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血管生成素样蛋白2纤维蛋白原样功能域多克隆抗体制备及分析 被引量：2

Preparation and analysis of polycolonal antibody against ANGPTL 2 fibrinogen like domain
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摘要 目的:克隆血管生成素样蛋白2(angiopoietin like protein2,ANGPTL2)cDNA;构建ANGPTL2纤维蛋白原样功能域原核表达载体;表达得到ANGPTL2纤维蛋白原样功能域融合蛋白;制备针对ANGPTL2纤维蛋白原样功能域的多克隆抗体,为全面分析研究ANGPTL2的功能及其与糖尿病肾病微血管病变的关系创造条件。方法:利用RT-PCR方法从肾脏总RNA中扩增ANGPTL2全长cDNA;以其为模板扩增编码ANGPTL2纤维蛋白原样功能域的cDNA片段,并进而将其克隆至原核表达载体pET-15b上,构建成ANGPTL2纤维蛋白原样功能域的原核表达载体pET-ANGPTL2;将pET-ANGPTL2导入BL21(DE3)菌内,通过IPTG诱导表达重组蛋白;利用重组蛋白5'端带有Histag的特点,采用含Ni的树脂进行亲和吸附纯化,获取高纯度的重组蛋白;将重组蛋白免疫新西兰大白兔,制备兔抗ANGPTL2多克隆抗体;利用ELISA法测定和免疫组化分析对此多抗的效价和特异性进行分析。结果:取得了带有完整阅读框的ANGPTL2全长cDNA;构建成与预先设计完全一致的原核表达载体pET-ANGPTL2;成功地在大肠杆菌中进行了ANGPTL2纤维蛋白原样功能域融合蛋白的表达,纯化获取纯度很高的重组蛋白;经免疫兔获取高效价的针对ANGPTL2纤维蛋白原样功能域的多克隆抗体;ELISA检测和免疫组化分析表明,此抗体不仅具有很高的效价,而且具有很好的特异性。结论:我们成功地克隆了ANGPTL2cDNA,构建了其原核表达载体,进行了原核表达,成功取得高纯度的重组蛋白,并得到了高效价、高特异性针对ANGPTL2纤维蛋白原样功能域的多克隆抗体,为全面分析研究ANGPTL2功能及其与糖尿病肾病微血管病变的关系创造条件。 Objective：To clone Angiopoietin like protein 2 cDNA and construct prokaryote expression vector pETANGPTL 2, and get recombinant ANGPTL 2 protein and antibody against ANGPTL 2 fibrinogen hike domain. Methodology：RT-PCR and conventional cloning methods were used to amplify ANGPTL 2 cDNA, and construct expression vector pET-ANGPTL2. After pET-ANGPTL2 was introduced into BL21 （DE3） bacteria, the expression of recombinant ANGPTL 2 protein was induced by IPTG. Then the protein was purified using TALON Metal Affinity resin, and analyzed by SDS-PAGE. The purified protein was used to immunize New Zealand rabbits. The antiserum against ANGPTL 2 fibrinogen like domain was obtained and then characterized by ELISA,and immunochemistry methods. Results： Angiopoietin like protein 2 cDNA was successfully cloned, prokaryote expression vector pET-ANGPTL 2 was constructed, recombinant ANGPTL 2 protein was obtained and ANGPTL 2 fibrinogen like domain antibody, which has high titer, affinity and specificity, was successfully prepared. Conclusion：ANGPTL 2 cDNA has been cloned, ANGPTL 2 recombinant protein has been obtained and high quality ANGPTL 2 antibody has been prepared, which was useful for further investigation of ANGPTL 2 function and its relationship with diabetic nephropathy.
作者 郑敬民 刘志红 孙骅 贾忠辉 陈丹 黎磊石
机构地区 南京军区南京总医院解放军肾脏病研究所
出处 《肾脏病与透析肾移植杂志》 CAS CSCD 2005年第5期418-422,共5页 Chinese Journal of Nephrology,Dialysis & Transplantation
基金 国家自然科学基金课题资助项目(NO:30470801) 江苏省自然基金课题资助项目(BK2005086)
关键词 血管生成素样蛋白2 多克隆抗体 糖尿病肾病 纤维蛋白原 多克隆抗体 血管生成素 抗体制备 功能域 原核表达载体 全长CDNA 免疫组化分析 蛋白2 angiopoietin like protein 2 multiclonal antibody diabetic nephropathy
分类号 R392 [医药卫生—免疫学]
  • 相关文献

参考文献15

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共引文献34

同被引文献32

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肾脏病与透析肾移植杂志
2005年 第5期

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