| 杨 萌1
，高爽爽 1
，谢业扬 1
，段家文 1
，陆专灵 2，韦友传 1.卵形鲳鲹 JAK3 蛋白的原核表达与纯化[J].广东农业科学,2020,47(1):137-142 |
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卵形鲳鲹 JAK3 蛋白的原核表达与纯化 |
| Prokaryotic Expression and Purification of JAK3 Protein in Golden Pompano (Trachinotus ovatus) |
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| DOI：10.16768/j.issn.1004-874X.2020.01.019 |
| 中文关键词: 卵形鲳鲹 JAK3 蛋白 原核表达 蛋白纯化 |
| 英文关键词: Trachinotus ovatus JAK3 protein prokaryotic expression protein purification |
| 基金项目:国家自然科学基金（31860736）；广西创新驱动发展专项（桂科 AA17204094-8） |
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| 中文摘要: |
| 【目的】纯化获取卵形鲳鲹（Trachinotus ovatus）JAK3（TroJAK3）重组蛋白，为了解TroJAK3蛋
白功能、相互作用及抗体制备奠定基础。【方法】应用分子生物学技术构建重组质粒pET-32a-TroJAK3，转化
大肠杆菌BL21感受态细胞，经IPTG诱导表达，SDS-PAGE和Western blot检测TroJAK3重组蛋白表达情况。【结
果】PCR扩增片段长度为3 333 bp，经双酶切鉴定、测序确认序列和开放阅读框正确，结果显示成功构建重组质
粒pET-32a-TroJAK3。经终浓度为1 mmol/L IPTG 37℃诱导4 h后进行SDS-PAGE检测，结果表明TroJAK3重组蛋
白以包涵体形式大量表达，分子量约为140 ku。经Ni-IDA树脂柱纯化，获得纯化的重组蛋白。Western blot检测
结果显示有140 ku的条带，表明TroJAK3重组蛋白能被抗His抗体识别。【结论】成功构建了重组质粒pET-32aTroJAK3，纯化得到高纯度的TroJAK3融合蛋白。 |
| 英文摘要: |
| 【Objective】The purpose of this study was to obtain purified recombinant protein of Trachinotus ovatus
JAK3 (TroJAK3)to lay a foundation for understanding the function, interaction and antibody preparation of TroJAK3 protein.
【Method】In this study, the recombinant plasmid pET-32a-TroJAK3 was constructed by molecular biology,the BL21
competent cells of E. coli were transformed, and. SDS-PAGE and Western blot were used to detect the expression of
TroJAK3 induced by IPTG.【Result】The PCR amplification fragment was 3 333 bp in length, and the recombinant plasmid
pET-32a-TroJAK3 was successfully constructed, which was confirmed by double enzyme digestion, nucleotide sequencing
and open reading frame. After being induced with a final concentration of 1 mmol/L IPTG at 37℃ for 4 h, SDS-PAGE assay
showed that TroJAK3 recombinant protein was highly expressed in a form of inclusion body, with a molecular weight of about
140 ku.The purified recombinant protein was obtained by Ni-IDA resin column. Western blot detection showed that here
was a band of 140 ku, indicating that TroJAK3 could be recognized by anti-his antibodies.【Conclusion】The recombinant
plasmid pET-32a-TroJAK3 was successfully constructed and purified to obtain TroJAK3 fusion protein with high purity. |
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