猪繁殖与呼吸综合征病毒Nsp2蛋白原核表达纯化及其蛋白酶活性分析

Expression and purification of Porcine Reproductive and Respiratory Syndrome Virus Nsp2 protein and analysis of cleavage activity

摘要:

摘要：【目的】表达并纯化猪繁殖与呼吸综合征病毒非结构蛋白2（Nsp2），分析Nsp2的蛋白酶活性。【方法】本研究通过PCR分别扩增nsp2基因的N端和C端，利用原核表达载体pET21a(+)表达Nsp2蛋白的N端和C端(即Nsp2-N 和 Nsp2-C)，通过Ni-NTA琼脂糖亲和层析和凝胶过滤的方法纯化两个重组蛋白。预测Nsp2-N含有半胱氨酸蛋白酶结构域，本研究利用western blot检测其顺式酶切蛋白酶活性；并人工合成潜在的十肽底物，利用体外多肽酶切实验检测其反式酶切蛋白酶活性。成功获得Nsp2

Abstract:

Abstract: [Objective] To express and purify Porcine Reproductive and Respiratory Syndrome Virus Nsp2 protein and analyze the protease activity of Nsp2. [Methods] N-terminus and C-terminus of nsp2 gene were amplified by PCR and inserted into expression vector pET21a(+), respectively. The recombinant protein (Nsp2-N and Nsp2-C) were over expressed in E.coli BL21 and purified by Ni-NTA agarose affinity chromatogram and gel filtration. There is a cysteine protease domain (CP) in Nsp2-N through genetic alignment. In this study, the protease activity of Nsp2-N in cis was analyzed by western blot. Additionally, the predicted peptide substrate were synthesized, the protease activity in trans was analyzed by peptide cleavage assay in vitro. [Results] Two soluble recombinant protein were successfully expressed and purity reached up to 90% after purification. The putative protease domain of Nsp2-N couldn’t cleave the predicted substrate through cis cleavage and trans cleavage. [Conclusion] The putative protease domain in Nsp2-N may need other host factors to act as cofactor, which supply basis for further identification of biological activity and screening of anti-virus drug.