[抗戊型肝炎病毒中和单克隆抗体13D8单链抗体的构建与表达]

[Construction and expression of single-chain antibody of neutralizing monoclonal antibody 13D8 against hepatitis E virus].

作者信息

Li Li-feng, Luo Wen-xin, Gu Ying, Zhu Zi-heng, Wang Ying-bin, Zhang Jun, Xia Ning-shao

机构信息

The Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen University, Xiamen 361005, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Sep;20(5):556-9.

PMID:15367346

Abstract

AIM

To weaken the immunogenicity of the neutralizing monoclonal antibody (mAb) 13D8 against hepatitis E virus and express its scFv.

METHODS

The V(L) and V(H) genes were cloned by RT-PCR from hybridoma cells producing mouse mAb. And then V(H)-linker-V(L) fragment (scFv) was constructed and cloned into vector pTO-T7. The scFv protein was expressed in E.coli. The activity of expressed scFv was detected by ELISA and Western blot.

RESULTS

SDS-PAGE analysis showed that the scFv was highly expressed mostly in the form of inclusion body in E.coli, and the yield was up to 26.8% of the total bacteria protein. The results of indirect ELISA and Western blot showed that the expressed scFv could bind specifically to a recombinant protein in OFR2 region of HEV (NE2). The result of competitive ELISA demonstrated that the epitope recognized by the scFv was the same as that by mAb 13D8.

CONCLUSION

The scFv constructed from V(H) and V(L) genes of mAb 13D8 with immunological activity was successfully expressed.

摘要

目的

削弱戊型肝炎病毒中和单克隆抗体（mAb）13D8的免疫原性并表达其单链抗体片段（scFv）。

方法

通过逆转录聚合酶链反应（RT-PCR）从产生小鼠mAb的杂交瘤细胞中克隆轻链可变区（V(L)）和重链可变区（V(H)）基因。然后构建V(H)-连接肽-V(L)片段（scFv）并克隆到载体pTO-T7中。scFv蛋白在大肠杆菌中表达。通过酶联免疫吸附测定（ELISA）和蛋白质免疫印迹法（Western blot）检测表达的scFv的活性。

结果

十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，scFv在大肠杆菌中主要以包涵体形式高效表达，产量高达细菌总蛋白的26.8%。间接ELISA和Western blot结果表明，表达的scFv能与戊型肝炎病毒（HEV）OFR2区域的重组蛋白（NE2）特异性结合。竞争ELISA结果表明，scFv识别的表位与mAb 13D8相同。