猪繁殖与呼吸综合征病毒N蛋白的原核表达、纯化与结构分析

Purification and Structural Analysis of The Nucleocapsid of PRRS Virus Expressed from Escherichia coli

将猪繁殖与呼吸综合征病毒 (PRRSV)BJ 4毒株N基因克隆至原核表达载体pET2 8a中 ,得到重组表达载体pET2 8 N ,转化EscherichiacoliBL2 1(DE3)细胞 ,获得可溶性表达 ,表达量占菌体蛋白的 2 8%。经ProbandNi2 + 亲和层析获得重组蛋白P2 8 N ,圆二色谱 (CD)测定结果表明 ,P2 8 N重组蛋白螺旋占 2 6 1% ,折叠占 2 3 7% ,转角 19 8% ,卷曲占 30 3%。

The DNA fragment encoding the nucleocapsid protein (N) of PRRSV BJ4 strain were cloned into the BamHⅠ/EcoRⅠ 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%) α-helical structure, β-sheet (23.7%),β-turn (19.8%), and random coil (30.3%), respectively. Finally,the secondary stucture of N protein of PRRSV was deduced.