Study On Preparation Of HTNF-α Monoclonal Antibody And Construction And Expression Of Recombinant Gene Encoding Human-Mouse Chimeric Antibody

Rheumatoid arthritis(RA)is one of the chronic autoimmune diseases,which has become a serious threat to human health as its incidence rate is as high as 0.24%globally.The main syndromes of RA are cartilage and bone injuries.Although the definite pathogenesis of RA is unknown,it has been found that many factors are related to RA,including heredity,environment,immune abnormalities,and so on.Immune abnormality as a cause of RA is particularly attractive because of their close relationship.The imbalance among various mediators in the immune system causes immune abnormalities,which can lead to uncontrolled inflammation and cell damage.The cell damage in RA is reflected by the destruction of the synovium,cartilage and bone.In this process,tumor necrosis factor alpha(TNF-α)is particularly attractive due to its ability to lead to abnormal immune regulation and induce RA via its abnormal secretion.TNF-α is one of the cytokines which is widely distributed and highly conservative.The functions of TNF-a are achieved by stimulating many different kinds of cells.TNF-α inhibitors can block the biological activity of TNF-α by specific binding,which can produce a series of beneficial effects for RA,such as to control inflammation and ease the syndromes.Thus,as number of TNF-α inhibitors,such as infliximab,adalimumab and etanercept,have been developed as therapeutic drugs in clinical use.However,the high cost,emerging immune rejection and other issues have limited the application of these drugs.Therefore,new TNF-α inhibitors are highly desirable.In order to develop new TNF-a inhibitors,we have prepared several anti-TNF-α monoclonal antibodies via conventional technology of hybridoma and McAb by utilizing human TNF-a as the antigen to immunize Balb/c mice and SP2/0-Ag14 cell as the myeloma cell line.To reduce the immune rejection,anti-TNF-α McAb was humanized.This work has laid the foundation for subsequent development of effective TNF-α inhibitors.Firstly,hTNF-α was utilized to immune female Balb/c mice.Next,eight hybridoma cells which secrete monoclonal antibodies that can specifically bind to hTNF-α were obtained via conventional McAb technique.The desired hybridoma cells were characterized.Subtype analysis of McAb showed that all eight McAb were IgM kappa subtype.Three hybridoma cells were utilized to prepare ascites,from which monoclonal antibodies were purified by a protein L resin column employing its capacity to bind the immunoglobulin kappa light chains.The purified antibody was characterized by BCA to have the concentration of 2 mg/mL and by SDS-PAGE and Western blot to have high purity.In order to select a suitable hybridoma cell line for subsequent genetic engineering,the affinities of three purified monoclonal antibodies were tested by non-competitive ELISA,respectively.The Ka values of 2B9E10,3E4C11 and 4H5A10 were 1.6 × 108,7.698 × 108 and 5.9 × 107 M-1,respectively.2B9E10 and 3E4C11 were high affinity McAb.Thus,3E4C11 which has the highest affinity was selected to perform genetic engineering.Subsequently,the genes for the light and heavy chain variable regions of the 3E4C11 antibody were extracted with universal primers,and then linked with a sufficient flexible linker(Gly4Ser)3 by SOE PCR to form a single chain Fv antibody fragments(ScFv)which was linked to the human IgG1 Fc gene sequence.The resultant gene was constructed into eukaryotic expression vector which was transfected into 293T cell for expression.Western blot showed that the target protein was expressed in cytoplasm.In conclusion,this research described not only the preparation of anti-TNF-αantibodies but also genetic engineering of the anti-TNF-α antibody.This work lays the foundation for the development of humanized anti-TNF-α antibodies as TNF inhibitors for RA treatment.