新型鸭呼肠孤病毒p18蛋白多克隆抗体区分强弱毒株的研究

Differentiation of virulent and attenuated strains of novel duck reovirus by anti-p18 polyclonal antibodies

摘要:

【背景】我们前期将新型鸭呼肠孤病毒(novel duck reovirus, NDRV)强毒株NP03在番鸭胚成纤维细胞(muscovy duck embryo fibroblast, MDEF)上进行传代，选育出NDRV弱毒株S，将强弱毒株p18蛋白进行比对发现弱毒p18蛋白C端缺失35个氨基酸。【目的】制备p18蛋白的多克隆抗体并鉴定NDRV强弱毒株p18蛋白的差异。【方法】采用RT-PCR方法扩增NDRV强弱毒株p18基因编码序列，克隆至原核表达载体pET-28a中诱导表达，用镍柱纯化目的蛋白，通过SDS-PAGE和Western Blot对纯化蛋白进行鉴定。进一步用纯化的蛋白免疫小鼠制备p18蛋白多克隆抗体，通过间接ELISA、Western Blot和间接免疫荧光试验(indirect immunofluorescence assay, IFA)对制备的多克隆抗体进行检测鉴定NDRV强弱毒株。【结果】对目的蛋白进行SDS-PAGE和Western Blot鉴定结果表明，分别获得了NDRV强弱毒株p18重组蛋白。对多克隆抗体进行间接ELISA检测，效价达1:51 200。Western Blot鉴定显示，制备的p18蛋白多克隆抗体能特异性检测NDRV强、弱毒株感染细胞后表达的p18蛋白，结果表明，强、弱毒株p18蛋白的分子量有明显差异，分别约为18 kDa和14 kDa。IFA结果表明，制备的p18蛋白多克隆抗体可以特异性识别NDRV强、弱毒复制时表达的p18蛋白。【结论】本研究制备的p18蛋白多克隆抗体可用于NDRV强弱毒株p18蛋白检测，并发现NDRV强、弱毒株p18蛋白存在明显差异，为深入研究NDRV p18蛋白生物学功能及致病机理奠定了基础。

Abstract:

[Background] In our previous study, we passaged the virulent strain NP03 of novel duck reovirus (NDRV) on muscovy duck embryo fibroblast (MDEF) cells and obtained an attenuated strain NDRV S. Compared with that of the virulent strain, the p18 protein of NDRV S misses 35 residues at the C-terminus. [Objective] To prepare anti-p18 polyclonal antibodies and identify the differences in p18 between the virulent and attenuated strains of NDRV. [Methods] RT-PCR was employed to amplify the p18 gene sequences of the virulent and attenuated strains of NDRV. The obtained sequences were then cloned into the prokaryotic expression vector pET-28a for induced expression. The target proteins were purified by nickel columns and identified by SDS-PAGE and Western Blot. The polyclonal antibodies were prepared by immunizing the BALB/c mice with the purified proteins, and the antibodies were detected and identified by indirect ELISA, Western Blot, and indirect immunofluorescence assay (IFA). [Results] The SDS-PAGE and Western Blot results showed that the recombinant p18 proteins of the virulent and attenuated strains of NDRV were obtained. The indirect ELISA result showed that the serum titer of the polyclonal antibodies was 1:51 200. The result of Western Blot demonstrated that the prepared polyclonal antibodies could specifically detect the p18 proteins expressed by cells infected with virulent and attenuated strains. The molecular weights of the p18 proteins of virulent and attenuated strains varied, being 18 kDa and 14 kDa, respectively. The IFA results confirmed that the prepared polyclonal antibodies could recognize p18 proteins expressed in cells infected with virulent and attenuated strains. [Conclusion] We successfully prepared the anti-p18 polyclonal antibodies and identified the significant differences in its composition between virulent and attenuated strains of NDRV. The findings lay a solid foundation for investigating the biological function of p18 as well as the pathogenic mechanism of NDRV.