| 莫超迪，覃呈欢，戴龙喜，韩鹏夫，纪月鑫，韦友传.黄喉拟水龟肺炎克雷伯菌外膜蛋白 A 的原核表达与纯化[J].广东农业科学,2023,50(3):137-143 |
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黄喉拟水龟肺炎克雷伯菌外膜蛋白 A 的原核表达与纯化 |
| Prokaryotic Expression and Purification of OmpA of Klebsiella pneumoniae Isolated from Mauremys mutica |
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| DOI：10.16768/j.issn.1004-874X.2023.03.016 |
| 中文关键词: 黄喉拟水龟 肺炎克雷伯菌 外膜蛋白 A 原核表达 重组蛋白 蛋白纯化 |
| 英文关键词: Mauremys mutica Klebsiella pneumoniae outer membrane protein A prokaryotic expression recombinant protein 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（20171286）；广西大学横向课题（20160284） |
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| 中文摘要: |
| 【目的】克隆黄喉拟水龟肺炎克雷伯菌外膜蛋白 A（KpOmpA）基因，构建重组质粒并诱导其表达，纯化获取重组蛋白 KpOmpA，为黄喉拟水龟抗肺炎克雷伯菌的相关疫苗研究奠定基础。【方法】以黄喉拟水龟肺炎克雷伯菌 DNA 为模板，克隆获得 KpOmpA 基因后与 pET-32a 表达载体连接；将构建的重组质粒 pET-32aKpOmpA 转化大肠杆菌 BL21（DE3）感受态细胞，经异丙基 -β-D- 硫代半乳糖苷（IPTG）诱导，通过 SDSPAGE 电泳检测 KpOmpA 重组蛋白的表达情况，并对重组蛋白进行可溶性分析；经 Ni-IDA 琼脂糖纯化树脂柱纯化 KpOmpA 重组蛋白，应用 Western blot 方法鉴定纯化蛋白。【结果】KpOmpA 基因 CDS 序列全长为 1 071 bp，推定编码 356 个氨基酸，与其他肺炎克雷伯菌的 OmpA 氨基酸序列同源性超过 99%，高度保守。重组质粒 pET-32a-KpOmpA 经双酶切、测序确认基因序列无移码或突变，表明重组质粒成功构建。转化后的 BL21 感受态细胞经终浓度为 1 mmol/L IPTG 在 37 ℃下诱导 4 h 后进行 SDS-PAGE 检测，结果表明，KpOmpA 重组蛋白大量表达，以包涵体形式存在，分子量约为 57 kD。经 Ni-IDA 琼脂糖纯化树脂柱纯化，获得较高纯度的重组蛋白。Western blot 检测结果显示有 57 kD 的条带，表明 KpOmpA 重组蛋白能被小鼠抗 His-tag 单克隆抗体识别。【结论】成功构建了重组质粒 pET-32a-KpOmpA，在大肠杆菌表达系统中诱导其表达，并纯化得到较高纯度的 KpOmpA 重组蛋白，为制备预防肺炎克雷伯菌感染的疫苗奠定基础。 |
| 英文摘要: |
| 【Objective】The study was conducted to clone the outer membrane protein A (KpOmpA) gene of Klebsiella pneumoniae isolated from Mauremys mutica, construct the recombinant plasmid and induce its expression, purify and obtain the recombinant protein KpOmpA, which would lay a foundation for preparing the vaccine to prevent K. pneumoniae infection. 【Method】Using the DNA as the template, KpOmpA gene was cloned and connected with pET-32a expression vector, and the constructed recombinant plasmid pET-32a-KpOmpA was transformed into the competent cells of E. coli BL21 (DE3). After induction by IPTG, the expression of recombinant protein KpOmpA was detected by using SDS-PAGE and the dissolubility was analyzed. Then the KpOmpA was purified by Ni-IDA agarose resin column. In addition, purified recombinant protein was identified by Western blot.【Result】The CDS sequence of KpOmpA was 1 071 bp in length with presumptive encoding 356 amino acids. The OmpA was highly conserved which shared more than 99% sequence homology with those from other K. pneumoniae strains. The pET-32a-KpOmpA was identified and confirmed by double enzyme digestion and sequencing. The sequencing result confirmed that there was no code shift or mutation, indicating that the recombinant plasmid was successfully constructed. The transformed competent cells of E.coli BL21 were induced by IPTG with the final concentration of 1 mmol/L at 37 ℃ for 4 h, SDS-PAGE assay showed that KpOmpA was expressed abundantly in the form of inclusion bodies, with a molecular weight of about 57 kD. The recombinant protein was purified and obtained by Ni-IDA agarose resin column. Western blot results show that there was a 57 kD band, indicating that recombinant protein KpOmpA could be recognized by His-tag mouse monoclonal antibody.【Conclusion】The recombinant plasmid pET-32a-KpOmpA was successfully constructed and expressed in E. coli expression system. The recombinant protein KpOmpA with high purity was obtained, laying a foundation for preparing the vaccine to prevent K. pneumoniae infection. |
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