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猪传染性胃肠炎病毒S基因抗原位点B与C之间
片段的克隆与原核表达
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张春叶1，孙英健1，沈红1，李焕荣1*，吴国娟1，于同泉2
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（1.北京农学院 动物科学技术系，北京102206；2.农业应用新技术北京市重点实验室，北京102206）
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摘要：
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将RT-PCR扩增的猪传染性胃肠炎病毒S基因抗原位点B与C之间的目的片段经克隆、双酶切后连接于表达载体，经酶切、PCR和测序鉴定的阳性重组质粒转化BL21(DE3)，用IPTG诱导重组菌株表达其融合蛋白。结果显示，目的片段的大小为357bp，序列分析表明，S基因与其他猪传染性胃肠炎病毒相应基因具有很高的同源性；Western-blotting检测表明，分子质量约34ku的融合蛋白具有良好的反应原性。 |
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关键词：
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猪；传染性胃肠炎病毒；S基因；抗原位点B；抗原位点C；克隆；原核表达 |
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中图分类号：S 852.659.6：Q 786 文献标识码：
A 文章编号：1673-4696(2008)06-0510-04 |
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Cloning and prokaryotic expression of the fragment between antigenic
sites B and C of S gene of transmissible gastroenteritis virus |
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ZHANG Chun-ye1,SUN Ying-jian1,SHEN
Hong1,LI Huan-rong1,WU Guo-juan1,YU
Tong-quan2 |
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（1.Department of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China;
2.Beijing Key Laboratory of New Technology of Agricultural Application,Beijing 102206,China） |
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Abstract:The fragment between antigenic sites B and C of S gene of swine transmissible gastroenteritis virus(TGEV) was amplified by RT-PCR and cloned into the prokaryotic expression vector after digestion.The positive recombinant plasmid,which was identified by enzymatic digestion,PCR and sequencing,was transformed into BL21 strain and the recombinant transformants were induced with IPTG.The prokaryotic expression and the immunologic specificity of the expressed protein were analyzed by SDS-PAGE and Western-blotting.The results showed that the target fragment was 357bp in length and it had a relatively high homology with the corresponding fragment from other TGEV strains in nucleotide and deduced amino acid sequences.Western-blotting analysis showed that the fusion protein of 34ku in molecular mass had good reactogenicity. |
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Key words: swine;TGEV;S gene;antigenic site B;antigenic site C;cloning;prokaryotic expression |
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