| 摘要: |
| 【目的】克隆鸡纤维蛋白原样蛋白2（fibrinogen-like protein 2,FGL2）基因，并进行生物信息学分析和原核表达，为鸡FGL2蛋白的功能研究奠定基础。【方法】采用逆转录PCR（reverse transcription PCR,RT-PCR）技术扩增鸡FGL2基因，克隆至pMD19-T载体，测序后对鸡FGL2基因及其编码的蛋白进行生物信息学分析。构建原核表达质粒pET-32a-FGL2，将其转化BL21（DE3）大肠杆菌感受态细胞，利用IPTG进行诱导表达，对表达产物进行SDS-PAGE和Western-blot分析。【结果】成功克隆了鸡FGL2基因的CDS区，序列全长为1 320 bp，编码439个氨基酸。生物信息学分析显示，鸡FGL2氨基酸与火鸡、雉鸡、珍珠鸡、鹌鹑等禽类的同源性高，达96%以上；与哺乳类动物的同源性较低，其中与犬的同源性仅有64.8%。FGL2蛋白由439个氨基酸组成，理论分子质量为50.24 ku，分子式为C2 221H3 451N615O673S22，理论等电点（pI）为8.63，为不稳定的亲水性分泌型蛋白，无跨膜区。成功构建了原核表达质粒pET-32a-FGL2，其在大肠杆菌BL21（DE3）可表达以包涵体形式为主的FGL2融合蛋白，分子质量约为70 ku。【结论】成功克隆出了1 320 bp的鸡FGL2基因，明确了其编码蛋白的生物学信息，经诱导表达后获得FGL2重组蛋白。 |
| 关键词: 鸡 FGL2基因 生物信息学分析 原核表达 |
| DOI： |
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| 基金项目:国家自然科学基金项目(31702207) |
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| Cloning, analysis and prokaryotic expression of FGL2 gene in chicken |
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GUO Yage,LIU Jialong,QI Zhiying,et al
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| Abstract: |
| 【Objective】The fibrinogen-like protein (FGL2) gene in chicken was cloned and bioinformatics analysis and prokaryotic expression were carried out to lay a foundation for studying the function of FGL2 protein in chickens.【Method】Reverse transcription PCR (RT-PCR) technology was used to amplify the FGL2 gene in chickens,and it was cloned to pMD19-T vector.Bioinformatics analysis was conducted on the gene and encoded proteins.The prokaryotic expression plasmid pET-32a-FGL2 was constructed,converted into BL21(DE3) E. coli competent cells,and induced by IPTG.The expression products were then analyzed by SDS-PAGE and Western-blot methods.【Result】The CDS region of the FGL2 gene was successfully cloned,with a total sequence length of 1 320 bp encoding 439 amino acids.Bioinformatics analysis showed that the homology of chicken FGL2 amino acids with turkeys,pheasants,guinea fowl,quail and other birds was as high as more than 96%,while that with mammals was low,of which it was only 64.8% with dogs.FGL2 protein was composed of 439 amino acids,with theoretical molecular mass of 50.24 ku,molecular formula of C2 221H3 451N615O673S22 and theoretical isoelectric point (pI) of 8.63.It was an unstable hydrophilic secreted protein with no transmembrane region.The pET-32a-FGL2 plasmid was successfully constructed,and SDS-PAGE electrophoresis and Western-blot detection showed that the prokaryotic expression plasmid pET-32a-FGL2 could express FGL2 fusion protein in the form of inclusion bodies in Escherichia coli BL21 (DE3),with a size of about 70 ku.【Conclusion】The chicken FGL2 gene with size of 1 320 bp was successfully cloned,the bioinformatics of its encoding proteins was clarified,and the FGL2 recombinant protein with good immune response was obtained after induction expression. |
| Key words: chicken FGL2 gene bioinformatics analysis prokaryotic expression |
