【摘要】 目的利用毕赤酵母表达系统(Pichia pastoris)高效表达HPV16L1蛋白。方法按照Pichia pastoris密码子偏爱性合成HPV16L1基因,构建pAO819r-16L1表达载体,电转化至GS115菌株中,经MD板和不同浓度G418筛选,挑取高拷贝整合菌株,甲醇诱导目的蛋白的表达,用HPV16L1抗血清,经Western blot检测蛋白的特异性。结果重组质粒pAO819r-16L1在甲醇营养型酵母菌株中经甲醇诱导后可表达HPV16L1蛋白,在试管中的表达量超过10mg/ml,经Western blot验证,该蛋白可与抗血清特异结合,在相对分子质量55000附近出现目的条带,证明该蛋白确为HPV16L1蛋白。结论利用毕赤酵母表达系统可高效表达HPV16L1蛋白,为研制HPV疫苗奠定基础。更多还原

【Abstract】 Objective To highly express L1 protein of human papillomavirus 16(HPV16)in Pichia pastoris.Methods Synthesize HPV16L1 gene according to Pichia pastoris-preferred codon and insert into pAO819r.Transform GS115 strain with the constructed recombinant plasmid pAO819r-16L1 by electrotransformation and select high copy integrated strains by MD plate culture and G418 screening for expression under induction of methanol.Identify the expressed product by Western blot with HPV16L1 antiserum.ResultsThe expressed protein,with a relative molecular mass of 55 000,showed specific reaction with HPV16L1 antiserum.The expression level was more than 10 mg/ml.Conclusion HPV16L1 protein was highly expressed in Pichia pastoris,which laid a foundation of development of HPV vaccine.更多还原