嗜水气单胞菌UDP-乙酰葡萄糖胺-4-差向异构酶基因的克隆表达及酶的性质

Cloning and expression of UDP N-acetylgalactosamine 4-epimerase gene from Aeromonas hydrophila and characteristics of the enzyme

通过水生动物源性嗜水气单胞菌(Aeromonas hydrophila,Ah)强毒株J-1株与弱毒株MR-1株抑制差减杂交得到gneJ差减片段,该片段与创伤弧菌(Vibrio vulnificus)和人源嗜水气单胞菌的UDP-乙酰葡萄糖胺-4差向异构酶基因有较高的同源性。扩增完整的gneJ基因,进行分布检测并将其克隆至质粒pET32a(+),在大肠杆菌(Escherichia coli)BL21中进行异源表达,SDS-PAGE结果显示,重组表达蛋白的分子量约59.7kD,与理论推测值基本相同。酶活性分析表明,该重组蛋白可将UDP-乙酰葡萄糖胺转化为UDP-乙酰半乳糖胺,确证gneJ基因编码蛋白为UDP-乙酰葡萄糖胺-4-差向异构酶。WesternBlot分析显示,嗜水气单胞菌J-1株胞外产物的兔抗血清可识别该重组蛋白,表明该蛋白与天然蛋白有相同的抗原性。以重组蛋白为免疫原,对小鼠进行动物保护实验,结果显示,该重组蛋白对小鼠有60%的保护率,证明UDP-乙酰葡萄糖胺-4差向异构酶可作为亚单位疫苗的侯选成分。

The gneJ gene was subtracted from SSH between high virulent strain J-1 and low virulent strain MR-1 of aquatic .4eromonas hydrophila strains. It was highly homologous to wbpP gene of Vibrio vulnificus and gne gene of Ah AH-3. Full length of gneJ gene was amplified by PCR, and its distribution was detected. Then it was cloned into plasmid pET32a （＋） and transformed into E.coli BL21, induced by IPTG and expressed. The results of SDS-PAGE showed that recombinant protein molecular weight was 59.7 kD. Enzyme activity assay showed that the recombinant protein was a UDP N-acetylgalactosamine 4-epimerase, which could catalyze the inter conversion of UDP-GlcNAc and UDP-GalNAc. So the gneJ gene was identified to encode a UDP N-acetylgalactosamine 4-epimerase. The result of recombinant protein Western Blot with Ah J-1 antiserum revealed that gneJ recombinant protein had similar antigenicity to savageness protein. The mice were immunized with the recombinant protein, then challenged by Ah J-1 strain （50LD50）, showing that the relative percentage survival （RPS） of recombinant protein to the immunized mice was 60%. The research suggests that gneJ gene commonly present in the pathogenic Ah and play an important role in pathogenesis. [Journal of Fishery Sciences of China, 2008, 15 （1） ： 106-112]