摘要
目的合成适合酵母表达的乙型肝炎病毒C基因,在毕赤酵母中高效表达重组乙型肝炎病毒核心抗原(HBcAg)。方法选用酵母的偏爱密码子对野生型乙型肝炎病毒C基因进行优化改造,采用基因搭桥及聚合酶链反应,制备合成基因,插入酵母表达载体pPICZA。携带有合成C基因的重组载体转化毕赤酵母菌株KM71H,经甲醇诱导表达HBcAg。结果酶切电泳及DNA测序证实合成的C基因正确克隆到表达载体中;聚丙烯酰胺凝胶电泳及ELISA显示优化后的乙型肝炎病毒C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C基因能明显提高HBcAg在毕赤酵母表达系统中的表达量。
Objective To synthesize hepatitis B virus （HBV） C gene which is suitable for yeast protein expression and express hepatitis B virus HBeAg in Pichia pastoris. Methods Using synonymous eodons preferred by yeast usage on protein expression to replace some native eodons of wild - type HBV C gene, the synthetic C gene （synC- gene） was achieved by a reeursive PCR （rPCR）. The synC -gene was inserted into the yeast expression vector pPICZA. The recombinant plasmid was transformed into KM71H yeast by eleetroporation. The yeast transformant induced by methanol expressed the HBcAg. Results The restriction analysis and DNA sequencing confirmed that the synC - gene was inserted to pPICZA. SDS - PAGE and ELISA indicated that the HBeAg was expressed by the yeast transformant. The expression level of HBeAg in the strain containing synC - gene was much higher than the strain containing native C - gene. Conclusion The HBcAg expression level can be promoted significantly by optimizing the codons of HBV native C -gene in Pichia pastoris.
出处
《海南医学》
CAS
2009年第11期12-16,共5页
Hainan Medical Journal
