非洲猪瘟病毒CP312R基因编码蛋白的原核表达及多克隆抗体的制备与应用

Prokaryotic expression of protein encoded by CP312R gene of African swine fever virus & application and preparation of its polyclonal antibody

为制备ASFV CP312R基因编码蛋白的多克隆抗体,利用PCR方法从非洲猪瘟病毒(African swine fever virus,ASFV)的灭活样品中扩增出924 bp的CP312R基因全长序列,利用同源重组法构建出原核表达质粒pCold-Ⅰ-ASFV-CP312R,将其转化至原核表达感受态细胞BL21中,经1 mmol/L的IPTG低温条件下诱导表达CP312R编码蛋白并经过镍柱亲和层析纯化后,利用SDS-PAGE对重组蛋白进行表达鉴定和反应原性分析,表达蛋白的相对分子质量约为34 kDa,通过Western blot分析,该蛋白能被ASFV抗体特异性识别。将所得的蛋白经处理纯化后免疫小鼠3次,得到含多克隆抗体的血清。利用该抗体检测实验室构建并拯救的表达ASFV CP312R基因编码蛋白的重组猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV),结果显示该多抗具有良好的反应原性和特异性,并且证明了ASFV CP312R基因编码蛋白可在PRRSV活载体中稳定表达。

To prepare polyclonal antibody against protein encoded by African swine fever virus(ASFV) CP312R,the 924 bp full-length sequence of CP312R gene was amplified from inactivated samples of ASFV by PCR,and the prokaryotic expression plasmid pCold-Ⅰ-ASFV-CP312R was constructed by homologous recombination and was transformed into competent cell BL21.The expression of pCP312R protein was induced by 1 mmol/L IPTG at low temperature for 16 h,and then the recombinant protein was identified and analyzed by SDS-PAGE.The molecular weight of the expressed protein was about 34 kDa,which was specifically recognized by ASFV antibody by Western blot analysis.The obtained protein was purified by nickel column affinity chromatography,and the mice were immunized for 3 times to obtain sera containing polyclonal antibody.Then,the antibody was used to detect the recombinant porcine reproductive and respiratory syndrome virus(PRRSV) expressing the protein encoded by ASFV CP312R gene which was constructed and rescued in our lab.The experimental results showed that the polyclonal antibody had good reactivity and specificity,and it was also proved that ASFV pCP312Rcould be stably expressed in PRRSV live vector.