乙型肝炎病毒S基因密码子最佳化及在毕赤酵母中的高效表达

目的 制备适合酵母表达的乙型肝炎病毒S基因,在毕赤酵母中高效表达重组乙型肝炎病毒S蛋白.方法 选用酵母偏爱的同义密码子替换野生型S基因的酵母稀有密码子,采取基因搭桥法及递归式聚合酶链反应,制备合成基因,捕入酵母表达载体pPICZB.携带有合成S基因的重组质粒转化毕赤酵母菌株KM71 H,经甲醇诱导表达乙型肝炎病毒S蛋白.结果 酶切电泳及DNA测序证实合成的S基因正确克隆到表达载体中;聚丙烯酰胺凝胶电泳及免疫印迹显示优化后的乙型肝炎病毒S基因在毕赤酵母中的重组蛋白表达量远高于野生型基因.结论 密码子优化的S基因能明显提高重组S蛋白在毕赤酵母表达系统中的表达量.

Codous optimization and high-level expression of hepatitis B virus S gene in Pichia pastoris

Objective To synthesize hepatitis B virus S gene which is suitable for yeast protein expression and express hepatitis B virus S protein in Pichia pastoris. Methods Synonymous codons preferred by yeast usage on protein expression were used to replace some native codons of wild-type HBV S gene.Synthetic S gene (synS-gene) was achieved by a recursive PCR (rPCR). The synS-gene was inserted into the yeast expression vector pPICZB. Recombinant plasmid was transformed into KM71H yeast by electroparation. The yeast transformant induced by methanol expressed the S protein. Results The restriction analysis and DNA sequencing confirmed that the synS-gene was inserted into yeast pPICZB in correct orientation. SDS-PAGE and ELISA indicated that the S protein was expressed by the yeast transformant. The expression level of S protein in the strain containing synS-gene was more higher than the strain containing wild-type S-gene. Conclusion Codons optimization can promote the yield of recombinant S protein in Pichia pastoris.