Construction Of Recombinant Eukaryotic Expression Vector Of Anti-IL-33 Human ScFv-Fc And Selection Of High-expression Cell Stains

Interleukin-33(IL-33)is a new member of the IL-1 family and the ligand of orphan ST2 molecules.IL-33 is widely expressed in multiple tissues and cells,and mainly involved in regulating Th2 immune and inflammatory response.It has obvious correlation with many allergic and autoimmune diseases.Some researchers have confirmed that treating with anti-IL-33 murine antibody or inhibiting IL-33 signaling pathways could relieve the symptoms of allergic rhinitis and rheumatoid arthritis.It indicates that IL-33 is a potential target for treatment of allergic and autoimmune diseases.Antibody drugs have undergone four phases which are murine antibody,human-mouse chimeric antibody,humanized antibodies and fully human antibody.Because of the adverse reactions of human anti mouse antibody,therapeutic antibodies have developed toward the humanized antibody and the fully human antibody.To connect single chain antibody fragments(scFv)and IgG Fc fragment by gene recombi-nation technology,the expressed fusion protein can combine with the specific antigen,and has a variety of function of Fc,such as CDC,ADCC effect,and longer serum half-life.So far fully human anti IL-33 scFv-Fc fusion antibody has not been reported.Objective:To construct anti-human IL-33 scFv-Fc recombinant vectors,express the activated scFv-Fc in Chinese hamster ovary cells(CHO)and select high-expression cell lines.Method:1.Construction of pcDNA/SP-scFv-Fc vector.? The IgG1 Fc was amplified and inserted into eukaryotic expression plasmid pcDNA3.1 to construct recombinant vector pcDNA3.1/Fc.? The signal peptide(SP)was synthesized and inserted into plasmid pcDNA3.1/Fc to construct recombinant vector pcDNA3.1/SP-Fc.? The human anti-IL33 scFv which was selected in our previous work was inserted into plasmid pcDNA3.1/SP-Fc to construct recombinant vector pcDNA3.1/SP-scFv-Fc.2.Construction of PMH3 EN/SP-scFv-Fc vector.SP-scFv-Fc fragment was cut from recombinant vector pcDNA3.1/SP-scFv-Fc and was inserted into plasmid PMH3 EN to construct recombinant vector PMH3 EN/SP-scFv-Fc.3.Transfection of CHO cells and selection of high-expressing cell lines.? cultivation of CHO cells and selection of suitable concentration of G418.? The CHO cells were transfected by the two kinds of recombinant plasmids with correct inserted SP-scFv-Fc sequences.To identify the level of transcription and translation of target SP-scFv-Fc by RT-PCR and Western blot.? selection of high-expre-ssion of cell lines,and identification of the scFv-Fc fusion proteins.Results:1.The sequencing results showed that the recombinant vector pcDNA3.1/SP-scFv-Fc and PMH3 EN/SP-scFv-Fc were succe-ssfully constructed and the size of inserted SP-scFv-Fc was about 1560 bp.2.The two sets of constructed scFv-Fc recombinant vetctors were transfected into CHO cells.After selection with G418,the total RNA of the transfected cells and non-tansfected cells was extracted and the RT-PCR was runed to detect the transcriptional level.The results showed that the SP-scFv-Fc was successfully transcripted in CHO cells.3.The transfeted cells were lysised and the western blot results showed that the single bands were showed with the size of 67 kDa in the two sets of transfected cells.The total expression level of scFv-Fc in recombinant vector PMH3 EN/SP-scFv-Fc was higher than that in vector pcDNA3.1/SP-scFv-Fc.4.The stable transfection cells were cultured in serum-free cell medium and the level of expressed scFv-Fc in cultural supernatant were detected by western blot.The results indicated that the soluble fusion scFv-Fc with bioactivity was secretory expressed stably in PMH3 EN/SP-scFv-Fc vector,but no band was showed in pcDNA3.1/SP-scFv-Fc vector because of the low level of expression.5.The stably expressed cell lines were cultured in soft agar plates and single cell strains were picked to expression.The results showed that the expression levels were obviously different in different cell stains and obviously higher in PMH3 EN vector than that in pcDNA3.1.Conclusion:1.The recombinant expression vetors of pcDNA3.1/SP-scFv-Fc,PMH3 EN/SP-scFv-Fc were sucessuly constructed.2.The high-expression cell strains were successfully selected and the scFv-Fc were secreted into cell cultural supernatant and has the binding activity with antigen IL-33.3.The expression level was obviously higher in PMH3 EN/SP-scFv-Fc than that in pcDNA3.1/SP-scFv-Fc.