猪繁殖与呼吸综合征病毒N蛋白原核表达及多克隆抗体的制备

摘要

本研究将猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)HuN4株的N基因克隆至原核表达载体pCold-Ⅰ中,经IPTG诱导,在大肠杆菌中表达了重组His-N蛋白,蛋白分子量为14 kDa,能被PRRSV阳性血清特异性识别.经超声裂解和SDS-PAGE检测后,重组蛋白主要以可溶性形式表达.随后利用磁珠纯化重组蛋白,采用背部皮下多点注射免疫BALB/c小鼠,经四次免疫后获得多克隆抗体.Western blot和间接免疫荧光检测结果表明,制备的多克隆抗体具有良好的免疫反应活性,能够与真核表达或病毒的N蛋白发生特异性反应.%In this study, the N gene of highly pathogenic Porcine reproductive and respiratory syndrome virus(PRRSV) strain HuN4 was cloned into prokaryotic expression vector pCold-Ⅰ and the recombinated protein was expressed after IPTG induction. The expressed N protein was 14 kDa in size and could be specificly recognized by PRRSV positive serum. Using ultrasonication and SDS-PAGE anlysis, the recombinant portein mainly expressed in a soluble form. The expressed His-N was purified by nickel magnetic beads and immunized to 8-week BALB/c mice. After 4 times immunization, the prepared anti-N polyclonal antibodies(pAb) was analyzed by Western blot and indirect immunofluorescence assay. The results showed that the prepared pAb showed good immunoreactivities to viral or eukaryotic expressed N protein.