抗PIWIL2蛋白多克隆抗体的制备、鉴定及初步应用

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.

Preparation,identification and application of polyclonal antibodies against human PIWIL2 protein

Objective: To prepare rabbit polyclonal antibodies against human PIWIL2 (HIWI2) protein, identify its properties and investigate the digestive tissue distribution of PIWIL2 using tissue chip. Methods: PIWIL2 peptide was synthesized using chemical method, and conjugated to Keyhole limpet hemocyanin (KLH) as an immunogen. And then PIWIL2-KLH conjugation was injected into rabbits subcutaneous to produce polyclonal antibodies. The specificity and sensitivity of antibodies were identified by ELISA and Western blot after purification using affinity chromatography. PIWIL2 was then immune stained on the tissue chip to study its distribution in digestive normal and tumor tissue. Results: Rabbit antibodies against human PIWIL2 were prepared after injection of PIWIL2-KLH conjugation. These antibodies were confirmed to specially recognize PIWIL2 peptides analyzed by ELISA and Western blot. PIWIL2 protein was stained positively in the cytoplasm of smooth muscle cell in the digestive normal and tumor tissue. Conclusion: The polyclonal antibodies against human PIWIL2 protein have been successfully prepared, which may be used as a marker in differential diagnosis from smooth muscle tumor and be a useful tool for further study in the pathway of miRNA/ RNA.