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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 screen Pichia pastoris efficiently expressing HBsAg and prepare the target protein. Methods: DNA was extracted from the sera of patients diagnosed with hepatitis B virus (HBV). The HBV gene was amplified by PCR and cloned into pPICZA, an intracellular expression vector of Pichia pastoris. The recombinant plasmids pPICZA-S and pPICZA-SH were constructed and linearized by SacI. After that, LiCl was chemically transformed into yeast strains GS115, X-33, KM71H and SMD1168. Results: The highest antigen content per unit volume of induced GS115 engineered bacteria was obtained. The addition of 0.1% casein amino acid in the induction medium inhibited the hydrolysis of the target protein, which was in favor of the expression of the target protein and roughly estimated the expression level 15.3mg / L, the best harvest time is 72h. Conclusion: SDS-PAGE and Western-blot analysis showed that the resulting product was hepatitis B surface antigen S protein.