摘要
为制备MHCⅠ基因工程疫苗的基础材料,构建了鸡MHCⅠ分子重组质粒并进行原核表达,进而制备鸡MHCⅠ分子的多克隆抗体。应用PCR方法,克隆鸡MHCⅠα和β2m基因,构建重组载体pETMHCⅠα和pET-MHCⅠβ2m,经PCR、双酶切和测序鉴定后,将重组质粒在大肠埃希菌Rosetta中进行诱导表达,融合蛋白纯化后,接种昆明小鼠制备多克隆抗体,血清稀释后用免疫印迹法(Western blot)分析。结果表明,鸡MHCⅠα和β2m基因在大肠埃希菌中成功表达,融合蛋白分子质量分别约为52.1ku和33.0ku;制备的鼠抗鸡MHCⅠα和β2m链多克隆抗体,经Western blot检测证实抗体特异性较强,可进一步用于鸡MHCⅠ分子的研究。
To explore MHC Ⅰ function as DNA vaccine, chicken MHC Ⅰ genes were expressed by using Escherichia coli prokaryotic expression system, and polyclonal antibodies against the recombinant protein MHCⅠ were prepared. Chicken MHC Ⅰα and β2m genes were cloned,the recombinant plasmid pET-MHC Ⅰα, pET-MHC Ⅰ β2m were constructed, and confirmed by PCR amplification, double enzyme digestion and DNA sequencing. Next, the recombinant plasmids were expressed in E. coli Rosetta induced by using IPTG. After purification of the recombinant protein,the polyclonal antibodies against the recombinant protein were prepared in Kunming mice. The reactivity of the prepared polyclonal antibodies were determined by Western blot. The results revealed that the MHC Ⅰα and β2m genes were successfully expressed in E. coli,and the fusion proteins were about 52.1 ku and 33.0 ku, respectively. The polyclonal antibodies had the specific reactinogenicity, it was proved by Western blot. All this made it possible to do further studies on chicken MHC Ⅰ molecule.
出处
《动物医学进展》
北大核心
2016年第5期38-42,共5页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31302044)
安徽省科技攻关项目(11010302119)
安徽省教育厅自然科学研究重点项目(KJ2013A203)
关键词
鸡MHCI类分子
原核表达
多克隆抗体
chicken MHC Ⅰ molecule
prokaryotic expression
polyclonal antibody
