猪链球菌国内分离株精氨酸脱亚氨酶的克隆表达及其活性分析

Cloning,expression and characterization analysis of the arginine deiminase of Streptococcus suis of China isolates

摘要:

根据GenBank上精氨酸脱亚氨酶(arginine deiminase,AD)序列AF546864,设计并合成一对引物,用PCR检测29株猪链球菌和7株马链球菌兽疫亚种的ad基因,发现29株猪链球菌均能检出此基因,而7株马链球菌兽疫亚种均未检出。扩增出的猪链球菌2型(SS2)强毒株HA9801的ad基因片段,定向克隆至pBAD/Myc-HisC严紧型质粒并转化TOP10。阳性重组菌经L-阿拉伯糖诱导,表达出分子量约47000Da的重组蛋白。经镍柱亲和层析,获得纯化的重组酶。活性分析显示,该酶具有巯基酶和金属酶的特征,其最适反应温度为37℃,最适pH值为6.5。Western blot分析显示,该酶能与SS2-HA9801全菌制备的兔抗血清发生特异性反应,表明其具有一定的免疫原性。检测该酶有助于进一步分析猪链球菌可能的毒力因子。

Abstract:

PCR analysis demonstrated the presence of the ad gene in all 29 S.suis strains tested, but none of the seven S.equi subsp. zooepidemicus strains. The fragment of ad gene of virulent isolate SS2-HA9801 was later cloned into pBAD/Myc-HisC vector via restriction endonuclease and then transformed into host strain TOP10. A recombinant protein of 47000Da was highly expressed after induced by L-ararose and purified by Ni-nitrilotriacetic acid affinity chromatography. Western blotting demonstrated that the recombinant protein can reacted to the polyclonal antibody raised against whole-cell protein of SS2-HA9801, which suggested that it possessed some immunogenicity and may be important for further research. Enzymatic assay revealed that the optimum temperature for its activity is 37℃ and pH is 6.5. Studies with class-specific inhibitors supported the assignment of a sulfhydryl enzyme with some metallo class characteristics.