激光生物学报, 2018, 27 (2): 161, 网络出版: 2018-06-29   

利用DNA免疫技术制备GATA1蛋白多克隆抗体

Preparation of Polyclonal Antibody Against GATA1 Protein by DNA Immunization
作者单位
1 湖南师范大学蛋白质化学及鱼类发育生物学教育部重点实验室, 心脏发育研究中心, 湖南 长沙 410081
2 湖南师范大学第一附属医院, 湖南 长沙410005
摘要
转录因子GATA1对正常红系细胞的分化起着关键作用, 其缺失导致原成红细胞的凋亡, 其过表达则能抑制红系细胞晚期的分化。本研究为了进一步利用斑马鱼研究gata1基因在血液血管发育过程中的功能, 通过反转录PCR(RT-PCR)扩增了斑马鱼gata1基因编码区序列, 构建了真核表达质粒pCAGGS-P7/gata1, 采用质粒DNA免疫技术的方法免疫了BALB/c小鼠, 制备了斑马鱼GATA1蛋白多克隆抗体。实验结果表明: 构建的真核表达重组质粒pCAGGS-P7/gata1 DNA具有良好的免疫原性, 在免疫小鼠后所获得的抗血清抗体效价达1∶500。Western blot和免疫荧光检测表明, 抗血清能特异型地识别GATA1蛋白。斑马鱼GATA1抗体成功制备为进一步的功能研究奠定了重要基础。
Abstract
Transcription factor GATA1 plays a key role in the differentiation of normal erythroid cells, GATA1 deletion caused the apoptosis of primary erythrocytes, Overexpression of GATA1 prevents late differentiation of erythrocytes. This experiment is aimed at studying how to effect the development of blood vascular. In order to prepare GATAI polyclonal antibody, the study amplificated gata1 coding region of zebrafish by reverse transcription PCR, constructed pCAGGS-P7/gata1 eukaryotic expression plasmid and adopted DNA Immunization technique to immunize mice. Detection of target protein expression by Western blot and immunofluorescence technology. Experimental results showed that the eukaryotic recombinant expression plasmid pCAGGS-P7/gata1 was successfully constructed. The recombinant plasmid has good immunogenicity, Antiserum obtained after immunization of mice, Antibody titer up to 1∶500, Western blot and immunofluorescence confirmed that antiserum can specifically recognize GATA1 protein, A firm foundation for the further study of gata1 function.

郭芬, 曹灵慧, 陈宇霖, 欧柏青, 吴秀山. 利用DNA免疫技术制备GATA1蛋白多克隆抗体[J]. 激光生物学报, 2018, 27(2): 161. GUO Fen, CAO Linghui, CHEN Yulin, OU Baiqing, WU Xiushan. Preparation of Polyclonal Antibody Against GATA1 Protein by DNA Immunization[J]. Acta Laser Biology Sinica, 2018, 27(2): 161.

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