Construction, Expression And Bioactivity Detection Of Single Chain FV RP215

Objective: Tumor called“fist killer”has been threathening humanhealth and breast cancer is one of the most common cancer of the female.Nowadays the anti-tumor drugs are non-targeting drugs, which kill tumorcells and also do harm to normal human tissues and organs. RP215is amouse-anti-human monoclonal antibody, and its antigen CA215specifallyexpressed in almost tumor tissues and cells. Moreover RP215justspecifically bond tumorous IgG and found no effection with IgG secretedby normal B cells. So we suppose RP215should be one of the most idealanti-cancer antibody drugs. This experiment used genetic engineeringantibody technology to reshape RP215monoclonal antibody into smallantibody RP215-scFv by connecting its VH and VL via a linker anddertermine its bioactivity in breast cancer, aiming to lay the foundation onexploring new antibody targeting drug against breast caner.Methods: VH and VL genes were augmented from hybridoma cellOC-3-VGH, secreting RP215-mAb, by RT-PCR, and then were jointtogether by a flexible linker via splicing overlap extension PCR(SOE-PCR)to get the RP215-scFv gene. After verified by double restricted enzyme cutand DNA sequencing, it was cloned into pET32a(+) vector and then expressed in E.coli BL21(DE3). The supernant of IPTG-inducedrecombinant protein was purified by Ni-NTA to obtain the target protein.SDS-PAGE was used to assess its molecular weight and purification.Western blot, cell immunofluorescence (IF), ELISA and competitiveELISA were used to determine the immunoreactivity of RP215-scFv inbreast cancer cell. The flow cytometer were used to detect cell cycledistribution and the function of inducing apoptosis in breast cancer cell.Results:1. Double enzyme cut and DNA sequencing analysis showedRP215-scFv prokaryotic expression vector was successfully constructed,named pET32a(+)-RP215-scFv.2. SDS-PAGE analysis showed itspurification was88%approximately and Western blot showed itsmolecular weight was about44KD，consistent with theoretical value.3.Indirect immunofluorescence showed RP215-scFv could recognize thesuperior tumorous antigen CA215localized on breast cancer cell（sMCF7，MB468, MB231, BT549and SK-BR-3）.4. ELISA and competive ELISAshowed RP215-scFv have good immunoactivity specifically binding withCA215.5.Flow cytometry showed RP215-scFv could induce breast cancercells arrest at G0/G1period,but inducing apopotosis function withoutstatistically significant difference.Conclusion: Our results illustrated that RP215-scFv has excellentimmunoactivity and localized accurately on breast cancer cells inmembrane-bound form,which may serve as an effective diagnosis method and anticancer drugs for breast cancer in the future.