[从大肠杆菌表达的猪繁殖与呼吸综合征病毒核衣壳的纯化及结构分析]

[Purification and structural analysis of the nucleocapsid of PRRS virus expressed from Escherichia coli].

作者信息

Huang Jin-Hai, Yang Han-Chun, Guo Xin, Chen Yan-Hong, Zha Zhen-Lin

机构信息

Key Laboratory of Preventive Veterinary Medicine of Ministry of Agriculture, College of Veterinary Medicine, China Agirictural University, Beijing 100094, China.

出版信息

Wei Sheng Wu Xue Bao. 2004 Dec;44(6):737-40.

PMID:16110950

Abstract

The DNA fragment encoding the nucleocapsid protein (N) of PRRSV BJ4 strain were cloned into the BamH I / EcoR I sites of pET28a vector to construct the expression plasmid pET28-N by designing special primers. The soluble protein (P28-N) were obtained by introducing the expression plasmid into E. coli BL21 (DE3) host cell, and the amount of recombinant protein reached to 28% of the total mass of bacterial protein. PET28-N were purified by nickel-affinity column of Proband resin. The circular dichroism (CD) analysis showed that the purified PET28-N shared a significant (26%) alpha-helical structure, beta-sheet (23.7%), beta-turn (19.8%), and random coil (30.3%), respectively. Finally,the secondary structure of N protein of PRRSV was deduced.

摘要

通过设计特异性引物，将编码猪繁殖与呼吸综合征病毒（PRRSV）BJ4株核衣壳蛋白（N）的DNA片段克隆到pET28a载体的BamH I / EcoR I位点，构建表达质粒pET28-N。将该表达质粒导入大肠杆菌BL21（DE3）宿主细胞，获得可溶性蛋白（P28-N），重组蛋白量达到细菌总蛋白质量的28%。用Proband树脂的镍亲和柱对PET28-N进行纯化。圆二色性（CD）分析表明，纯化后的PET28-N分别具有显著的（26%）α-螺旋结构、β-折叠（23.7%）、β-转角（19.8%）和无规卷曲（30.3%）。最后推导了PRRSV N蛋白的二级结构。