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为了实现人乳头瘤病毒(Humanpa pillomavirus,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SacII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HPV16L1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(pH7.0)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12h补加甲醇至终浓度为1%(V/V),37oC、200r/min条件下诱导培养72h后,HPV16L1蛋白的最高表达量为78.6mg/L。本研究为多形汉逊酵母源HPV16L1疫苗的研制奠定了基础。
In order to achieve efficient expression of the human papillomavirus (HPV) 16 subtype capsid protein L1 in Hansenula polymorpha, based on the amino acid sequence of the L1 protein and the codon preference of Hansenula polymorpha The coding sequence of L1 protein was optimized, and the complete coding sequence was synthesized, named HPV16L1. The promoter of MOXp and the terminator AOXTT were used as expression regulatory elements. URA3, a uracil-related gene, was used as a screening marker to construct recombinant plasmid pYMOXU-HPV16 of HPV16L1. The plasmid pYMOXU-HPV16 was digested with SacII to be linearized, and the H. polymorpha strain H-ura3 was electrotransformed. The recombinant strains were screened for the complement of auxotrophy. The PCR amplification and HPV16L1 protein expression analysis showed that the recombinant Hansenula strain HP-U-16L stably expressing high level of L1 protein was obtained. The initial optimization of fermentation conditions in shake flask showed that induction culture was carried out with YPM (pH7.0) as the basal medium, the inoculum size was controlled so that OD600 of the initial culture medium was 1.0, methanol was added every 12h to a final concentration of 1% (V / V), 37 ℃, 200r / min under the conditions of induction culture 72h, the highest expression of HPV16L1 protein was 78.6mg / L. This study laid the foundation for the development of the polyphase Hansenula-derived HPV16 L1 vaccine.