PEDV N蛋白的原核表达纯化及B细胞抗原表位预测分析

本研究旨在对猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)N基因进行克隆构建、表达与其B细胞抗原表位性质的预测。将PEDV的N基因进行PCR扩增,克隆到原核表达载体pET-28a(+)中,经酶切验证获得阳性克隆,将阳性克隆在表达菌E. coli BL21(DE3)中表达。通过对表达条件及纯化条件的优化,获得高纯度表达蛋白。利用生物信息同源模型化软件Swiss-Pdb Viewer建立PEDV-N蛋白的3D结构,并根据生物信息学软件DNA-Star预测PEDV-N蛋白的二级结构、抗原性、亲水性和表面可及性分析,综合分析预测其B细胞抗原表位。经预测PEDV-N蛋白氨基酸序列中存在13个B细胞优势抗原表位区域。研究结果将进一步为PEDV-N蛋白体外表达产物的应用及研制基因工程疫苗提供理论基础。

Prokaryotic expression and purification of PEDV N protein and analysis of B cell antigen epitope

The aim of this study was to clone and construct the porcine epidemic diarrhea virus (PEDV) N gene and to predict the epitope properties of its B cells. The N gene of PEDV was amplified by PCR and cloned into the prokaryotic expression vector pET-28a(+).The positive clone was obtained by enzyme digestion, and the positive clone was expressed in the expression strain E. coli BL21 (DE3). High purity expressed proteins were obtained by optimizing expression conditions and purification conditions. The 3D structure of PEDV-N protein was established by bioinformatics homology modeling software Swiss-Pdb Viewer and the secondary structure, antigenicity, hydrophilicity and surface probability of PEDV-N protein were predicted according to bioinformatics software DNA-Star, and the antigen epitopes of its B cells were predicted by comprehensive analysis. It is predicted that there were 13 B cell dominant epitopes in the amino acid sequence of PEDV-N protein. The research results will further provide a theoretical basis for the application of PEDV-N protein expression products in vitro and the development of genetically engineered vaccines.