摘要
目的:构建含抗HIV-1外膜蛋白gp120单链抗体(scFv)基因的酵母表达载体,实现目的蛋白的分泌性表达,并检测其生物活性。方法:采用基因工程和重组DNA技术,从质粒pET28-scFv中切下目的基因片段,定向克隆入毕赤酵母分泌型表达载体pPIC9的相应位点,构建重组酵母表达质粒pPIC9-scFv。BglⅡ线性化后,电转化入受体酵母菌GS115中,筛选阳性重组子,进行甲醇诱导表达,并用RT-PCR、SDS-PAGE、双抗体夹心Dot-ELISA法分析目的蛋白表达情况。结果:通过表型筛选、PCR鉴定获得阳性重组酵母工程菌,RT-PCR、SDS-PAGE分析表明目的蛋白得到很好表达,表达量可占培养液上清总蛋白量的18%,双抗体夹心Dot-ELISA法检测,表达产物能够特异识别重组HIV-1gp120抗原蛋白并与之发生特异性免疫反应,证明表达的重组scFv具有良好的抗体活性。结论:成功地实现了scFv基因的分泌性表达,为抗AIDS靶向治疗及进一步研究其抗HIV活性提供了依据。
AIM： To construct the vector and express anti-HIV-1 envelope glycoprotein single chain Fv fragment in Pichia pastoris. METHODS： The target gene was digested from plasmid pET28-scFv and cloned into pichia pastoris vector via gene engineering and DNA recombination techniques. The recombinant plasmid was linearized and transferred into Pichia pastoris strains GS11.5 by electroporation. After positive recombinant was selected and expression was induced by methanol, the target protein was analyzed by RT-PCR, SDS-PAGE and double-antibody sandwich ELISA. RESULTS： High copies of transformant were obtained by phenotype determining and PCR amplification. RT-PCR and SDS-PAGE demonstrated the target protein was successfully expressed. And the yield account for about 18 percent of the total cell proteins. Double-antibody sandwich ELISA analysis proved that the recombinant scFv had good biological activities since it could be recognized and induce special immune respond with gp120 antigen. CONCLUSION： The scFv was expressed successfully. This research will lay the foundation for AIDS target therapy and further study of anti-HIV activities.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2007年第12期1144-1146,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
吉林省科技厅资助项目(20060570)
关键词
HIV-1
外膜蛋白
单链抗体
酵母分泌表达
HIV-1
envelope glycoprotein
single chain Fv fragment （ scFv ）
secretory expression in pichia pastoris
