牛结节性皮肤病病毒P32蛋白的原核表达及多克隆抗体制备

Prokaryotic expression of lumpy skin disease virus P32 protein and preparation of polyclonal antibodies

为原核表达牛结节性皮肤病病毒(LSDV)P32蛋白并制备其多克隆抗体,本研究基于GenBank中LSDV-P32基因序列,使用多种生物信息学工具对P32蛋白的理化性质及生物学特性进行分析,选择富含B细胞表位的区段连接至pCold-TF载体,优化表达条件,重组蛋白纯化后利用SDS-PAGE和Western blot鉴定。将纯化后的P32蛋白免疫獭兔,制备多克隆抗体,利用ELISA、IFA和Western blot对多克隆抗体进行特性鉴定。结果显示:本研究利用原核表达系统成功表达了可溶性LSDV-P32蛋白,最佳表达条件为诱导温度15℃、IPTG终浓度0.1 mmol/L、摇床转速120 r/min、诱导时间30 h;制备的兔源多克隆抗体ELISA效价可达1∶409600,可应用于ELISA、Western blot和IFA试验。本研究为LSDV-P32蛋白功能的研究及相关检测试剂研发提供了一定技术支撑。

To express bovine Lumpy Skin Disease Virus(LSDV)P32 protein and the preparation of its polyclonal antibodies,the LSDV-P32 gene sequence in GenBank was downloaded,followed by bioinformatics analysis of the physicochemical and biological properties of the P32 protein and ligation of the B-cell epitope-rich region to the pCold-TF vector.After optimizing the expression conditions,purified recombinant protein was identified by SDS-PAGE and Western blot.Purified protein was used to immunize rex rabbits to prepare polyclonal antibodies against P32 protein,and properties of polyantibodies were identified by ELISA,indirect immunofluorescence assay(IFA)and Western blot.The results showed that the soluble LSDV-P32 protein was successfully expressed,and the optimal expression conditions were 120 r/min shaking for 30 h at 15℃with 0.1 mmol/L IPTG.The rabbit-derived polyclonal antibodies with a titer of 1∶409600 in ELISA,and can be used for ELISA,Western blot,and IFA.This study provides technical support for the functional study of LSDV-P32 protein and the development of related detection reagents in future.