文章摘要
李 琛,林 红,刘新建,王忠灿,周镇先,陈乐如,管晓虹,朱 进.人源抗狂犬病毒免疫型抗体库的构建及特异性抗体筛选与鉴定[J].南京医科大学学报,2010,(5):575~578616
人源抗狂犬病毒免疫型抗体库的构建及特异性抗体筛选与鉴定
Construction and screening of human immunized phage-display antibody libraries against rabies virus
投稿时间:2009-11-03  
DOI:10.7655
中文关键词: 噬菌体展示  狂犬病毒  人源scFv抗体库
英文关键词: phages display  rabies virus  human scFv antibody library
基金项目:国家“863”资助项目(2007AA02Z418);南京军区“122”工程资助
作者单位
李 琛 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京 210029 
林 红 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京 210029 
刘新建 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京 210029 
王忠灿 南京军区军事医学研究所,江苏 南京210002 
周镇先 南京市第二医院检验科,江苏 南京 210003 
陈乐如 南京军区军事医学研究所,江苏 南京210002 
管晓虹 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京 210029 
朱 进 南京军区军事医学研究所,江苏 南京210002 
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中文摘要:
      目的:利用噬菌体展示技术,构建人源抗狂犬病毒单链抗体库,筛选特异性的抗狂犬病毒糖蛋白人源单链抗体(scFv)并对其进行初步鉴定?方法:从接种狂犬疫苗的志愿者外周血中提取总RNA,RT-PCR扩增VH和VL基因,并利用重叠扩增PCR将VH和VL拼接为scFv?将纯化后的scFv克隆至噬菌体载体pComb3XSS构建人源抗狂犬病毒单链抗体库,以狂犬病毒糖蛋白(RABVG)为抗原,从抗体库中筛选抗RABVG的scFv?通过phage-ELISA验证噬菌体单链抗体的结合特异性,将阳性克隆转化E.coli TOP10F′进行表达?结果:构建了人源抗狂犬病毒抗体库,抗体库的库容为3.29×109?经过5轮筛选,获得43株与RABVG特异结合的噬菌体单链抗体,其中15个A450值较高的克隆序列分析结果显示,有3个正确的单链抗体基因?单链抗体基因转化TOP10F′构建工程菌,表达纯化后的单链抗体,经ELISA检测能够与RABVG特异性结合?结论:从构建的人源免疫型抗狂犬病毒单链抗体库筛选出的能与RABVG特异性结合的scFv,为进一步制备抗RABVG的治疗性人源化抗体奠定了基础?
英文摘要:
      Objective:To construct human single chain variable fragment (scFv) antibody library using phage display technology,and screen out special antibodies against the glycoprotein of rabies virus(RABVG) . Methods:The total RNA was isolated from the donors vaccinated with rabies vaccines and was used to amplify VH and VL genes by RT-PCR. VH and VL genes were joined together by overlap PCR to form scFv. The purified scFv gene repertoires were cloned into the phage vector pComb3XSS to construct the primary phage library. Panning against RABVG was performed for 5 rounds and the phage library was identified. The positive recombinant phages identified by ELISA were used to infect E.coli TOP10F′ for soluble scFv. Results:A recombinant phage antibody library against RABV was successfully constructed. 43 clones were found to bind to RABVG. 15 clones that had higher A450 values were detected with DNA sequencing and 3 of them were confirmed. The scFv gene was transformed into E.coli TOP10F′ for expression and purification. The purified scFv was proved to specifically bind to RABVG from the ELISA results. Conclusion:A human phage-display antibody library has been successfully constructed, and the selected scFv fragment can specifically bind to RABVG. It could be used in further studies of generation of human anti-RABVG antibodies for clinical application.
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