| 摘要: |
| [目的]实现牛口蹄疫病毒AsiaⅠ型江苏分离株VP1基因在大肠杆菌中的表达,为VP1蛋白的深入研究奠定基础.[方法]根据GenBank公布的AsiaⅠ型口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)核苷酸序列(登录号:AF030244),设计并合成1对特异性引物,应用RT-PCR方法扩增出牛Asia Ⅰ型FMDV江苏分离株VP1基因,将其连接人pMD18-T载体,测序后克隆入原核表达载体pET-32a(+)中,构建原核表达载体pET-VP1。阳性重组质粒转化感受态大肠杆菌BL21(DE3),用IPTG进行诱导表达,收集表达产物并进行SDS-PAGE电泳,利用West-ern-blot分析表达蛋白的抗原性.[结果]牛AisaⅠ型FMDV VP1基因在大肠杆菌中获得高效表达,表达产物的相对分子质量约为43 ku,并能被牛AsiaⅠ型FMDV阳性血清识别,有一定的生物学活性,表达量约占菌体蛋白总量的37.7%.[结沦]牛AsiaⅠ型FMDV VP1基因表达成功,且表达产物具有一定的生物学活性. |
| 关键词: AsiaⅠ型口蹄疫病毒 VP1基因 原核表达 |
| DOI： |
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| 基金项目:江苏省农业三项工程计划项目（SX(2005)069） |
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| Cloning and Expression in E. coli for the VP1 Gene of Foot-and-Mouth Disease Virus Asia Ⅰ type isolated in Jiangsu Province |
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| Abstract: |
| 【Objective】 The study was done in order to express the VP1 gene of FMDV AsiaⅠtype isolated in Jiangsu Province in E.coli and for the future research of the VP1 protein. 【Method】 With a pair of specific primers designed according to the relevant nucleotide sequence of FMDV Asia Ⅰ Ind63/72 (NC004915) strain,the VP1 gene of FMDV AsiaⅠtype which was isolated in Jiangsu province was amplified with RT-PCR method.The PCR product was cloned into the pMD18-T vector and then sequenced.Following that it was cloned into plasmid pET-32a (+) to obtain a prokaryotic expression vector pET-VP1.【Result】 With the positive plasmid transformed into the host cell E.coli BL21 (DE3),the VP1 gene was successfully expressed with the induction of 1.0 mmol/L IPTG and the expression of the target protein accounted for 37.7% of E.coli total proteins.Western-blot analysis showed that the expression product had good immunogenicity.【Conclusion】 The VP1 gene of cattle AsiaⅠtype FMDV could be highly expressed in the E.coli BL21.The expression product weight was 43 ku,and Western-blot analysis showed that the expression product had immunogencity. |
| Key words: Foot-and-Mouth Disease Virus VP1 gene Prokaryotic expression |
