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肠毒素性大肠杆菌K99-ST1融合基因的克隆
及原核表达
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李书光，管宇，肖跃强，王金良，沈志强*
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(山东省滨州畜牧兽医研究院 山东绿都生物科技有限公司，山东 滨州256600)
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摘要：
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为有效预防肠毒素性大肠杆菌(ETEC)引起的犊牛和羔羊腹泻，将PCR扩增获得的ETEC K99菌毛抗原基因和人工合成的ST1基因片段，借助pUCm-T载体，构建了重组质粒pUCm-K99-ST1，从而获得了融合基因K99-ST1；使用EcoRⅠ+SalⅠ双酶切将该融合基因定向插入表达载体pET-30a中，成功构建了表达载体pET-K99-ST1，将其转入表达菌株BL21(DE3)中，经IPTG诱导，获得31ku的蛋白； Western-blotting结果显示，该融合蛋白可与K99阳性血清发生特异性反应。 |
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关键词：
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大肠杆菌；K99-ST1融合基因；融合蛋白 |
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中图分类号：S 852.612：Q 786 文献标识码：
A 文章编号：1673-4696(2008)02-0111-05 |
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Cloning and prokaryotic expression of the fusion gene expressing
antigen K99 and ST1 of enterotoxigenic Escherichia coli |
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LI Shu-guang，GUAN Yu,XIAO Yue-qiang,WANG Jin-liang,SHEN Zhi-qiang
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(Shandong Binzhou Animal Science & Veterinary Medicine Academy/
Shandong Lüdu Biotechnology Company Limited,Binzhou 256600，China) |
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Abstract: To prevent calf and lamb diarrhea caused by enteotoxigenic Escherichia coli(ETEC),the K99 gene amplified by PCR and the synthesized nucleic acid fragment of ST1 were cocloned into pUCm-T vector to construct recombinant plasmid pUCm-K99-ST1,and then the fusion gene K99-ST1 was obtained.The fusion gene K99- ST1 was inserted into expression vector pET-30a to construct recombinant expression vector pET-K99- ST1.The pET-K99- ST1 was transformed into expression strain BL21(DE3) and induced with IPTG.A 31ku fusion protein was expressed from BL21(DE3,pET-K99-ST1).The fusion protein specifi-cally reacted with K99 positive sera in Western-blotting test. |
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Key words: Escherichia coli;K99-ST1 fusion gene;fusion protein |
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