摘要：
基于生物信息学、化学合成技术和免疫学技术,利用预测的抗原肽进行抗体的制备已被广泛地应用.本研究探讨了利用预测的抗原肽制备针对PRRSV N蛋白的特异性抗体.首先,利用生物学软件对高致病性猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)N蛋白进行抗原性预测,获得一个位于蛋白N端16个氨基酸的候选抗原肽.通过BLAST比对,发现该抗原肽在不同北美型毒株中高度保守.其次,采用Fmoc固相法进行抗原肽合成,并将合成的抗原肽通过MBS法偶联到KLH载体蛋白上.随后,将偶联好的抗原肽与弗氏完全佐剂或不完全佐剂混合,免疫新西兰大白兔,制备抗血清,并利用IgG亲和层析的方法纯化抗体.最后,利用ELISA、Western blot和间接免疫荧光法(indirect immunofluorescence assay,IFA)对抗体的特异性进行检测,结果显示,利用偶联好的抗原肽包被ELISA板,检测抗体的滴度,纯化抗体的滴度超过105;Western blot结果显示,该抗体(1:2000)可以特异地识别PRRSV感染的Marc145细胞中的N蛋白,重要的是该抗体可以与不同毒株反应;IFA检测显示,该纯化的抗体(1:100)可以检测PRRSV感染的细胞.因此,利用生物信息学软件预测的PRRSV N蛋白的抗原肽,能有效地应用于抗PRRSV N蛋白抗体的制备,制备的抗体不仅为研究PRRSV N蛋白奠定基础,而且为研究PRRSV与细胞相互作用提供了一个有效的工具.%To date, prediction of antigen peptides based on bioinformatics, chemical synthetic and immunological techniques has been commonly used for generation of antibodies. Here, we determined the possibility of generation of specific antibody against PRRSV N protein by using predicted antigen peptide. First, one antigen peptide (16 amino acids) close to N terminal of was predicted from N protein of a highly pathogenic PRRSV strain. Then, the BLAST analysis showed that this peptide was conserved in different North American PRRSV strains. Secondly, this antigenic peptide was synthesized by using Fmoc solid phase method and coupled to the KLH vector protein by MBS method. Subsequently, New Zealand white rabbits were immunized with the conjugated antigenic peptide emulsified with Freund's complete or incomplete adjuvant. The antiserum was collected from the immunized rabbit and antibodies were purified using IgG affinity chromatography. The purified antibodies showed good signals at dilution of over 105 in ELISA. When used at dilution of 1:2000 in Western blotting, the antibodies reacted with N protein from Marc-145 cells infected with different PRRSV strains. The dilution of 1: 200 of this antibody preparation was used in IFA for detection of PRRSV in infected cells. Collectively, the antigen peptide of the PRRSV N protein was predicted using bioinformatics software and used for generation of antibodies, which showed good reactivity in ELISA, Western blot and IFA.
