| Receptor tyrosine kinase-like orphan receptor 1（ROR1）is a group of transmembrane proteins belonging to the family of receptor tyrosine kinases（RTKs）.ROR1 plays a key role in embryonic muscle and skeletal development but is rarely expressed in most normal mature tissues.Study found that ROR1 is overexpressed in a variety of tumors,such as chronic lymphocytic leukemia,neuroblastoma,breast cancer,kidney cancer and ovarian cancer,and promotes tumor cell growth,enhances tumor cell invasion and metastasis ability.The incidence of ovarian cancer ranked third in the female reproductive system malignancy,second only to cervical cancer and endometrial cancer,but the mortality rate is highest in the first gynecological malignancies.The clinical features of ovarian cancer are occult onset,strong invasion and metastasis,rapid progress,and poor prognosis.The main clinical treatment program of ovarian cancer is cytoreductive surgery combined with chemotherapy.Although patients of ovarian cancer may initially well respond to the treatment program,mostly patients would relapse due to highly resistant.In addition,obvious side effects can not be ignored.This project uses genetic engineering technology to prepare and express chimeric anti-ROR1 Fab antibody,and to explore its biological characteristics.Functional assays were employed to study this antibody on the biological behavior of ovarian cancer in vitro.Objectives:1.To prepare and express chimeric anti-ROR1 Fab antibody using genetic engineering techniques.2.To explore the impacts of chimeric anti-ROR1 Fab antibody on the biological behavior of ovarian cancer in vitro.Methods:1.Construction and expresstion of the chimeric monoclonal anti-ROR1 Fab antibody Monoclonal antibody cell fusion technology was used to screen anti-ROR1 positive monoclonal cells.Total RNA was extracted using RNA extraction kit,and single-stranded cDNA was obtained by reverse transcription.Using this as a template,the variable heavy and light chain sequences（VL and VH）of the anti-ROR1 Fab antibody were amplified by PCR and then compared with the database.The primers are redesigned based on the correct nucleic acid sequence.Then,the heavy and light chain variable and constant regions were amplified by PCR,and the antibody light chain variable region（VL）attached with the light chain constant region（CL）,leaving the heavy chain variable region（VH）connected to the heavy chain constant region（CH1）,the light（L）and recombinant heavy（Fd）chains were obtained.After the restriction enzyme digestion,the light chain（L）,the recombinant heavy chain（Fd）and the expression vector pETDUET-1 were digested,respectively,and the purified products were collected to leave the light chain（L）and the recombinant heavy chain（Fd）connected with the expression vector pETDUET-1to construct a recombinant plasmid pETDUET-1-ROR1-cFab,respectively.The recombinant plasmid pETDUET-1-ROR1-cFab was transformed into E.coli BL21and induced by IPTG.The chimeric anti-ROR1 Fab antibody was purified by a protein purification system.2.Identification of chimeric monoclonal anti-ROR1 Fab antibody SDS-PAGE and Western blot were employed to test the expression of the anti-RORl antibody Fab.ELISA and Biacore X100 were used to identify the specifically bind and high affinity of anti-ROR1 Fab antibody.3.In vitro experiments of chimeric monoclonal anti-ROR1 Fab antibody Western blot,FACS and immunofluorescence were employed to detect the expression of ROR1 protein on A2780 and Iose386 cells.CCK-8,healing assay,Transwell and apoptosis experiments were used to identify anti-ROR1 Fab antibody on the biological behavior of ovarian cancer.Results:1.The recombinant prokaryotic expression vector pETDUET-1-ROR1-cFab was successfully constructed without any mutations and non-functional gene after sequencing.Then,pETDUET-1-ROR1-cFab was transformed into E.coli BL21,induced with IPTG at 16°C overnight,and purified by protein system.2.SDS-PAGE and Western blot indicated that the anti-ROR1 Fab antibody was obvious band approximately 27 kDa and mainly expressed in cell supernatant.ELISA showed that anti-ROR1 Fab antibody could specifically bind ROR1.Biacore X100illustrated that the affinity constant of anti-ROR1 Fab antibody was 3.233×10-8.3.Western blot,FACS and immunofluorescence declared that ROR1 is highly expressed in A2780 cells and low in Iose386 cells.CCK-8,healing assay,Transwell and apoptosis experiments explained that anti-ROR1 antibody Fab could inhibit A2780 cell proliferation and migration,as well as promoting its apoptosis at higher concentrations.Conclusion:In this experiment,the anti-ROR1 antibody Fab was successfully prepared and verified that the antibody can specifically bind to ROR1 with high affinity.In vitro experimental studies show that anti-ROR1 antibody Fab can inhibit the malignant biological behavior of ROR1-positive ovarian cancer A2780 cells.The monoclonal antibodies obtained by the monoclonal antibody cell fusion technology may be a promising candidate for the targeted therapy of ovarian cancer. |
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