甲型流感H3抗原在毕赤酵母系统中的优化表达与纯化

Optimized Expression and Purification of Influenza H3 Antigen in Pichia pastoris

选用甲型流感H3N2亚型的代表株作为研究对象,基于对H3蛋白二级结构的分析预测和密码子使用频率曲线,对其基因序列进行了科学的密码子优化设计。以密码子优化的力度与局部二级结构混乱度成反比为原则,兼顾表达效率和正确的结构与功能,将优化后的H3序列克隆至表达载体pPICZα,电转入毕赤酵母系统中。经甲醇诱导使H3蛋白在毕赤酵母中高效分泌表达,通过聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blot)鉴定其表达,同时筛选高拷贝菌株。最后对该菌株培养发酵,30℃甲醇诱导72 h后收集发酵的上清液,采用10 kD膜包过滤对其浓缩,经钴柱亲和层析等手段得到了H3蛋白的单一条带,并通过Western blot确认。纯化获得的H3抗原溶液本身不表现出血凝活性,但是与镍柱填料结合富集后表现出一定程度的血凝活性。本研究为流感病毒H3抗原在毕赤酵母系统中的重组表达和后续疫苗研发提供了基础。

In this study,a representative H3N2 influenza A strain was selected.Based on the analysis and prediction of the secondary structure of H3 protein and the codon usage frequency curve,the codons of H3 gene were optimized scientifically.Based on the principle that the intensity of codon optimization is inversely proportional to the disorder degree of local secondary structure,both expression efficiency and correct structure are considered.Then the optimized H3 sequence was cloned into the pPICZαexpression vector and transferred into Pichia pastoris by electroporation.After methanol induction,the H3 protein was expressed and secreted efficiently in P.pastoris.The expression of H3 was identified by coomassian bright blue staining and Western blot,and high copy strains were screened.Finally,the strain was cultured and fermented in shaking flask.After induced by methanol at 30℃for 72 h,the supernatant of fermentation was collected and concentrated by 10 kD membrane filtration.The purification of H3 recombinant protein was obtained by cobalt column affinity chromatography,and confirmed by coomassian bright blue staining and Western blot.The purified H3 antigen solution did not show haemagglutination activity,however,the nickel column bound H3 showed some to a certain extent.Our study laid a foundation for the recombinant production of H3 in P.pastoris and future vaccine development.