诺如病毒GII.6 P粒子原核表达与纯化

Prokaryotic Expression and Purification of Norovirus GII.6 P Particle

原核表达的诺如病毒(Noroviruses,NoV)衣壳蛋白亚基P粒子与No V有相同的抗原类型,可以代替NoV在体外进行结合,这对于研究该病毒与宿主和环境载体结合的相关机理有重大意义。本研究成功构建GII.6 P粒子基因表达载体,并对原核表达体系中诱导剂浓度、诱导温度及诱导时间进行优化。结果表明表达载体在22℃、2×10^(-4)mol/L IPTG诱导22 h后表达量最高。随后,在亲和层析的基础上结合阴离子交换以及凝胶过滤层析对表达产物进行纯化,最终获得高纯度GII.6 P粒子。

The P particles,subunits of Norovirus（No V） capsid protein,show the same antigen type as No Vs and are capable of work as an alternative in vitro binding study,which is of great significance to study the mechanism of the binding of No Vs with their hosts. Hence,in this study,the No V GII. 6 P gene was cloned into p GEX-4 T-1 plasmid,then the recombinant GII. 6 P was expressed simultaneously in different strains of E. coli,BL21（DE3）,Rosetta and Origami B for further optimization of the expression system. The results showed that the expression level of recombinant plasmid p GEX-4 T-1-GIL. 6 P after 22 ℃ and induced for 22 h at concentration of 2 × 10^（-4） mol/L IPTG reached the highest. Subsequently,the soluble expression level of BL21 strain was higher than that of other strains. Soon after,the GII.6 P particles were purified based on affinity chromatography combined with anion exchange and gel filtration chromatography,finally obtained high-purity of GII.6 P particles.