CAJ | 학술논문

目的建立一种简单易行的从HIV-1感染者中筛选包膜糖蛋白(envelope glycoprotein,Env)特异性单克隆抗体的方法.方法采集HIV-1感染者抗凝全血,分离外周血单个核细胞,利用生物素标记的HIV Gp120抗原与B细胞膜上的IgG特异性结合,然后用链霉亲和素标记的磁珠分选出能够产生Env特异性抗体的记忆性B细胞.用单细胞RT-PCR法从记忆性B细胞中扩增抗体的重链可变区基因与轻链可变区的基因并克隆到表达载体中,将携带重链基因的质粒与携带轻链基因的质粒共转染293T细胞,获得HIV-1特异性人单克隆抗体,并进行抗体结合活性的鉴定.结果从1例我国HIV-1感染者记忆性B细胞中筛选出3株对HIV-1 Env抗原有结合活性的单克隆抗体.结论利用生物素标记抗原与记忆性B细胞特异结合,并用亲和素标记的磁珠对细胞进行分选,结合单细胞RT-PCR技术可以成功筛选出抗原特异性的单克隆抗体.
목적 건립일충간단역행적종HIV-1감염자중사선포막당단백(envelope glycoprotein,Env)특이성단극륭항체적방법.방법 채집HIV-1감염자항응전혈,분리외주혈단개핵세포,이용생물소표기적HIV Gp120항원여B세포막상적IgG특이성결합,연후용련매친화소표기적자주분선출능구산생Env특이성항체적기억성B세포.용단세포RT-PCR법종기억성B세포중확증항체적중련가변구기인여경련가변구적기인병극륭도표체재체중,장휴대중련기인적질립여휴대경련기인적질립공전염293T세포,획득HIV-1특이성인단극륭항체,병진행항체결합활성적감정.결과 종1례아국HIV-1감염자기억성B세포중사선출3주대HIV-1 Env항원유결합활성적단극륭항체.결론 이용생물소표기항원여기억성B세포특이결합,병용친화소표기적자주대세포진행분선,결합단세포RT-PCR기술가이성공사선출항원특이성적단극륭항체.
Objective To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals.Methods Human B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads.Gp120 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane.The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating,counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs.The antibody genes were amplified by single cell RT-PCR and nested PCR,cloned into eukaryotic expression vectors and transfected into 293T cells.The binding activity of recombinant antibodies to Env were tested by ELISA.Results Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual.Conclusion We can obtain Env-specific antibody by biotin labbled antigen,magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.