[真核表达载体的构建及抗重组人碱性成纤维细胞生长因子嵌合抗体的表达]

[Construction of eukaryotic vector and expression of chimeric antibody against rh-bFGF].

作者信息

Xiang Jun-jian, Li Hong-qing, Deng Ning, Yang Hong-yu, Tong Yi-gang, Li Jian-min

机构信息

Lab. of Molecular Immunology and Antibody Engineering, College of Life Sciences and Technology, Jinan University, Guangzhou 510632, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Mar;20(2):163-7.

PMID:15191716

Abstract

AIM

To express a human-mouse chimeric antibody against rh-bFGF antigen in eukaryotic cells.

METHODS

The VL and VH genes were amplified and cloned from the hybridoma 1F11 secreting anti-rh-bFGF mouse monoclonal antibody (mAb) and the CL and CH genes were amplified and cloned from the plasmid pMDHC and pMDLC. They were inserted into eukaryotic expression vectors after sequencing. The recombinant plasmids were then transformed into CHO-dhfr- cells for expression. The humanization and antigen specificity of expressed products were identified by indirect ELISA. The relative molecular mass (Mr) of expressed products was determined by SDS-PAGE.

RESULTS

The cloned antibody genes were identified to be functional by sequencing. The chimeric antibody could be detected in the culture supernatant of transformed CHO cells. The humanization and specificity of the chimeric antibody to rh-bFGF were confirmed by ELISA.

CONCLUSION

The mouse-human chimeric antibody against rh-bFGF has been expressed successfully in eukaryotic cells, which lays the foundation for its further clinical research.

摘要

目的

在真核细胞中表达抗重组人碱性成纤维细胞生长因子（rh-bFGF）抗原的人-鼠嵌合抗体。

方法

从分泌抗rh-bFGF小鼠单克隆抗体（mAb）的杂交瘤1F11中扩增并克隆轻链可变区（VL）和重链可变区（VH）基因，从质粒pMDHC和pMDLC中扩增并克隆轻链恒定区（CL）和重链恒定区（CH）基因。测序后将它们插入真核表达载体。然后将重组质粒转化到中国仓鼠卵巢二氢叶酸还原酶缺陷型（CHO-dhfr-）细胞中进行表达。通过间接酶联免疫吸附测定（ELISA）鉴定表达产物的人源化和抗原特异性。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定表达产物的相对分子质量（Mr）。