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吉布森巴贝虫顶膜抗原-1基因的表达及表达
产物的生物学活性
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杨俊1，周金林2，周望平1，玄学南2，肖兵南1，邱美珍1
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（1.湖南省畜牧兽医研究所，湖南 长沙410131；
2.带广畜产大学 国家原虫病研究中心，日本 带广080-8555）
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摘要：
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通过免疫筛选法，从构建的犬吉布森巴贝虫cDNA基因表达文库中筛选到1个cDNA克隆，此cDNA核苷酸全长2108bp，包含1个完整的开放阅读框，编码蛋白的分子质量约为62ku。经过分析，此蛋白（BgAMA-1）与其他巴贝虫属的顶膜抗原-1具有较高的同源性。为了研究其生物学活性，以此cDNA为模板，利用PCR扩增BgAMA-1的全基因，并将其克隆到pGEX-4T-3载体上，构建了原核表达载体pGEX-4T-3/BgAMA-1。通过Western-blotting对表达并纯化的重组蛋白的生物学活性进行分析。结果显示，构建的pGEX-4T-3/BgAMA-1能在大肠杆菌DH5α中表达，所获的重组抗原BgAMA-1在Western-blotting上仅与吉布森巴贝虫感染犬血清发生反应，证实其具有种特异性，在诊断方面可能有一定的应用价值。 |
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关键词：
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吉布森巴贝虫；顶膜抗原-1；原核表达 |
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中图分类号：S 852.723：Q 786
文献标识码：
A 文章编号：1673-4696(2008)02-0128-05 |
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Cloning and expression of Babesia gibsoni apical membrane antigen-1
gene and analysis of biological activity of the expressed
recombinant protein |
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YANG Jun1,ZHOU Jin-lin2,ZHOU
Wang-ping1，XUAN Xue-nan2,XIAO Bing-nan1，QIU
Mei-zhen1 |
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(1.Hunan Institute of Animal and Veterinary Sciences,Changsha 410131,China;2.National Research Center for
Protozoan Diseases,Obihiro University of Agriculture and Veterinary Medicine,Obihiro 080-8555,Japan) |
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Abstract: One cDNA clone was identified by immunoscreening from a cDNA expression library prepared from Babesia gibsoni merozoite mRNA and its complete nucleotide sequence was 2108bp in length,including one single open reading frame and encoding a 62ku protein which had high homology with that of apical membrane antigen-1(AMA-1) of other species.This protein was designated as B.gibsoni AMA-1(BgAMA-1).To analyze the biological activity of the recombinant BgAMA-1,the full-length BgAMA-1 gene was amplified from the cDNA clone by PCR and cloned into prokaryotic expression plasmid pGEX-4T-3 to construct expression vector pGEX-4T-3/BgAMA-1.The pGEX-4T-3/BgAMA-1 was expressed in Escherichia coli DH5α and the expressed product BgAMA-1 reacted only with the sera from dogs infected with B.gibsoni in Western-blotting test.The result revealed that the BgAMA-1 was a species-specific antigen and might have potential as reagent for the diagnosis of B.gibsoni infection. |
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Key words: Babesia gibsoni;apical membrane antigen-1;prokaryotic expression |
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