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 vi-rus,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.
Prokaryotic expression of protein encoded by CP312R gene of African swine fever virus & application and preparation of its polyclonal antibody
To prepare polyclonal antibody against protein encoded by African swine fever virus(AS-FV)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 ex-pression 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 chromatogra-phy,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 vi-rus(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 reactivi-ty and specificity,and it was also proved that ASFV pCP312R could be stably expressed in PRRSV live vector.
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