乙型肝炎病毒合成e基因在毕赤酵母中的表达及产物免疫学特性  

Expression of hepatitis B virus e gene synthesized in Pichia pastoris and the immunological characterization of the product

作　　者：李朝霞[1] 席云[1] 洪国强[1] 胡波[1] 梁敏坚[1] 许珏[1] 李林[1] 

机构地区：[1]中山大学附属第三医院检验科,广州510630

出　　处：《中华检验医学杂志》2006年第9期807-812,共6页Chinese Journal of Laboratory Medicine

基　　金：广东省自然科学基金(021858)

摘　　要：目的合成适合酵母表达的乙型肝炎病毒(HBV)e 抗原基因,在毕赤酵母中表达出高免疫反应性和特异性的重组乙型肝炎病毒 e 抗原(HBeAg)。方法采取基因搭桥法及 Taq 酶聚合反应,选用酵母菌偏爱的同义密码子替换野生型 e 基因的部分密码子,制备合成 e 基因,插入 pPICZαA酵母菌表达载体。携带有合成 e 基因的 pPICZαA转化毕赤酵母菌株 GS115,经甲醇诱导表达 HBeAg。结果酶切电泳及 DNA 测序证实合成的 e 基因已正确克隆到表达载体中;聚丙烯酰胺凝胶电泳(SDS-PAGE)、immunoblt 及 ELISA 显示 HBeAg 在毕赤酵母中高效分泌表达,表达量约为63mg/L,培养上清液 ELISA 测定效价1:81920,最大吸光度值达2.8;重组 HBeAg 与乙型肝炎病毒核心抗体间无交叉反应。结论毕赤酵母表达系统高效分泌表达出与乙型肝炎病毒核心抗原间无交叉抗原性、具有良好免疫反应性的 HBeAg。Objectives To synthesize hepatitis B virus e gene which was suitable for yeast protein expression and express hepatitis B virus e antigen （HBeAg） in Pichia pastoris. Methods Using synonymous codons preferred by yeast usage on protein expression to replace some native codons of wild-type HBV e gene, the synthetic e gene （syneAg-gene） was achieved by a recursive PCR （rPCR）. The syneAg- gene was inserted into the yeast expression vector pPICZaA. The recombinant plasmid was transformed into GS115 yeast by electroporation. The yeast transformant induced by methanol expressed the HBeAg. Results The restriction analysis and DNA sequencing confirmed that the syneAg-gene was inserted to yeast pPICZαA in correct orientation. SDS-PAGE, immunoblot and ELISA indicated that the secreted form of HBeAg was expressed by the yeast transformant. The expression level of HBeAg in the strain containing syneAg-gene was 63 mg/L. The titer of the recombinant HBeAg in culture supernatant was 1：81 920 and the maximum OD （ optical density） value of absorbance was 2. 8 by ELISA. No cross reactivity between Pichia pastoris-derived HBeAg and anti-HBc antibody was found. Conclusion The recombinant HBeAg with high degree of specificity and immunoreactivity is expressed efficiently in Pichia pastoris.

关 键 词：肝炎e抗原 乙型 基因表达 毕赤酵母 酶联免疫吸附测定 

分 类 号：R392[医药卫生—免疫学]