抗人整合素αvβ3单链抗体片段的构建与表达

[Construction and expression of anti-human integrin alphavbeta3 scFv].

作者信息

Wang Chen, Hou Li-hua, Zhang Yan-ming, Li Jian-min, Liao Zhen-lin, Du Gui-xin, Chen Wei, Sun Qi-hong, Tong Yi-gang

机构信息

Institute of Microbiology & Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Mar;20(2):159-62.

PMID:15191715

Abstract

AIM

To construct single chain antibody (scFv) gene of mAb E10 against human integrin alphavbeta3.

METHODS

The VH and VL genes were amplified from hybridoma cells secreting mAb E10 by RT-PCR and connected with the use of linker (Gly4Ser)3 to assemble scFv gene. The scFv gene was cloned into prokaryotic expression vector pTIG-TRX and expressed in E. coli BL21 (DE3).

RESULTS

SDS-PAGE analysis showed the expressed recombinant protein with relative molecular mass (Mr) being 31,000. Western blot confirmed that the protein was labeled with His6. scFv protein was expressed as soluble protein under the condition of a small amount of IPTG induction and culture at lower temperature. The purity of the protein purified through Ni-NTA agarose metal affinity resin column was over 91%. The purified protein could bind to the human integrin alphavbeta3 by ELISA confirmation.

CONCLUSION

scFv against human integrin alphavbeta3 has been successfully constructed and expressed,which lays the foundation for further clinical research.

摘要

目的

构建抗人整合素αvβ3单克隆抗体E10的单链抗体（scFv）基因。

方法

通过RT-PCR从分泌单克隆抗体E10的杂交瘤细胞中扩增VH和VL基因，并使用接头（Gly4Ser）3连接以组装scFv基因。将scFv基因克隆到原核表达载体pTIG-TRX中，并在大肠杆菌BL21（DE3）中表达。

结果

SDS-PAGE分析显示表达的重组蛋白相对分子质量（Mr）为31,000。Western印迹证实该蛋白用His6标记。在少量IPTG诱导和较低温度培养条件下，scFv蛋白以可溶性蛋白形式表达。通过Ni-NTA琼脂糖金属亲和树脂柱纯化的蛋白纯度超过91%。经ELISA证实，纯化后的蛋白可与人整合素αvβ3结合。