猪瘟病毒Erns蛋白原核表达及其多克隆抗体制备

Prokaryotic expression of Erns protein of classical swine fever virus and preparation of its polyclonal antibody

  • 摘要: 【目的】 制备猪瘟病毒(Classical swine fever virus,CSFV)Erns蛋白多克隆抗体,为进一步研究Erns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Erns蛋白结构。以真核表达载体pCMV-HA-Erns为模板,PCR克隆CSFV Erns基因,构建原核表达载体pET-32a-Erns。将pET-32a-Erns转化大肠杆菌BL21(DE3)感受态细胞,添加IPTG诱导Erns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Erns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】 Erns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;Erns基因片段大小为681 bp,其重组蛋白主要以包涵体形式表达;Erns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Erns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Erns蛋白水平。Erns蛋白多克隆抗体的制备有助于更好地理解Erns蛋白在CSFV感染和免疫过程中的作用,对深入研究Erns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。

     

    Abstract: 【Objective】 The study aimed to prepare polyclonal antibody against the Erns protein of the classical swine fe‐ ver virus(CSFV),which could provide theoretical basis for further exploring the structure and function of the Erns protein,as well as for elucidating the pathogenic mechanisms of CSFV. 【Method】 Bioinformatics methods were used to predict the structure of the Erns protein. The CSFV Erns gene was cloned by PCR using the eukaryotic expression vector pCMVHA-Erns as a template,and the prokaryotic expression vector pET-32a-Erns was constructed. The pET-32a-Erns vector was then transformed into Escherichia coli BL21(DE3)receptor cells,IPTG was added to induce the expression of Erns protein,confirming the expression form of Erns protein. The target protein was purified by Ni-NTA affinity chromatography to obtain high-purity Erns 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 Erns protein, and their titer and specificity were assessed. 【Result】 The Erns protein lacked signal peptide and transmembrane domains, making it a hydrophilic protein that contained multiple B-cell epitopes. The size of the Erns gene fragment was 681 bp,and its recombinant protein was primarily expressed as inclusion bodies. The polyclonal antibodies against the Erns 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 Erns protein possess high titer and strong specificity,making them suitable for detecting the expression levels of the Erns protein in cells using ELISA and Western blotting. The preparation of Erns protein polyclonal antibodies contributes to a better under‐ standing of the role of Erns protein in CSFV infection and immune processes. This has significant implications for further research into the function of Erns protein,the biological characteristics of CSFV,and the development of prevention and diagnostic methods for CSFV.

     

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