高尔基体蛋白73基因片段的克隆表达及抗重组蛋白单克隆抗体的制备

[Cloning and expressing of golgi protein73 gene fragment and preparation of monoclonal antibodies against the recombinant protein].

作者信息

Guo Jing-Xia, Wei Hong-Shan, Song Yong-Ji, Zhao Jing, Liu Ai-Xia, Liu Jia, Chen Lin, Xu Jun, Li Bo-An, Mao Yuan-Li

机构信息

Center of Clinical Laboratory, The No. 302 Hospital of The PLA, Beijing 100039, China.

出版信息

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2013 Aug;27(4):301-3.

PMID:24579482

Abstract

OBJECTIVE

To clone and express human Golgi glycoprotein73 protein, and prepare the monoclonal antibody (mAb) against the protein.

METHODS

GP73 gene was amplified from HepG2 cells by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-GP73 and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot was used to detect specificity of mAbs.

RESULTS

The prokaryotic plasmid expressing the recombinant protein was constructed, and the GP73 recombinant protein was expressed and purified. Five hybridoma cell lines that secreted anti-GP73 mAbs were obtained. 2 of 5 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with GP73 protein.

CONCLUSION

The GP73 recombinant protein is highly purified and has strong antigenicity. The anti-GP73 mAbs were prepared successfully.

摘要

目的

克隆并表达人高尔基体糖蛋白73（GP73），并制备抗该蛋白的单克隆抗体（mAb）。

方法

采用RT-PCR从肝癌细胞系HepG2中扩增GP73基因，将其与pQE31连接构建重组质粒pQE-GP73，转化至大肠杆菌BL21。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达的蛋白，用6×组氨酸标签进行纯化，并用于免疫BALB/c小鼠。通过细胞融合技术制备特异性单克隆抗体。采用蛋白质免疫印迹法（Western Blot）检测单克隆抗体的特异性。