E-cadherin前体蛋白抗体的制备与鉴定

Abstract

[中文文摘]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. [英文文摘]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 o f E-cadherin can not be detected at endo genous levels.In order to get qualified antibody agianst E-cadher in pr e, Ps t I-Pv u II fragment (amino acids 31 to 164) N-terminal in the precursor region of E-cadher in 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 har vested and an anti-Pre-E-cadherin polyclonal antibody was purified. The precursor of E-cadher in was successfully detected with great sensit ivity and specificity by western blotting using t his anti- Pre-E-cadher in antibody in the lysate of 293T cells transfected with pCMV5-c-myc E-cadherin. Fur thermore, the antibody lays a good foundation for fur ther research on the function of E-cadherin pre, especially on its role in signal transduction pathways.