乙型肝炎病毒S基因在毕赤酵母中的表达及在乙型肝炎病毒表面抗体检测中的应用

Expression of hepatitis B virus S gene in pichia pastoris and application of the S protein synthesized in immunoassay of HBsAb

目的 构建含有乙型肝炎病毒S基因的表达载体 ,在毕赤酵母中表达出高免疫反应性的重组乙型肝炎表面抗原 (HBsAg)S蛋白 ,用以制备乙型肝炎表面抗体 (HBsAb)酶免试剂盒。 方法采用聚合酶链反应从含乙型肝炎病毒全基因序列 (adw)的质粒PHBV1中扩增出S基因 ,并将其插入pPICZB质粒。携带有S片段的pPICZB质粒转化毕赤酵母菌株KM71H ,甲醇诱导重组酵母细胞表达S蛋白。表达产物包被聚丙乙烯反应板 ,用酶联免疫吸附实验 (ELISA)检测HBsAb。结果 酶切电泳及DNA测序证实乙型肝炎病毒S基因已正确克隆到表达载体中 ;聚丙烯酰胺凝胶电泳显示HBsAg在毕赤酵母中表达 ;表达量约占总蛋白的 2 5 %～ 35 %。表达产物用ELISA测定效价达 1∶2 5 6 0 0 ;Westernblot显示与正常人血清无交叉反应 ;用纯化产物作为包被抗原对卫生部临床检验中心HBsAb质控物及血清标本进行检测 ,灵敏度达 10mIu/ml,特异性达 10 0 % ,重复性及线性良好 ,与国产商品试剂检测结果一致。结论 毕赤酵母表达系统高效表达出具有良好免疫反应性和特异性的HBsAg。

Objective To express hepatitis B virus S gene in Pichia pastoris and apply the S protein of HBsAg synthesized in immunoassay of HBsAb. Methods HBV S gene was amplified by PCR from plasmid PHBV1, which contained HBV (adw subtype) whole sequence and inserted into the yeast expression vector pPICZB. The recombinant plasmid was transformed into KM71H yeast by electroporation. The yeast transformant induced by methanol expressed the S protein. The S protein synthesized was coated to microplate for detection of HBsAb by sandwich ELISA. Results The restriction analysis and DNA sequencing confirmed that HBV S gene had been cloned to yeast expression vector pPICZB correctly. SDS PAGE and Western blot revealed the existence of the S protein synthesized and there was no cross reaction between all proteins coming from Pichia pastoris and normal sera. The expression level of S protein in this strain was about 35% of total cell protein as judged by SDS PAGE scanning of protein. The titer of the recombinant HBsAg (rHBsAg) in the cell lysate was 1∶25 600 by ELISA. The results of detecting HBsAb with the recombinant S protein as the coated antigen indicated that the sensitivity reached to 10 mIu/ml, the specificity was 100%, nice duplication and linearity. A good correlation was obtained between rHBsAg origin reagent and commercial blood origin kit in random samples measurement. Conclusion The recombinant S protein with high degree of immunoreactivity and specificity to HBsAb was expressed efficiently in Pichia.pastoris. The product can be applied to develop the Immunoassays of HBsAb in the qualitative and quantitative detection.