建立非放射性磷酸脂質水解□A2細胞表面分子接受器之檢測系統

Abstract

哺乳動物的第一類磷酸脂質水解□A2（Group I phospholipase A2，簡稱PLA2-I）透過其細胞表面分子接受器影響許多生理作用，例如：前列腺素合成、受精作用、細胞增生、平滑肌收縮和發炎反應等等。為了瞭解PLA2-I如何與其細胞表面分子接受器作用及作用詳細途徑，我們欲發展一簡便、安全、非放射性的偵測方式。利用生物素與卵白素之間能形成極強的共價鍵之特性，我們以基因重組方法設計了帶有生物素標記的融合蛋白質，以供卵白素進行辨識之用；我們選殖了豬胰臟的PLA2-I基因片段並將其與可做生物素修飾的生物□基載體蛋白（biotinyl-carboxylase carrier protein，簡稱BCCP）剪接形成融合蛋白質，所建構之融合蛋白質表現質體可在大腸桿菌BL21(DE3)中大量表現並形成包涵體；經嘗試數種培養液與培養溫度等條件後，在M9低限培養液、16℃低溫培養的條件下，我們可得到微量水溶性融合蛋白質，並經由SDS-PAGE電泳圖與西方墨點轉印法確認。經GST親和性管柱純化後，融合蛋白質分子量與預期相近，其純度約為50%，對應受質專一活性則較野生型PLA2-I 為高。而在利用卵白素進行辨識作用時，我們發現卵白素可成功辨識包涵體中的融合蛋白質但未能偵測到水溶性融合蛋白質。未來，我們將利用融合蛋白質與PLA2-I接受器之間的作用進一步偵測PLA2-I接受器在不同組織或細胞中之分布、含量與活性。

Mammalian pancreatic group I phospholipase A2 (pPLA2-I) has its specific receptor on a variety of mammalian cells and various biological responses, e.g. synthesis of prostaglandins, fertilization, cell proliferation, smooth muscle contraction and inflammatory response, are elicited by PLA2-I via its receptor. For understanding molecular mechanisms leading to these cellular responses mediated by PLA2-I receptor, we try to develop a new, handy, safe and non-radioactive detection system. Avidin-biotin interaction technique provides a simple and sensitive detection method and is widely used. We constructed a expression vector pGEX-5X/PB encoding porcine pPLA2-I cDNA fragment with a truncated-biotinyl-carboxylase carrier protein (BCCP) fusion tag. The fusion protein was over-expressed in Escherichia coli BL21(DE3) and formed insoluble inclusion bodies. To improve fusion protein solubility, various expression conditions of the fusion protein were tested. In M9 minimum medium and 16℃ culture, little amount of soluble fusion protein was detected by western blotting analysis. After purification of bacterial lysates using the affinity column, 50 percent purity fusion protein was collected. The activity assay suggests that the fusion protein is functional. Though the biotinylation detection of insoluble fusion protein was successful, purified fusion protein couldn’t be seen.