抗HEV中和抗体13D8的单链抗体的构建及表达

目的 :降低HEV中和性单抗 (mAb) 13D8的鼠源性 ,表达其单链抗体 (scFv)。方法 :从分泌 13D8鼠mAb的杂交瘤细胞中 ,通过RT PCR克隆mAb的VL、VH 基因 ,并进一步组装成VH linker VL 型的scFv片段。将scFv片段克隆到pTO T7载体中 ,在大肠杆菌中进行表达。用ELISA、Westernblot检测scFv的活性。结果 :SDS PAGE表明 ,13D8的scFv在E .coli中得到高效表达 ,表达量达菌体总蛋白的 2 6 .8%左右 ,表达产物主要以包涵体的形式存在。间接ELISA和Westernblot检测表明 ,表达的 13D8的scFv能与HEVOFR2区中一段重组蛋白(NE2 )特异结合。竞争ELISA表明 ,scFv与原鼠mAb识别的为同一表位。结论 :成功地表达出具有免疫学活性的 13D8的scFv。

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

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.