抗果蝇Trio蛋白抗体的制备及鉴定  

Preparation of anti-drosophila Trio antibody

作　　者：缪峰[1] 吕雯雯[1] 丁洁[1] 袁榴娣[1] 

机构地区：[1]东南大学发育与疾病相关基因教育部重点实验室,江苏南京210009

出　　处：《东南大学学报（医学版）》2008年第4期255-258,共4页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目(30670444)

摘　　要：目的:制备兔抗果蝇Trio蛋白的抗体。方法:以RT-PCR扩增Trio的1 173 bp的基因片段并克隆入pET28a(+)和pGEX-4T-1(His)6C表达载体中,表达并纯化Trio-His、Trio-GST融合蛋白。用纯化的Trio-His融合蛋白免疫家兔产生抗Trio的抗体,用Trio-GST亲和层析法进行纯化并采用Western blot法检测抗体生物学活性。结果:表达的Trio-His蛋白以包涵体的形式存在,用His亲和层析法分离纯化后得到较纯的Trio-His融合蛋白,以纯化的Trio-His免疫家兔制备的兔抗Trio的抗体,经Western blot分析显示该抗体可与果蝇S2细胞Trio特异性结合。结论:获得具有良好特异性的兔抗Trio抗体,为进一步研究Trio的功能奠定了基础。Objective To prepare the rabbit antibody against Trio protein. Methods Trio cDNA was amplified by RT-PCR, and then was subcloned into the fusion expression vectors pET28a（ ＋ ） and pGEX-4T-1 （His）6C. After being expressed in E. coli BL21, the truncated Trio protein was purifed and used to immunize rabbit. Purified antibody were obtained through affinity chromatography column with the expressed GST-Trio protein. The specificity of the puri- fied antibody was characterized by Western blot. Results The trio gene was successfully inserted into pET28a（ ＋ ） and pGEX-4T-1 （His）6C. After induction, the fusion protein was expressed in the form of inclusion body. The purified fusion protein was obtained by affinity chromatography. After immunizing rabbits, the antibody against Trio was obtained, showed that the antibody had a good specificity through Western blot analysis. Conclusion The rabbit antibody against Trio has been successfully prepared, which lays the foundation for further study of it＇s function.

关 键 词：Trio基因 原核表达 抗体制备 

分 类 号：Q786[生物学—分子生物学]