人乳头瘤病毒16亚型L1蛋白在多形汉逊酵母中的优化表达

Optimized expression of the L1 protein of human papillomavirus in Hansenula polymorpha

摘要:

为了实现人乳头瘤病毒(Human papillomavirus, HPV)16亚型衣壳蛋白L1在多形汉逊酵母(Hansenula polymorpha)中的高效表达, 根据L1蛋白的氨基酸序列及多形汉逊酵母的密码子偏爱性, 对L1蛋白的编码序列进行优化设计, 合成了完整的编码序列, 命名为HPV16L1。以甲醇诱导型启动子MOXp和终止子AOXTT为表达调控元件, 以尿嘧啶合成相关基因URA3为筛选标记, 构建了HPV16L1的重组表达质粒pYMOXU-HPV16。用Sac II酶切质粒pYMOXU-HPV16使其线性化, 电转化多形汉逊酵母菌株H-ura3, 依据营养缺陷互补筛选重组菌株。通过PCR扩增及HPV16 L1蛋白表达量分析表明已获得稳定高表达L1蛋白的重组汉逊酵母菌株HP-U-16L。摇瓶发酵条件的初步优化表明, 以YPM (pH 7.0)为基础培养基进行诱导培养, 控制接种量使初始培养液OD600为1.0, 每隔12 h补加甲醇至终浓度为1% (V/V), 37oC、200 r/min条件下诱导培养72 h后, HPV16 L1蛋白的最高表达量为78.6 mg/L。本研究为多形汉逊酵母源HPV16 L1疫苗的研制奠定了基础。

Abstract:

The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pMOXZα-A vector to generate plasmids pMOXZ-HPV16N and pMOXZ-HPV16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPV16L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37°C for 72 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by H. polymorpha.