The Production And Application Research Of Camel Anti Human TNF-? VHH Antibody

Aims:Tumour Necrosis Factor-?(TNF-?)is an inflammatory cytokine produced by macrophages,monocytes and other types of cells during acute inflammation.TNF-? have pleiotropic actions such as leading to necrosis or apoptosis,resistance to infection and cancers,repair of tissue damage and so on.But excessive expression of TNF-? is considered to be responsible for autoimmune disease such as rheumatoid arthritis,Crohn's disease,ulcerative colitis,ankylosing spondylitis,Psoriasis,scleroderma,and so on.So it is an effective target for the treatment of these diseases.Currently,five TNF-? inhibitors are available for clinical use.However,the application of monoclonal antibodies on a wide scale suffered from a number of technical limitations and drawbacks such as:expensive production,low tissue penetration,the potential immunogenicity of the mouse antibody.A more cost-effective production method or alterative anti-TNF-? therapy is,therefore,needed.As compared to antigen binding derivatives of conventional antibodies,the single domain nature of VHH gives rise to several unique features such as high expression yields and ease of purification,highly soluble and stable,high-affinity binders.This research aims to produce a TNF-?-specific nanobody that can effective neutralizes TNF-?,blocks the TNF-? signal path and suppresses excessive TNF-? induced autoimmune disease.Methods:?.Construction of anti-TNF-? VHH-T7 display library:1.The human TNF-? cDNA were synthesized and cloned into Pet28a expression vector.TNF-? protein were expressed in BL21(DE3)E.coli cells and purified using HisTrap columns.The activity of TNF-? protein was analyzed in L929 cellls.2.Camel HCAb were purified using Protein G and Protein A affinity chromatography.Mouse anti camel HCAb polyclonal antibody were generated by immuning mouse with camel HCAb.3.After immuning camel with TNF-?,total RNA isolated from peripheral blood lymphocytes was used as template for first strand cDNA synthesis using oligo(dT)primers.The cDNA was used as a template to amplify the VHH-encoding sequences by nest PCR.VHH-encoding sequences were cloned into T7 phage vector and obtain expression in-frame with the 10B protein such that recombinant fusion proteins are displayed.The phage display anti-TNF-?VHH library was constructed by package reaction in vitro and plate proliferation.Plaque assay and PCR-sequencing were used to evaluate the library.II.Screening the anti-TNF-a VHH-T7 Library by Biopanning:The Nanobodies against TNF-a were selected by phage display.The anti-TNF-a VHH expressing phages were selected through the affinity of TNF-a antigen.The positive clones expressing TNF-a-specific nanobodies were identified by ELISA.TNF-a specific VHH antibody were cloned into Pet28a expression vector and expressed in BL21(DE3)E.coli cells and purified using HisTrap columns.?.Characteristics of TNF-a-specific Nanobodies:Binding affinity and thermostability of TNF-a-specific nanobodies were analyzed by ELISA.A neutralization assay against TNF-a was performed in L929 cells.Results:1.The purity of TNF-a protein analyzed by SDS-PAGE is more than 90%.The LD50 of TNF-a is 3ng/ml in L929 cells.2,HCAb was successfully isolated from camel sera.The mouse anti-HCAbs polyclonal antibody was also obtained.3.The size of the library was 4×106pfu,which was determined by plaque assay.The correct insertion rate of the anti-TNF-? VIIH-T7 Library was nearly 98%by PCR analysis.The diversity of anti-TNF-? VHH-T7 Library is excellent by analysing VHH sequence.4.Three TNF-a-specific nanobodics were determined by ELISA.The binding affinity of these three TNF-a-specific nanobodics was listed as follows:TNF-a NB9>TNF-a NB7>TNF-a NB1.Nanobodies after 96h treatment with 37?,the binding activities tested by ELISA were still more than 80%.All of these three TNF-a nanobodies showed inhibitory activity aganist TNF-? in vitro.The LD50 of TNF-? NB9 is 20 nM;The LD50 of TNF-? NB1 is 100 nM;The LD50 of TNF-? NB7 is 450 nM.Conclusion:I.Binding affinity of TNF-a NB9 with TNF-? is the highest among these three TNF-a specific nanobodies.?.Thermostability of TNF-? NB 1,TNF-NB 7 and TNF-? NB 9 are all excellent.?.All of these three TNF-?-sp?cific nanobodies showed inhibition activity aganist TNF-a in vitro.Among them,the TNF-? NB9 was the best one.?.TNF-a NB9 is a high-yield,water-soluble,excellent-thermostability fragment with the highest inhibitory activity against TNF-a in vitro.We provide the TNF-a NB9 which was a novel antibody for the treatment of TNF-?-incduced inflammation at low cost.