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运用噬菌体表面呈现技术,从禽流感病人恢复期血中获得淋巴细胞,通过基因工程手段,构建了人源抗H5N1禽流感病毒基因工程抗体文库。用纯化的人源H5N1禽流感病毒颗粒(A/Anhui/1/2005)及重组血凝素蛋白HA(A/VietNam/1203/2004)对Fab噬菌体抗体库进行富集筛选,成功地获得了抗禽流感病毒H5N1血凝素蛋白HA的人源单抗Fab段基因,并在大肠杆菌中获得有效表达。通过序列测定确定抗体轻重链型别,然后将阳性克隆的轻链和重链Fd段基因分别克隆入全抗体表达载体pAC-L-Fc后转染昆虫Sf9细胞,利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达。用ELISA、IFA和流式细胞术对所获人源单抗的功能特性进行鉴定。结果表明,我们获得了2株特异性针对H5N1禽流感病毒血凝素蛋白HA而与甲1型和甲3型人流感病毒无交叉反应的人源单抗(AVFluIgG01、AVFluIgG03)。微量中和试验结果表明,除A/Guangdong/1/2006外,AVFlu-IgG01能够广泛地中和HA基因进化上属于Clade2的中国南方、北方及中部地区的H5N1禽流感病毒分离株,同时还对属于CladeⅠ的越南H5N1分离株A/VietNam/1203/2004具有中和活性;AVFluIgG03虽然不能中和A/VietNam/1203/2004,但是对属于Clade2的所有中国H5N1分离株均具有中和作用。人源中和性抗禽流感病毒H5N1基因工程全抗体的获得不仅为高致病性禽流感病毒H5N1的预防和治疗带来了希望,同时也为其疫苗研制提供了新的思路。
Using phage surface display technology, lymphocytes were obtained from blood recovered from bird flu patients, and a genetically engineered antibody library of human anti-H5N1 bird flu virus was constructed through genetic engineering. Fab phage antibody library was enriched and screened with purified human H5N1 bird flu virus particles (A / Anhui / 1/2005) and recombinant hemagglutinin HA (A / VietNam / 1203/2004) The human monoclonal antibody Fab fragment gene of avian influenza virus H5N1 hemagglutinin HA was efficiently expressed in E. coli. The light and heavy chains of the antibody were identified by sequence analysis. The positive clones were cloned into the full-length antibody expression vector pAC-L-Fc and then transfected into insect Sf9 cells. The baculovirus / insect cell system Achieve the secretion of full-body antibody expression. The functional properties of the obtained human monoclonal antibodies were identified by ELISA, IFA and flow cytometry. The results showed that we obtained two human monoclonal antibodies (AVFluIgG01, AVFluIgG03) specific for HA of H5N1 avian influenza virus without cross-reaction with human influenza A (H1N1) and human influenza A (H3N) viruses. In addition to A / Guangdong / 1/2006, AVFlu-IgG01 was able to neutralize the H5N1 avian influenza virus isolate in southern China, northern and central China evolved to HA gene evolution in Clade2 in addition to A / Guangdong / 1/2006. Vietnam H5N1 isolate A / VietNam / 1203/2004 belonging to Clade I has neutralizing activity; AVFluIgG03, although not able to neutralize A / VietNam / 1203/2004, neutralizes all Chinese H5N1 isolates belonging to Clade2. The gain of H5N1 genetically engineered antibody against human neutralizing avian influenza virus not only provided hope for the prevention and treatment of H5N1, but also provided a new idea for vaccine development.