摘要
【目的】制备猪瘟病毒(Classical swine fever virus, CSFV)E^(rns)蛋白多克隆抗体,为进一步研究E^(rns)蛋白结构和功能及解析CSFV的致病机理提供理论依据。【方法】采用生物信息学方法预测E^(rns)蛋白结构。以真核表达载体pCMV-HA-E^(rns)为模板,PCR克隆CSFV E^(rns)基因,构建原核表达载体pET-32a-E^(rns)。将pET-32a-E^(rns)转化大肠杆菌BL21(DE3)感受态细胞,添加IPTG诱导E^(rns)蛋白表达,确定Erns蛋白表达形式。利用Ni-NTA亲和层析法纯化蛋白,以获得高纯度的E^(rns)蛋白。采用背部皮下多点注射法免疫小鼠,经4次免疫后采集小鼠血液,分离血清制备获得Erns蛋白多克隆抗体,并检测其效价和特异性。【结果】E^(rns)蛋白不含信号肽和跨膜结构域,为亲水性蛋白,含有多个B细胞抗原表位;E^(rns)基因片段大小为681 bp,其重组蛋白主要以包涵体形式表达;E^(rns)蛋白多克隆抗体对原核表达的重组蛋白效价为1∶64000,对真核表达的重组蛋白效价为1∶1000,且能特异性识别CSFV。【结论】制备获得的E^(rns)蛋白多克隆抗体具有效价高和特异性强等特点,可用于ELISA和Western blotting检测细胞中E^(rns)蛋白水平。E^(rns)蛋白多克隆抗体的制备有助于更好地理解E^(rns)蛋白在CSFV感染和免疫过程中的作用,对深入研究E^(rns)蛋白功能、CSFV生物学特性及猪瘟的防治和诊断方法的开发具有重要意义。
【Objective】The study aimed to prepare polyclonal antibody against the E^(rns) protein of the classical swine fever virus(CSFV),which could provide theoretical basis for further exploring the structure and function of the E^(rns) protein,as well as for elucidating the pathogenic mechanisms of CSFV.【Method】Bioinformatics methods were used to predict the structure of the E^(rns) protein.The CSFV E^(rns) gene was cloned by PCR using the eukaryotic expression vector pCMV-HA-E^(rns) as a template,and the prokaryotic expression vector pET-32a-E^(rns) was constructed.The pET-32a-E^(rns) vector was then transformed into Escherichia coli BL21(DE3)receptor cells,IPTG was added to induce the expression of E^(rns) protein,confirming the expression form of E^(rns) protein.The target protein was purified by Ni-NTA affinity chromatography to obtain high-purity E^(rns) protein.Mice were immunized using the back subcutaneous multiple injection method,and after 4 immunizations,blood was collected to isolate serum for the preparation of polyclonal antibodies against the E^(rns) protein,and their titer and specificity were assessed.【Result】The E^(rns)protein lacked signal peptide and transmembrane domains,making it a hydrophilic protein that contained multiple B-cell epitopes.The size of the E^(rns) gene fragment was 681 bp,and its recombinant protein was primarily expressed as inclusion bodies.The polyclonal antibodies against the E^(rns) protein had titers of 1∶64000 for the prokaryotic expressed recombinant protein and 1∶1000 for the eukaryotic expressed recombinant protein,and they could specifically recognize CSFV.【Conclusion】The prepared polyclonal antibodies against the E^(rns)protein possess high titer and strong specificity,making them suitable for detecting the expression levels of the E^(rns) protein in cells using ELISA and Western blotting.The preparation of E^(rns) protein polyclonal antibodies contributes to a better understanding of the role of E^(rns) protein in CSFV infection and immune processes.This has significant implications for further research into the function of E^(rns) protein,the biological characteristics of CSFV,and the development of prevention and diagnostic methods for CSFV.
作者
宋丹丹
金奕欣
黄袁慧
王文锋
闵开骏
张传亮
罗廷荣
李晓宁
SONG Dan-dan;JIN Yi-xin;HUANG Yuan-hui;WANG Wen-feng;MIN Kai-jun;ZHANG Chuan-liang;LUO Ting-rong;LI Xiao-ning(College of Animal Science and Veterinary Medicine,Guangxi University,Nanning,Guangxi 530004,China;Guangxi Engineering Research Center of Veterinary Biologics/Guangxi Key Laboratory of Livestock and Poultry Breeding and Disease Prevention and Control/Key Laboratory of Animal Disease Prevention and Control in Guangxi Universities,Nanning,Guangxi 530004,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2024年第9期2835-2842,共8页
Journal of Southern Agriculture
基金
国家自然科学基金项目(32360869,31660722)
广西自然科学基金项目(2023GXNSFDA026043,2021GXNSFAA 220007)。
关键词
猪瘟病毒
E^(rns)蛋白
原核表达
多克隆抗体
classical swine fever virus(CSFV)
E^(rns)protein
prokaryotic expression
polyclonal antibody
