毕赤酵母菌高效表达乙肝表面抗原的研究

The high expression of HBsAg in Pichia pastoris

目的:筛选高效表达HBsAg的毕赤酵母菌,制备目的蛋白。方法:从已确诊的乙肝病人血清中提取DNA,PCR扩增HBVS基因,将其分别克隆入毕赤酵母胞内表达载体pPICZA中。构建重组质粒pPICZA-S和pPICZA-SH,经SacI线性化后,LiCl化学法转化入酵母菌株GS115、X-33、KM71H和SMD1168。结果:诱导表达后的GS115工程菌单位体积的培养基所得的抗原含量最高,诱导培养基中加入0.1%酪蛋氨基酸后,可抑制目的蛋白的水解,有利于目的蛋白的表达,粗略估算表达量为15.3mg/L,最佳收获时间为72h。结论:经SDS-PAGE和Western-blot分析表明,所得产物为乙肝表面抗原S蛋白。

Objective： To secrete the Piehia pastoris strain which has the highest expression of HBsAg. Methods： The HBV S gene was amplified by PCR and cloned into the expression vector pPICZA. Then the reconstructed plasmid pPICZA-S and pPICZA-SH were transformed into the following Pichia pastoris strains： GS115 X-33,KM71H and SMD1168 through LiC1 chemical method. Results： The results indicated that the highest expressed Pichia pastoris strain is GS 115 with expression level of 15.3 mg/L and the best harvest time point is 72 hours after induced, because adding the 0.1% casamino acid into inductive medium can restrict the hydrolysis of desired proteins and enhance their expression. Conclusion： HbsAg analyzed by SDS-PAGE and Western-blot was expressed after induced with methanol.