佳表达条件；通过15 L发酵罐发酵，SP Sepharose Fast Flow、Q Sepharose Fast Flow和Superdex 200 pg纯化及复性HPV16 L2E7融合蛋白，制备的融合蛋白纯度可达95%以上，经SDS-PAGE、Western blotting鉴定证实，制备的HPV16 L2E7蛋白加CpG佐剂对小鼠移植瘤生长具有明显的抑制作用，有75% (6/8) 的小鼠不成瘤，为后续的疫苗产业化奠定基础。

Abstract:

HPV16 L2E7 is a fusion protein used for therapeutical vaccine targeting HPV virus. To increase its expression in Escherichia coli, we optimized the codon usage of HPV16 l2e7 gene based on its codon usage bias. The optimized gene of HPV16 sl2e7 was cloned into three different vectors: pGEX-5X-1, pQE30, ET41a, and expressed in JM109, JM109 (DE3) and BL21 (DE3) lines separately. A high expression line was selected with pET41a vector in BL21 (DE3) cells. After optimization of the growth condition, including inoculation amount, IPTG concentration, induction time and temperature, the expression level of HPV16 L2E7 was increased from less than 10% to about 28% of total protein. HPV16 L2E7 protein was then purified from 15 L culture by means of SP Sepharose Fast Flow, Q Sepharose Fast Flow and Superdex 200 pg. After renaturing, HPV16 L2E7 protein with ≥95% purity was achieved, which was confirmed via SDS-PAGE gel and Western blotting. The combined use of purified HPV16 L2E7 and CpG helper has shown clear inhibition of tumor growth in mice injected with tumor cells, with six out of eight mice shown no sign of tumor. This study lays a solid foundation for a new pipeline of large-scale vaccine production.