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| 抗PIWIL2蛋白多克隆抗体的制备、鉴定及初步应用 |
| 朱风尚1,黄志刚1,胡莺2,张小燕2,陈锡美1,孟逊2,郜恒骏1,2* |
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| (1.同济大学附属同济医院消化内科,上海 200065*2.生物芯片上海国家工程研究中心,上海 201203) |
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| 摘要: |
| 目的:制备兔抗人PIWIL2(P-element-induced wimpy testis like 2,或称HIWI2)的多克隆抗体,并鉴定其特异性,初步探讨其在人正常及肿瘤组织中的分布。方法:人工合成特异性PIWIL2多肽,以马来酰胺活化匙孔血蓝蛋白(Keyhole limpet hemocyanin,KLH)为载体构建多肽免疫原,致敏大白兔,制备兔抗人PIWIL2多克隆抗体。亲和层析法纯化抗体,ELISA和Western印迹鉴定特异性,人组织芯片行PIWIL2的免疫组化研究。 结果:通过构建PIWIL2多肽-KLH载体复合物致敏大白兔,制备出兔抗人PIWIL2蛋白多克隆抗体。ELISA及Western印迹证实兔抗人PIWIL2抗体可特异性识别PIWIL2多肽。人组织芯片免疫组化染色显示,PIWIL2在肿瘤细胞的表达具有组织特异性,大多数正常和癌组织的平滑肌细胞胞质中呈阳性染色。 结论:成功制备出兔抗人PIWIL2多克隆抗体,为后续研究奠定了基础。 |
| 关键词: PIWIL2蛋白 多克隆抗体 组织芯片 |
| DOI：10.3724/SP.J.1008.2008.01034 |
| 投稿时间：2008-02-11修订日期：2008-07-10 |
| 基金项目: |
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| Preparation,identification and application of polyclonal antibodies against human PIWIL2 protein |
| ZHU Feng-shang1,HUANG Zhi-gang1,HU Ying2,ZHANG Xiao-yan2,CHEN Xi-mei1,MENG Xun2,GAO Heng-jun1,2* |
| (1. Department of Gastroenterology,Tongji Hospital,Tongji University,Shanghai 200065,China*2. National Engineering Center For BioChip in Shanghai,Shanghai 201203) |
| Abstract: |
| Objective:To prepare rabbit polyclonal antibody against human P-element-induced wimpy testis like 2 (HIWI2) protein,identify its properties and investigate its distribution in normal and tumor tissues by means of tissue chip. Methods: PIWIL2 peptide was synthesized chemically and conjugated to Keyhole limpet hemocyanin (KLH) as an immunogen. Then the PIWIL2-KLH conjugation was injected into rabbits subcutaneously to produce polyclonal antibodies. The specificity and sensitivity of antibodies were identified by ELISA and Western blotting after purification by affinity chromatography. PIWIL2 was then immuno-stained on the tissue chip to study its distribution in the normal and tumor tissues. Results: Rabbit’s antibodies against human PIWIL2 were prepared after the injection of PIWIL2-KLH conjugation subcutaneously. These antibodies were identified as PIWIL2 peptides by ELISA and Western blotting assay. PIWIL2 protein expression was tissue-specific in tumor tissues,with PIWIL2 protein found in the cytoplasm of smooth muscle cells of most normal and tumor tissues. Conclusion: The polyclonal antibodies against human PIWIL2 protein have been successfully prepared,which provides a basis for further study on the role of PIWIL2 in the pathway of miRNA/RNA. |
| Key words: PIWIL2 protein polyclonal antibody tissue chip |
