花生主要变应原Ara h2的克隆及原核表达

被引：2

作者：

董慧

梁秉绍

黄艳梅

李飞

陈吟霜

周珍文

机构：

[1] 广州医科大学广州市妇女儿童医疗中心

来源：

现代生物医学进展 | 2017年 / 17卷 / 04期

基金：

广东省自然科学基金;

关键词：

花生; 变应原; Ara h2; 克隆; 表达;

D O I：

10.13241/j.cnki.pmb.2017.04.006

中图分类号：

Q943.2 [植物基因工程];

学科分类号：

071007 ; 090102 ;

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Eco RⅠ、HindⅢ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。

引用

页码：624 / 627

页数：4

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