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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.
OBJECTIVE: To prepare anti-idiotype single-chain variable region antibody scFv (anti-IddsFv) against hepatitis C virus (HCV) envelope protein E2 (E2) and lay a foundation for the development of anti-IddscFv vaccine against HCVE2.Methods: Surface-mount technique, the anti-HCVE2 monoclonal antibody was solid-phase coated on the Nunc plate and randomly selected 53 clones from the phage single chain variable region antibody library after 5 rounds of “adhesion-elution-amplification” The positive clones were screened by enzyme-linked immunosorbent assay (ELISA), cross-reaction and competition inhibition assay, and the positive clones with anti-idiotypic antibody single chain variable fragment (anti-IddscFv) , And sequence analysis of the coding sequence of HCVE2-specific anti-IdscFv.Results: The phage single chain variable region antibody library was screened by 5 cycles of “adhesion-elute-amplification” Phage were 12-fold more enriched than the first round, and the binding activity of the anti-IddscFv and HCVE2 monoclonal antibodies contained in the fifth round of the supernatants after the screening was determined by enzyme-linked immunosorbent assay (ELISA), of which 18 Strain ELISA ELISA absorbance (A450nm) value is higher (E11A450nm0.928, E14A450nm1.152, E17A450nm1.136, E28A450nm1.163, E53A450nm0.965). After these phage antibodies were cross-reacted with bovine serum albumin (BSA), five of them were confirmed to have weak cross-reactivity (E11A450nm0.044, E14A450nm0 .062, E17A450nm0.166, E28A450nm0.012, E53A450nm0.069), combined with two A ELISA repeat experiment A values and competitive inhibition test results, and finally identified one (E28) positive clones.Plasmid, DNA sequencing, DNA With a size of 768bp.Conclusion: The anti-IddsFv of McAb HCVE2 can be successfully obtained by using the phage antibody library technology, and the experimental results have created the conditions for the research on the prevention and treatment of hepatitis C with anti-IddsFv.