E-cadherin前体蛋白抗体的制备与鉴定  

Preparation and Identification of the Antibody Against the Precursor Protein of E-cadherin

作　　者：王继峰[1] 叶志云[1] 林舒勇[1] 

机构地区：[1]厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室,福建厦门361005

出　　处：《厦门大学学报（自然科学版）》2011年第1期97-100,共4页Journal of Xiamen University：Natural Science

基　　金：福建省自然科学基金资助项目(2008J0109)

摘　　要：E-cadherin是一种重要的肿瘤转移抑制因子,它的功能异常能够导致肿瘤细胞的转移.细胞内成熟的E-cadherin由其前体蛋白剪切而成,目前还没有针对该前体蛋白的特异性抗体.为了得到能用于检测内源性E-cadherin前体蛋白(E-cadherin pre)的抗体,对E-cadherin基因用限制性内切酶PstⅠ和PvuⅡ进行双酶切,获取E-cadherin基因的N-端片段,将其重组入原核表达载体pGEX-4T-2中,在大肠杆菌BL21(DE3)菌株中表达重组蛋白,经谷胱甘肽-琼脂糖珠子纯化后,得到较高纯度的E-cadherin前体抗原.用该抗原免疫新西兰兔,获得抗血清,进一步纯化血清得到抗E-cadherinpre的多克隆抗体.用Western blot检测了外源性和内源性E-cadherin pre,发现该抗体效价高且特异性强;免疫荧光实验表明该抗体可以用于细胞染色,为深入研究E-cadherin pre在细胞内的功能奠定了基础.E-cadherin,an important tumor suppressor which was defected highly correlates with tumor metastasis was derived from its precursor protein.As for the commercial unavailability,the precursor form of E-cadherin can not be detected at endogenous levels.In order to get qualified antibody agianst E-cadherin pre,Pst Ⅰ-Pvu Ⅱ fragment（amino acids 31 to 164） N-terminal in the precursor region of E-cadherin was ligated to pGST-parallel vector,expressed in the E.coli.strain BL-21.The fusion protein was purified by GST beads,and rabbits were immunized.9 weeks later,the sera of the rabbits were harvested and an anti-Pre-E-cadherin polyclonal antibody was purified.The precursor of E-cadherin was successfully detected with great sensitivity and specificity by western blotting using this anti-Pre-E-cadherin antibody in the lysate of 293T cells transfected with pCMV5-c-myc E-cadherin.Furthermore,the antibody lays a good foundation for further research on the function of E-cadherin pre,especially on its role in signal transduction pathways.

关 键 词：E-CADHERIN PRE 抗体 pGST E-CADHERIN PRE 

分 类 号：R392.11[医药卫生—免疫学]