Generation of ENO1 Single Chain Variable Fragment and Its Inhibitory effect on cervical cancer cells

Objective Enolase l (a-enolase, ENO1) is highly expressed in a variety of tumor cells, including cervical cancer. The eno1 monoclonal antibody could inhibit the invasion and migration of cervical cancer cells;however, it could not enter into cells. To overcome the limination, this study prepared ENO1 Single-Chain Variable Fragment (anti-ENO1 scFv) and validated its in vitro anti-cervical cancer cell effect to address the issue of antibody entry into cells. Methods Firstly, the nucleotide sequence of anti-ENO1 scFv was inserted into pET-30a prokaryotic expression vector by restriction enzyme digest sites (Nde I and HindIII); Then,the expression was induced by isopropyl β-D- thiogalactoside(IPTG) in E.coli BL21(DE3) cells. Second,Ni–NTA chromatography was used for the purification, and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Thirdly, the anti-cervical cancer effect of anti-ENO1 scFv in vitro was studied through cell proliferation assay, colony formation assay, wound healing assay, and Transwell assay. Results The recombinant plasmid pET30a-ENO1 scFv was successfully constructed. SDS-PAGE analysis showed that the expression of anti-ENO1 scFv could be located in the periplasmic space and extracellular space of BL21 and about 180 ug/mL purified anti-ENO1 scFv was obtained. Cell experiments showed that anti-ENO1 scFv could inhibit the activity of ENO1, significantly reduce the content of pyruvate and lactic acid, inhibit cell proliferation, invasion and migration, and clone formation of cervical cancer cells ( p <0.05). Conclusion The present study demonstrated that anti-ENO1 scFv can specifically block the expression of ENO1 on the cell membrane and inhibit the activity of ENO1 glycolytic enzyme in tumor cells, and it is expected to become a potential anti-tumor drug for cervical cancer.