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目的 :原核表达人肝再生磷酸酯酶 2 (PRL 2 )与谷胱甘肽S转移酶 (GST)和 6个串联组氨酸 (6×His)的融合蛋白 ,并制备GST PRL 2特异性鸡卵黄抗体。方法 :将人PRL 2cDNA的全长蛋白编码序列 ,克隆入两种原核表达载体pGEX 4T 2和pET2 1a中 ,在大肠杆菌BL2 1中诱导表达融合蛋白。用Glu tathioneSepharose 4B和Ni NTAagarose亲和柱分别纯化目的蛋白。以纯化的GST PRL 2融合蛋白免疫产蛋母鸡制备多克隆抗体 ,应用 6×His PRL 2对抗体进行亲和层析纯化 ,纯化产物用Westernblot进行分析。结果 :得到高表达量的融合蛋白 ,经亲和层析柱纯化后获得较高纯度的GST PRL 2和 6×HisPRL 2融合蛋白。以GST PRL 2融合蛋白免疫鸡得到抗PRL 2的多克隆抗体 ,Westernblot证实 ,经 6×HisPRL 2亲和层析纯化的抗体 ,能够识别 6×His PRL 2和GST PRL 2融合蛋白 ,但不同GST蛋白起反应 ,表明具有较高的特异性。结论 :利用原核表达的人PRL 2融合蛋白制备的抗PRL 2多克隆抗体具有较好的特异性 ,为研究PRL 2蛋白在细胞信号转导过程中的作用提供了重要的技术和材料保障
OBJECTIVE: To prokaryotic express the fusion protein of human liver regenerated phosphatase 2 (PRL 2), glutathione S transferase (GST) and 6 tandem histidine (6 × His), and to prepare GST PRL 2 specific chicken Yolk antibody. METHODS: The full-length coding sequence of human PRL 2 cDNA was cloned into two prokaryotic expression vectors, pGEX 4T 2 and pET2 1a, and the fusion protein was induced in E. coli BL21. Purify the target protein with Glu tathione Sepharose 4B and Ni NTA agarose affinity column. The polyclonal antibody was prepared by immunizing laying hen with purified GST PRL 2 fusion protein. The antibody was purified by affinity chromatography using 6 × His PRL 2, and the purified product was analyzed by Western blot. Results: The fusion protein with high expression level was obtained. The purified GST PRL 2 and 6 × HisPRL 2 fusion proteins were purified by affinity chromatography. The polyclonal antibody to PRL2 was obtained by immunizing chickens with the GST PRL2 fusion protein and Western blot confirmed that the 6xHisPRL2 affinity purified antibodies recognized the 6xHis PRL2 and GST PRL2 fusion proteins but different GST The protein reacts, indicating a high specificity. CONCLUSION: The anti-PRL 2 polyclonal antibody prepared by prokaryotic human PRL 2 fusion protein has good specificity and provides important technical and material support for the study on the role of PRL 2 protein in cell signal transduction