伪狂犬病病毒gB抗原的可溶性原核表达及纯化条件的优化

Prokaryotic expression of pseudorabies virus gB gene and optimization of protein purification conditions

为实现伪狂犬病病毒gB蛋白的可溶性表达,基于伪狂犬病病毒gB基因的序列分析及密码子优化,基因合成gB基因胞外区（含全部抗原表位）序列,亚克隆至原核表达载体pSMK中,获得表达载体pSMKgB。经大肠杆菌诱导表达,并对诱导温度、可溶性、IPTG加入时机、终浓度及诱导时间进行优化。结果表明,伪狂犬病病毒的gB蛋白在16℃、D600值为1.2、IPTG终浓度为0.8mmol/L的条件下,诱导20h,蛋白表达效果最好。经超声破碎,镍柱纯化可获得可溶性的gB蛋白。蛋白印迹法分析结果表明,纯化的目的蛋白能与抗组氨酸标签的抗体及猪伪狂犬病病毒阳性血清发生特异性反应,证实表达的蛋白具有较好的反应原性。本研究为后期制备猪伪狂犬病血清检测ELISA试剂盒奠定了基础。

In order to study the immunogenicity of pseudorabies virus gB protein,we constructed the prokaryotic expression system containing gB gene,and named as recombinant plasmid pSMK-gB. It was transformed into Escherichia coli and induced to express target protein. The induction temperature and time,protein solubility,and final concentration of IPTG were optimized. The results showed that the pseudorabies virus gB protein was expressed at the highest level,when temperature was 16℃,theD600 value was up to 1.2,IPTG concentration was 0.8mmol/L,and termination of induction at 20 h. In addition, the expressed protein was soluble. After purification of gB protein using nickel column,Western-blot analysis was used to confirm the target protein with anti-histidine tag specific antibody as primary antibody. This study will provide the condition for development of the gB-based ELISA kit in the future.