| 摘要: |
| 【目的】 构建猪传染性胸膜肺炎放线杆菌（Actionobacillus pleuropneumoniae，App）鞭毛蛋白（flic）编码基因的重组表达质粒，对其编码蛋白进行生物信息学分析,并将其在原核细胞中进行表达。【方法】 利用PCR方法扩增flic基因片段，将其克隆至pMD-18T载体后测序，利用生物信息学软件对其编码蛋白的二级结构及跨膜区进行预测。构建重组表达质粒pGEX-flic，转化大肠杆菌工程菌BL21，经IPTG诱导表达后，通过SDS-PAGE及Western-blo对表达蛋白进行鉴定。【结果】 获得的目的基因片段长度为528 bp，测序表明其序列与GenBank公布的一致；软件预测结果显示，该蛋白具有多个明显的二级结构成分及跨膜区;SDS-PAGE检测表明，在约50 ku处有一特异性条带；Western-blot检测表明，表达蛋白具有良好的抗原活性。【结论】 成功获得了App 的flic基因，其编码的蛋白质具有较多的α螺旋区和无规则卷曲区域，表达的鞭毛蛋白具有生物学活性。 |
| 关键词: 猪传染性胸膜肺炎放线杆菌 flic基因 鞭毛蛋白 生物信息学 |
| DOI： |
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| 基金项目:河南省科技厅科技攻关项目（0524040001） |
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| Cloning expression and bioinformation analysis of flic gene of Actionobacillus pleuropneumoniae |
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| Abstract: |
| 【Objective】 The study cloned the flic gene of App and performed sequencing and analysis of biological information.【Method】 Polymerase chain reaction PCR was used to amplify the flic gene from.The secondary structure and membrane-spanning regions of flic protein was predicted by biosoftware.Then the flic gene was cloned into the E.coli expression vector pGEX-KG,which was induced with IPTG and the fusion protein was analyzed by SDS-PAGE and Western-blot.【Result】 The 528 bp fragment of flic gene was successfully cloned into vector,the pGEX-flic recombinant plasmid was expressed and induced by IPTG and Western-blot method showed good antigenicity of the recombinant protein,and there were flexible regions such as coil region and turn region in flic protein.【Conclusion】 A confirmed flic gene has been obtained and analyzed,providing a good foundation for research on motility and ontogenesis of App furtherly. |
| Key words: Actionobacillus pleuropneumoniae filc gene flagellin bio informatics |
