丙型肝炎病毒包膜蛋白E2抗独特型人源单链可变区抗体的筛选与鉴定

Production and identification of anti-idiotypic antibody single chain vari-able fragments against HCV E2

目的:制备抗丙型肝炎病毒(HCV)包膜蛋白E2(E2)的抗独特型单链可变区抗体scFv(抗-IdscFv),为研制HCVE2的抗-IdscFv疫苗奠定基础.方法:采用噬菌体表面展示技术,将抗HCVE2单克隆抗体固相包被于Nunc板,从噬菌体单链可变区抗体库中经过5轮“黏附-洗脱-扩增”筛选过程,随机挑选出53个克隆,利用酶联免疫黏附法、交叉反应和竞争抑制实验,对其进行免疫学检测,获得与HCVE2单克隆抗体结合活性较强的抗独特型抗体单链可变区片段(抗-IdscFv)的阳性克隆,并对HCVE2特异性抗-IdscFv的编码序列进行序列测定分析.结果:对噬菌体单链可变区抗体库经过5轮“黏附-洗脱-扩增”的筛选后,结合到包被平皿的噬菌体与第一轮相比,富集了12倍.用酶联免疫黏附实验(ELISA)方法测定第五轮筛选后上清液中含有的抗-IdscFv与HCVE2单克隆抗体结合活性.其中有18株克隆ELISA的吸光度(A450nm)值较高(E11A450nm0.928,E14A450nm1.152,E17A450nm1.136,E28A450nm1.163,E53A450nm0.965).对这些噬菌体抗体进行与牛血清白蛋白(BSA)的交叉反应后,确定其中有5株交叉反应较弱(E11A450nm0.044,E14A450nm0.062,E17A450nm0.166,E28A450nm0.012,E53A450nm0.069),结合2次ELISA重复实验的A值及竞争抑制实验结果,最后确定1株(E28)阳性克隆.提取质粒,进行DNA序列测定,DNA大小为768bp.结论:用噬菌体抗体库技术能够成功地获得单抗HCVE2的抗-IdscFv,本实验结果为开展用抗-IdscFv防治丙型肝炎的研究创造了条件.

AIM: To screen anti-idiotypic single chain variable fragments(anti-Id scFv) against Hepatitis C virus envelope protein E2(HCV E2) monoclonal antibody so as to lay a foundation fordeveloping of anti-Id scFv vaccine of the Hepatitis C virus.METHODS: The recombinant phage antibody library wasconstructed by Hepatitis C virus envelope protein E2 mono-clonal antibody which was coated in a microtiter plate, afterfive rounds of biopanning, 53 clones specific to HCV E2antibody were determined with the enzyme-linkedimmunosorbent assay (ELISA). The specificity of anti-idiotypic scFv was identified by ELISA, cross reaction andcompetition inhibition assay. The DNA sequence of thepositive clone was determined.RESULTS:Specific anti-Id scFv against HCV E2 antibodywere enriched by 12 times after five rounds of panning withHCV E2 antibody. 53 clones were checked for their bindingactivities with ELISA and 18 clones were able to bind toHCV E2 antibody, and 5 clones had low A450 nm valuecombination with BSA.1 clone named E28 exhibited specificbinding to HCV E2 antibody identified by direct and compe-tition inhibition ELISA methods. The DNA sequence datashowed that the anti-Id scFv coding gene included 768 bp.CONCLUSIONS: The anti-Id scFv fragments to HCV E2monoclonal antibody can be successfully selected by re-combinant phage antibody technique, which paves a wayfor the study of prevention and treatment of hepatitis Cusing anti-Id scFv.