猪瘟病毒E2蛋白A3BCD主要抗原结构域的原核表达

Expression of the Main Antigenic Domain in A3BCD of E2 Glycoprotein of Classical Swine Fever Virus in Escherichia coli

目的:对猪瘟病毒E2蛋白A3BCD进行原核表达,以期获得具有抗原活性的表达产物。方法:利用反转录PCR(RT-PCR)和套式PCR(nPCR)技术从河南省近期流行猪瘟病毒野毒株中扩增得到E2基因,并将其克隆至pUCm-T载体,构建克隆载体pUCm-TE2;利用PCR方法扩增E2基因的主要抗原域A3BCD,克隆至pET-32a(+)载体,构建表达载体pET-32aE2-2,转化到大肠杆菌Rosetta(DE3)中,用IPTG进行诱导表达,对诱导产物进行SDS-PAGE和Western-blotting分析。结果:SDS-PAGE结果显示在相对分子质量约35000处出现预期条带,表达产物主要以包涵体形式存在;Western-blotting检测表明,表达产物能与猪瘟病毒阳性血清发生特异性反应,出现单一反应带;用8mol/L尿素(pH8.0)溶解包涵体,经Ni-NTA亲和层析法纯化,获得纯度较高的目的蛋白,ELISA检测表明能与猪瘟抗体阳性血清发生特异性反应。结论:重组质粒pET-32aE2-2转化大肠杆菌Rosetta(DE3),解除了由于稀有密码子造成的表达限制,表达量得到提高;表达产物为硫氧还蛋白和E2-2的融合蛋白,易于纯化,且具有活性,为研制猪瘟抗体ELISA检测试剂盒奠定了基础。

Objective： To express the main antigenic domain in A3BCD of E2 glycoprotein of classical swine fever virus （CSFV） in E.coli. Methods： E2 gene of prevalent virulent strain of CSFV from Henan province was amplified by RT-PCR and the nested PCR（nPCR）, and it was cloned into pUCm-T vector for sequencing. The main antigenic domain A3BCD of CSFV E2 glycoprotein was amplified by PCR, and it was cloned into pET-32a（＋） vector. The result plasmid named as pET-32a/E2-2 was transformed into E.coli Rosetta（DE3）. After induced by IPTG for hours, recombinant expression product was analyzed by SDS-PAGE and Western-blotting. Results： A specific expression band in form of inclusion body with a relative molecular weight 35000 was detected by SDS-PAGE. Result of Western-blotting showed that the expressed protein was recognized by the CSFV positive serum. The inclusion body was dissolved in 8.0 mol/L urea and subsequently purified by Ni-NTA affinity chromatography to obtain highly purified protein. Results of ELISA indicated that protein of interest could be recognized by the positive serum of CSF. Conclusion： The problem of the expression restrictions caused by rare codon is resolved, and high yield of antigenic fusion protein can be purified easily. The work provides a ground in research of CSF antibody ELLISA test kit.