| 董 慧,梁秉绍,黄艳梅,李 飞,陈吟霜,周珍文.花生主要变应原Ara h2的克隆及原核表达[J].现代生物医学进展英文版,2017,17(4):624-627. |
| 花生主要变应原Ara h2的克隆及原核表达 |
| Cloning and Prokaryotic Expression of Peanut Major Allergen Ara h2 |
| Received:May 04, 2016 Revised:May 26, 2016 |
| DOI：10.13241/j.cnki.pmb.2017.04.006 |
| 中文关键词: 花生 变应原 Ara h2 克隆 表达 |
| 英文关键词: Peanut Allergen Ara h2 Clone Expression |
| 基金项目:广东省自然科学基金项目（9151012001000009）；广东省科技厅一般项目（2014A020212013） |
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| 中文摘要: |
| 摘要 目的：在原核载体中克隆、表达花生主要变应原Ara h2，为其重组变应原应用研究奠定基础。方法：合成花生主要变应原Ara h2基因，设计特异性引物行PCR扩增，经EcoRⅠ、Hind Ⅲ双酶切后与做相应酶切的pET-28a载体连接，转化大肠杆菌BL21，提取质粒进行双酶切鉴定及测序分析，使用IPTG诱导融合蛋白表达，使用Ara h2特异性多克隆抗体对表达产物进行免疫印迹鉴定。结果：成功扩增了Ara h2基因，重组质粒双酶切见目的条带，基因测序显示Ara h2在正确开放阅读框中，基因长423bp，编码140个氨基酸，预测的等电点为5.3，分子量约16660.17 Da，基因比对分析显示其与相关报道的核苷酸序列一致性达100%。重组pET-28a-Ara h2/BL21经0.6 mmol/L IPTG诱导表达可见重组融合蛋白在相应分子量大量表达，使用Ara h2多克隆抗体免疫印迹法能检测到目的蛋白。结论：成功克隆、表达了花生主要变应原Ara h2，为其重组变应原应用研究奠定了基础。 |
| 英文摘要: |
| ABSTRACT Objective: To clone and express peanut major allergen Ara h2 in pET-28a expression plasmid, which make foundation for the recombinant peanut allergen application research. Methods: Peanut major allergen Ara h2 sequences were synthesized. The DNA fragment of Ara h2 was PCR amplified by designed specific primer, followed by EcoRⅠ, Hind Ⅲ digestion, and then ligated with pET-28a which was digested with the same enzymes. The recombinant plasmid was transformed into E.coli BL21 and identified by double enzyme digestion and sequence analysis. The expression of recombinant protein was induced by IPTG and confirmed by western-blot using Ara h2 specific multi-clone antibody. Results: Ara h2 gene with a length of 423bp was amplified, the recombinant plasmid was identified by double enzyme digestion. Sequences analysis showed that Ara h2 was in the correct open reading frame, and the predicted molecular weight is 16660.17 Da with 140 amino acids. The sequence comparison analysis showed that it was identical to Ara h2 gene of peanut allergen in GenBank. The recombinant pET-28a-Ara h2/BL21 was induced by 0.6 mmol/L IPTG, the objective recombinant protein was observed by SDS-PAGE and identified by Western-blot which using multi-clone antibody. Conclusion: Peanut major allergen Ara h2 was cloned and expressed in prokaryotic system, which laid a foundation for its' application research. |
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