猪PCSK9单克隆抗体的制备及其在PRRSV感染过程中的应用

Preparation of a Monoclonal Antibody to Porcine PCSK9 and its Application during PRRSV Infection

实验室前期shot-gun质谱结果表明PCSK9在PAM空细胞和PRRSV感染组PAM细胞间存在差异表达。为了深入研究PCSK9与PRRSV间的相互作用机制,本实验在体外合成猪PCSK9基因并克隆至原核表达载体pET-28a中,构建重组表达质粒pET-28aPCSK9。利用该原核表达系统获得了高效表达的重组蛋白PCSK9(约75kDa),进一步通过镍柱亲和层析技术纯化重组蛋白PCSK9作为免疫原,皮下免疫5-6周龄的BALB/c小鼠(100ng/只),获得了1株产生PCSK9特异性单克隆抗体的杂交瘤细胞株(2A2B12)。应用此单克隆抗体对猪繁殖与呼吸综合征病毒(PRRSV)感染PAM细胞后的内源性PCSK9变化情况进行了检测。Western blot和IFA结果表明本实验制备的PCSK9单克隆抗体具有良好的特异性,抗体针对的表位位于PCSK9的C端结构域(463-705 aa);此外,Western blot结果表明PRRSV感染可抑制PCSK9蛋白的表达,过表达PCSK9对PRRSV的复制有明显的抑制作用。本研究中研制的抗体可为今后猪源PCSK9与PRRSV的相互作用研究提供工具支持。

Previous studies of shot-gun mass spectrometry have demonstrated that proprotein convertase subtilisin/kexin type 9(PCSK9)is differently expressed in PAM cells with or without Porcine reproductive and respiratory syndrome virus(PRRSV)infection. In order to further explore the interaction mechanism between PCSK9 and PRRSV, pig-originated PCSK9 gene was synthesized in vitro and cloned into prokaryotic expression vector pET-28A followed by generating recombinant expression plasmid PET-28aPCSK9. PCSK9 protein of about 75 kDa was expressed as inclusion body at a high level in the prokaryotic expression system and purified by nickel column affinity chromatography. The purified PCSK9 protein was used to subcutaneously immunize BALB/C mice at 5-6week-age(100 ng/mouse) and then a hybridoma cell line secreting monoclonal antibody(MAb) 2A2B12 against PCSK9 was obtained after fusion and screening. The MAb 2A2B12 had good specificity as visualized in Western blot and IFA and then used to detect dynamic changes of endogenous PCSK9 in PAMs after PRRSV infection. The target site of MAb 2A2B12 was localized in the C-terminal domain(463-705 aa) of PCSK9. In addition, the results of Western blot using this MAb showed that PRRSV infection inhibited the expression of PCSK9. On the contrary, the presence of PCSK9 obviously inhibited the replication of PRRSV. The availability of the MAb provided a useful tool for further research on the interaction between porcine PCSK9 and PRRSV.