猪丁型冠状病毒N基因原核表达及多克隆抗体制备

Prokaryotic expression of porcine deltacoronavirus N gene and preparation of its polyclonal antibody

旨在研究猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)N蛋白的抗原性,为下一步诊断试剂盒的研发提供依据。试验通过RT-PCR的方法扩增PDCoV CH/GX/1468B/2017株完整的N基因并构建原核表达质粒pET32a-N,将pET32a-N转化至大肠杆菌BL21感受态细胞中,经诱导表达获得重组N蛋白,纯化后经Western blot方法检测其反应原性,免疫昆明小鼠制备重组N蛋白多克隆抗体并进行效价测定(间接ELISA法)和特异性验证(间接免疫荧光法)。结果显示:重组N蛋白在IPTG终浓度为1.0 mmol/L,37℃诱导表达6 h的条件下可获得最高表达量,该重组蛋白主要以可溶性蛋白的形式表达;Western blot结果显示,纯化后的重组N蛋白可以和PDCoV阳性猪血清发生特异性结合,具有良好的反应原性。制备的多克隆抗体的效价可达1∶64000,间接免疫荧光法结果表明其能特异性识别和结合PDCoV,说明重组N蛋白具有较好免疫原性。提示:PDCoV N蛋白抗原性好,可作为诊断试剂盒的候选抗原。

The aim of this study was to investigate the antigenicity of the porcine deltacoronavirus N protein and to lay a foundation for developing diagnostic kits.In this study,the complete N gene of a PDCoV CH/GX/1468B/2017 strain was amplified by RT-PCR,and the prokaryotic expression plasmid pET32a-N was constructed.pET32a-N was then transformed into E.coli BL21(DE3)cells and expressed by IPTG to obtain recombinant protein N.After purification,the reactivity of recombinant protein N was detected by Western blot.Next,the purified recombinant protein N was used to immunize Kun-ming mice to prepare a polyclonal antibody.The titer of the polyclonal antibody was detected by indirect ELISA,and its specificity was verified by indirect immunofluorescence.The results showed that the optimal induction condition for the highest expression of recombinant N protein was a final concentration of IPTG of 1.0 mmol/L,and induction at 37℃for 6 h.The recombinant protein mainly presented in the form of a soluble protein.The Western blot results showed that purified recombinant protein N could specifically bind to PDCoV positive serum and had good reactogenicity.The titer of the poly-clonal antibody was 1∶64000.The IFA results showed that the polyclonal antibody could specifically recognize and bind PDCoV virus.This study indicated that PDCoV N protein had good antigenicity and could be used as a candidate antigen of the diagnostic kit.