[抗人肝癌嵌合Fab抗体HAb18在毕赤酵母中的分泌表达]

[Secretory expression of chimeric Fab antibody HAb18 against human hepatocellular carcinoma in Pichia pastoris].

作者信息

Dong Hong-lin, Xing Jin-liang, An Jia-ze, Yang Xiang-min, Yao Xi-ying, Dou Ke-feng, Chen Zhi-nan

机构信息

Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2005 May;21(3):342-6.

PMID:15862155

Abstract

AIM

To express secretively chimeric Fab antibody HAb18 (cFab) against human hepatocellular carcinoma in Pichia pastoris.

METHODS

Genes encoding CL chain and Fd fragment of cFab antibody HAb18 were subcloned into vectors pPIC9K and pPICZalphaA, respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/CL and pPICZalphaA/Fd were transformed into the genome of Pichia pastoris GS115. Mut(+) multiple insert transformants were screened by G418 and Zeocin and then induced with 5 mL/L methanol to express cFab.

RESULTS

4 days after methanol induction, 26 mg/L of the cFab fragment was detected in the culture supernatant. Western blot proved that the expressed protein could specifically bind with HAb18GEF antigen.

CONCLUSION

The successful expression of cFab/HAb18 in Pichia pastoris lays the foundation for large-scale production and further application of the antibody.

摘要

目的

在毕赤酵母中分泌表达抗人肝细胞癌嵌合Fab抗体HAb18（cFab）。

方法

将编码cFab抗体HAb18轻链恒定区（CL）和重链可变区与恒定区（Fd）片段的基因分别亚克隆至载体pPIC9K和pPICZalphaA。经DNA序列分析确认后，将重组质粒pPIC9K/CL和pPICZalphaA/Fd转化至毕赤酵母GS115基因组中。通过G418和博来霉素筛选Mut(+)多拷贝插入转化子，然后用5 mL/L甲醇诱导表达cFab。