多拷贝重组HBsAg质粒的构建及在毕赤酵母中的高效表达

目的通过构建多拷贝数表达质粒,获得高表达乙型肝炎表面抗原(HBsAg)的毕赤酵母(Pichia pastoris)菌株。方法通过对单拷贝表达质粒进行改造,将表达盒(5′AOX-HBsAg-TT)重复导入载体,构建多拷贝数表达质粒。通过电转化、15L规模发酵和纯化,获得纯化HBsAg颗粒,并对表达产物的特异性、免疫原性及表达量与拷贝数间的关系进行分析。结果多拷贝数重组毕赤酵母的表达产物经ELISA、SDS-PAGE、Western blot检测,显示出良好的特异性,电镜观察证明表达产物能够自发装配成22nm类病毒颗粒(VLP),其表达量与拷贝数呈正相关,拷贝数提高未对菌体正常生长带来影响,HBsAg制备的免疫原能有效刺激小鼠产生保护性抗体。结论含多拷贝数表达质粒的工程菌可显著提高毕赤酵母HBsAg表达量。

Construction of Recombinant Plasmid with Multi-copy Expression Cassette for High Expression of HBsAg in Pichia pastoris

Objective To establish a recombinant Pichia pastoris strain with multi-copy expression cassette for high expression of HBsAg.Methods Insert multiple copies of expression cassette 5′ AOX-HBsAg-TT into single copy expression vector.Transform the constructed recombinant plasmid with multi-copy expresion cassette to Pichia pastoris GS115 strain by electroporation,screen positive clones and inoculate to BMGY medium for expression under induction of methanol.Purify the expressed product by hydrophobic and ion exchange chromatography.The procedure was further scaled up to a 15 L fermentation reactor.Analyze the specificity and immunnogencity of expressed product as well as the relationship between expression level and copy number.Results ELISA,SDS-PAGE and Westem blot showed that HBsAg with good specificity was highly expressed in Pichia pastoris.Electron microscopy proved the purified expressed product as virus-like particles at a mean diameter of 22 nm.The expression level was positively related to the copy number.However,the increase of copy number showed no significant influence on the nomal growth of recombinant Pichia pastoris strain.The expressed HBsAg induced protective antibody in mice effectively.Conclusion The construction of recombinant plasmid with multi-copy expression cassette increased the expression level of HBsAg significantly.