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猪囊尾蚴T24基因的克隆与表达
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李海龙1,2，景志忠1，姜悦平2，才学鹏1
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(1.中国农业科学院 兰州兽医研究所 家畜疫病病原生物学国家重点实验室 甘肃省动物寄生虫病重点
实验室，甘肃 兰州730046；2.石河子大学 动物科技学院，新疆 石河子832003)
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摘要：
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采用RT-PCR方法扩增猪囊尾蚴T24免疫原基因，将扩增产物与pGEM-T easy载体连接，重组质粒经PCR、酶切鉴定后进行测序；构建T24基因的pGEX-4T-1原核表达载体，经IPTG诱导表达后，进行SDS-PAGE、Western-blotting；用所表达的蛋白免疫小鼠，经ELISA检测血清抗体，验证其免疫原性。结果显示，所克隆的T24基因片段长716bp，含有1个678bp的开放阅读框，其编码226个氨基酸，与已报道的猪囊虫T24基因核苷酸序列同源性为100%；表达的融合蛋白大小为40ku，并能被猪囊虫阳性血清识别；免疫小鼠在免疫1周后即可检测到血清抗体，第30d达到较高水平，表明该融合蛋白具有较好的免疫原性。 |
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关键词：
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猪囊尾蚴；T24基因；克隆；表达；免疫原性 |
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中图分类号：S 852.734：Q 786
文献标识码：
A 文章编号：1673-4696(2006)07-0547-05 |
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Cloning and expression of T24 gene of Cysticercus cellulosae |
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LI Hai-long1,2,JING Zhi-zhong1,JIANG
Yue-ping2,CAI Xue-peng1 |
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(1.Key Laboratory of Animal Parasitology of Gansu Province/State Key Laboratory of Veterinary
Etiological Biology/Lanzhou Veterinary Research Institute，Chinese Academy of Agricultural Sciences,
Lanzhou 730046, China; 2. College of Animal Science and Technology, Shihezi University，Shihezi 832003, China) |
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Abstract: The T24 immunogen gene of Cysticercus cellulosae was amplified by RT-PCR, and ligated into pGEM-T easy vector.The recombinant plasmid was certified by PCR amplification, digestion with endonuclease and sequencing. A prokaryotic expression plasmid pGEX-T24 was constructed by inserting the T24 gene into pGEX-4T-1.The expression was induced by IPTG, the cultures were collected at different times, and the expressed protein was analyzed by SDS-PAGE and Western-blotting. The mice were immunized with the fusion protein, and antibody of the T24 protein was tested by ELISA. Results showed that the T24 cDNA was 716bp in length and had an ORF which was 678bp in length and encoded 226 amino acids. Homology of nucleotide sequence between the cloned T24 gene and the sequence from GenBank was 100%.The fusion protein of 40ku in size was recognized by positive serum of Cysticercus cellulosae and had strong immunogenicity.The serum antibody could be tested on day 7 post-immunization,and rea-ched peak on day 30 post-immunization,which showed that the fusion protein had strong immunogenicity. |
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Key words: Cysticercus cellulosae; T24 gene; cloning; expression; immunogenicity |
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