人源抗人类免疫缺陷病毒1型基因工程抗体的初步研究  

Primary researches on human genetically engineered antibodies to HIV-1

作　　者：许四宏[1] 杜润蕾[2] 王素婷[1] 宋爱京[1] 李秀华[1] 梁米芳[2] 王佑春[1] 

机构地区：[1]中国药品生物制品检定所,北京100050 [2]中国疾病预防控制中心病毒所

出　　处：《中华微生物学和免疫学杂志》2010年第11期1057-1062,共6页Chinese Journal of Microbiology and Immunology

基　　金：基金项目：“十一五”科技重大专项（2009zxlo004-801）

摘　　要：目的 构建噬菌体抗体库,筛选抗HIV-1抗体Fab,并对其进行鉴定.方法 采集无症状、HIV-1抗体滴度较高的HIV-1感染者全血,分离淋巴细胞并提取总RNA,经RT-PCR扩增人轻链基因和重链Fd基因.运用噬菌体展示技术,构建噬菌体抗体库,经过3轮＂吸附-洗脱-富集＂筛选,将获得的克隆进行诱导表达,表达产物经ELISA检测,筛选阳性抗HIV-1 Fab克隆 对获得的克隆进行抗体基因序列测定并分析序列同源性和互补决定区（CDRs） 对获得的阳性克隆与轻链克隆CL8进行链置换并检测链置换前后抗体亲和力的变化 对获得的克隆进行诱导表达及纯化,同时进行中和试验.结果 构建了库容为8×106的噬菌体抗体库 经3轮筛选,获得11株抗HIV-1 Fab克隆,测序和序列分析显示,获得了 10条轻链和8条重链,它们和已经申请专利的部分HIV-1 gp120抗体的基因序列有较高的同源性（轻链同源性为60%～90% 重链同源性为71%～85%） CDRs分析显示,11株抗体CDRHs均比较长（12～20aa） 链置换后的抗体亲和力并没有明显的改进 Fab诱导表达量均大于10 mg/L,利用重组HIV-1假病毒系统进行中和试验,结果显示,3株Fab具有一定的中和作用.结论 成功获得抗HIV-1抗体Fab,为进一步对其进行研究和应用奠定基础.Objective To pan and characterize anti-HIV-1 Fab by the phage antibody library technology. Methods Total RNA were extracted from lymphocytes which were isolated from peripheral blood collected from asymptomatic HIV-1 infected donors with high titer antibody against HIV-1. The genes of heavy chains Fd fragment and light chains of antibody were amplified by RT-PCR. The phagmids pComb3X cloned Fd and light chain genes were transformed into E. coli XL1-Blue by electroporation to construct phage Fab library. By three runs of ＂absorption-elution-neutralization-enrichment＂, the clones were induced by IPTG and characterized by ELISA. The positive clones were sequenced and analyzed the sequences. Subsequently, Fab antibodies of these positive clones were induced to expressed and purified, then the recombinant virus neutralization assay was performed. Results A phage Fab library was constructed with 8×106 members, and 11 positive clones were obtained by detecting IPTG-induced-expressing Fabs with ELISA. By analysis of the sequences, 10 light chain genes and 8 Fd genes were ensured to be obtained. Compared with the genes of anti-HIV-1 antibodies in HIV sequence database, the gene sequences we obtained were highly homologous to some patent genes of anti-HIV-1 gp120 antibodies in HIV sequence database（ light chains with 60%-90% identity, Fd with 71%-85% identity） The CDRs of these positive clones were determined by comparing the positive clone genes with antibodies＇ genes in V base database, furthermore, CDRH3 of these positive clones has the length of 12-22 aa. Strand shift had little effect to improving affinity of our Fab clones. Fab antibodies were induced to express at the concentration of 〉 10 mg/L. Three Fab antibodies neutralize HIV-1 virus to some extent. Conclusion The studies will provide the basis on further study on the anti-HIV-1 Fabs obtained successfully.

关 键 词：HIV-1 基因工程抗体 噬菌体展示 链置换 中和试验 

分 类 号：R5[医药卫生—内科学] R3[医药卫生—临床医学]