学术
摘 要:
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。Western Blot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。[著者文摘]
文章出处:
《中国水产科学》-2008年1期 -106-112页
栏目信息:
分 类 号:
文献标识码:
A
文章编号:
1005-8737-（2008）01-0106-07
Cloning and expression of UDP N-acetylgalactosamine 4-epimerase gene from Aeromonas hydrophila and characteristics of the enzyme
GONG Xia, LU Cheng-ping, YAO Huo-chun （Key Laboratory of Animal Disease Diagnostic and Immunology, Ministry of Agriculture, Nanjing 210095, China）
Abstract:
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][著者文摘]
Key words:
Aeromonas hydrophila; UDP N-Acetylgalactosamine 4-Epimerase; cloning and expression; antigenicity analysis
基金资助:
江苏省攻关（三药）项目（BE2004609）.
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