猫传染性腹膜炎病毒N蛋白表达及其多克隆抗体制备

Preparation and Application of N Protein Polyclonal Antibody of Feline Infectious Peritonitis Virus SH2021 Strain

本研究旨在体外原核表达猫传染性腹膜炎病毒(feline infectious peritonitis virus,FIPV)SH2021株核衣壳蛋白(nucleocapsid,N),制备兔源多克隆抗体。根据FIPV SH2021株N基因序列设计引物,构建pCold-I-N重组表达质粒。随后,将重组质粒转化至表达感受态细胞BL21(DE3),在终浓度为1.0 mmol·L^(-1)的IPTG和16℃的诱导条件下进行表达。最后,利用His层析柱进行纯化,纯化后的重组蛋白免疫新西兰大白兔制备多克隆抗体。结果表明,纯化获得重组N蛋白大小约为45 ku,制备的N蛋白兔多克隆抗体效价可达1∶512000,该抗体对N蛋白具有良好的反应原性。本研究成功所制备了FIPV N蛋白兔多克隆抗体,该抗体具有良好的反应原性和特异性,为FIPV抗原抗体检测以及研究N蛋白生物学功能研究提供重要工具。

This study aimed to express the nucleocapsid protein(N)of feline infectious peritonitis virus(FIPV)strain SH2021 in vitro and prepare rabbit polyclonal antibodies.Primers were designed based on the N gene sequence of FIPV strain SH2021 to construct the recombinant expression plasmid pCold-I-N.Subsequently,the recombinant plasmid was transformed into expression-competent cells BL21(DE3),and expression was induced under conditions of 1.0 mmol·L^(-1)IPTG and 16℃.Finally,purification was performed using a His column,and the purified recombinant protein was used to immunize New Zealand white rabbits to prepare polyclonal antibodies.The experimental results showed that the purified recombinant N protein had a size of approximately 45 ku,and the titer of the prepared rabbit polyclonal antibodies against the N protein reached 1∶512000.The antibodies exhibited good reactivity and specificity towards the N protein.This study successfully prepared rabbit polyclonal antibodies against the FIPV N protein,which showed good reactivity and specificity,providing important tools for FIPV antigen-anti-body detection and research on the biological functions of the N protein.