猪流行性腹泻病毒S蛋白的表达及其特异性单域抗体的制备

发布时间：2018-05-07 07:02

本文选题：猪流行性腹泻病毒 + 噬菌体抗体库　；参考：《内蒙古农业大学》2017年硕士论文

【摘要】：猪流行性腹泻(Porcine epidemic diarrhea,PED)是猪常见多发病,给养猪业造成非常严重的经济损失。目前,疫苗免疫是防控PED的主要措施,但是疫苗免疫存在免疫空白期,高密度养殖带来环境的深度恶化、疫苗免疫失败、抗生素滥用,以及某些猪场存在蓝耳病和圆环病毒病等对疫苗免疫的抑制,传统的PED活病毒(弱毒)疫苗自身存在着传播疾病的风险等因素,对该病的防控带来不利的影响。噬菌体展示技术是将抗体分了展示于噬菌体表面的技术,能够针对特定抗原进行筛选并得到特异性抗体。猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)的主要抗原表位均分布在病毒的纤突蛋白(S蛋白,Spike protein)上。利用噬菌体展示技术构建PEDV S蛋白噬菌体抗体库,并从抗体库中筛选和制备PEDV S蛋白特异性单域抗体,相应的单域抗体可用于PED诊断、治疗和预防。本研究中,我们利用PEDV免疫双峰驼,分离外周血淋巴细胞后提取总RNA,通过RT-PCR合成cDNA,再用单域抗体特异性引物进行三次扩增得到VHH基因。将VHH基因片段与pCANTAB5E载体连接,电转化E.coli TG1,成功构建了库容为5.49×106的初级单链抗体库。并将PEDV关键抗原蛋白S克隆到原核表达载体pET-28a中,重组质粒再转化到感受态细胞Transetta-DE3,进行可溶性蛋白表达和纯化。并以S蛋白作为抗原对抗体库进行3轮富集和筛选,Phage-ELISA筛选和获得了 S蛋白特异性单域抗体。
[Abstract]:Porcine epidemic diarrhea (PED) is a common disease in pigs, which causes serious economic loss to pig industry. At present, vaccine immunization is the main measure to prevent and control PED, but vaccine immunization exists in blank period, high density culture brings about deep deterioration of environment, vaccine immunization fails, antibiotics abuse. Some factors, such as blue ear disease, circovirus disease and so on, existed in some pig farms, and the traditional PED live virus (attenuated virus) vaccine itself had the risk of spreading disease, which had a negative effect on the prevention and control of the disease. Phage display is a technique that separates antibodies on phage surface and can screen specific antigens and obtain specific antibodies. The main antigenic epitopes of porcine epidemic diarrhea virus (PEV) were distributed on the spike protein of the virus. The phage antibody library of PEDV S protein was constructed by phage display technique, and the specific single domain antibody of PEDV S protein was screened and prepared from the antibody library. The corresponding single domain antibody can be used in the diagnosis, treatment and prevention of PED. In this study, we immunized Bactrian Camels with PEDV, isolated peripheral blood lymphocytes, extracted total RNAs, synthesized cDNAs through RT-PCR, and amplified VHH gene with single domain antibody specific primers for three times. The VHH gene fragment was ligated with pCANTAB5E vector and electrotransformed into E.coli TG1. A primary single-chain antibody library with a storage capacity of 5.49 脳 10 ~ 6 was successfully constructed. The key antigen protein S of PEDV was cloned into the prokaryotic expression vector pET-28a, and the recombinant plasmid was transformed into Transetta-DE3 cells for expression and purification of soluble protein. Using S protein as antigen, the antibody library was enriched and screened by Phage-ELISA for three rounds, and S-protein specific single-domain antibody was obtained.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28

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