Construction Of Ochratoxin A Recombinant Antibody And Its Expression In Mammalian Cells(Expi 293F)

Immunoassay technology plays an important role in ensuring food quality and safety.For the construction of most immunoassay systems,the preparation of monoclonal antibodies is the primary prerequisite and key raw material.In the current field of rapid detection of food safety,after obtaining hybridoma cells that can secrete specific antibodies,antibodies are mainly prepared in large quantities by animal ascites.However,this type of preparation is faced with virus contamination,animal welfare,and the risk of degradation and loss of antibody production capacity as the cell passage increases.In this study,the common food-borne mycotoxin ochratoxin A(OTA)was used as the research object.On the basis of the hybridoma cell lines of OTA monoclonal antibody obtained in the early stage of the laboratory,the serum-free in vitro culture hybridoma and its antibody preparation,OTA recombinant full-length antibody construction,OTA recombinant chimeric antibody construction and its expression in mammalian cell lines were studied in order to provide a variety of paths for the preparation of OTA antibodies.The main research results are as follows:1)The serum-free in vitro suspension culture of OTA hybridoma cells and the preparation of their antibodies were studied.The OTA hybridoma cells were domesticated in three commercial mediums(OPM,BAJ and YQ)with low serum(10%)by gradually reducing the serum content.The results showed that under the condition of OPM serum-free medium,the OTA hybridoma cells reached 106 cells/mL on the fourth day of suspension culture,which showed better suspension culture performance than the other two serum-free mediums.After 5 days of suspension culture,the OTA monoclonal antibody was obtained.The results of SDS-PAGE identification showed that the OTA monoclonal antibody was obtained.The OTA antibody prepared under serum-free culture conditions retained the complete IgG antibody structure.The results of indirect ELISA and indirect competitive ELISA showed that the titer of the antibody was 1:16000,the IC50 was 1.330±0.147 ng/mL,and the affinity constant Ka was 1.1 × 108 L/mo1,and retained the performance of specific binding to OTA antigen.2)The construction of OTA recombinant full-length antibody expression vector was studied.Through the extraction of total RNA,reverse transcription and degenerate PCR,the full-length genes of the heavy chain and light chain of OTA monoclonal antibody were successfully cloned and their amino acid sequences and basic structures were analyzed.The heavy chain gene sequence H was 1431 bp in size,encoding 477 amino acids.Two gene sequences(L1 and L2)were measured in the light chain.The size of L1 was 849 bp,encoding 283 amino acids.L2 was identified as an abnormal immunoglobulin chain gene,namely non-functional light chain.On this basis,pCDNA3.1 and pCDNA3.4 vectors were selected as the main skeleton,and the fulllength genes of OTA heavy chain and light chain were cloned into the above vectors respectively.Four expression vectors of OTA heavy and light chain antibodies were constructed,which were pCDNA3.1-OTA-H,pCDNA3.1-OTA-L,pCDNA3.4-OTA-H and pCDNA3.4-OTA-L,respectively.The results of colony PCR and DNA sequencing showed that the corresponding heavy and light chain antibody gene sequences in the constructed expression vectors were accurate and complete.3)The transient transfection expression of OTA recombinant full-length antibody was carried out based on mammalian cells.The constructed heavy and light chain vectors were co-transfected into Expi 293F cells and CHO-S cells at a mass ratio of 2:3,1:1,and 3:2,respectively.The cell culture medium was collected after transient transfection.SDS-PAGE results showed that the OTA recombinant full-length antibody based on pCDNA3.4 was effectively expressed in Expi 293F cells.The OTA recombinant full-length antibodies obtained by co-transfection of heavy and light chain vectors with mass ratios of 2:3,1:1,and 3:2 were named OTA-3.4-1,3.4-2,and 3.4-3,respectively.By ELISA assay,the above three OTA recombinant full-length antibodies OTA-3.4-1,3.4-2 and 3.4-3 all retained the specific binding ability to OTA,and had no cross-reaction with other my cotoxin DON,ZEN and AFB1 detection antigens.The IC50 were 1.085±0.106,2.278±0.204 and 5.336±0.182 ng/mL,respectively.The affinity constant of the recombinant full-length antibody OTA-3.4-3 with the highest sensitivity was 3.41 ×108 L/mol.4)The construction of OTA recombinant chimeric antibody expression was carried out.The heavy chain and light chain variable region genes of OTA antibody were cloned into the pFUSE vector,and the expression vectors of heavy and light chain fragments of OTA mouse-human chimeric antibody were constructed,which were pFUSE-OTA-H and pFUSE-OTA-L,respectively.The results of colony PCR and DNA sequencing showed that the corresponding heavy and light chain antibody gene sequences in the chimeric antibody expression vector were accurate and complete.On this basis,the constructed heavy and light chain vectors were co-transfected into Expi 293F cells at a mass ratio of 1:1,and the cell culture medium was collected after transient transfection.The results of SDS-PAGE showed that the recombinant chimeric antibody of OTA was effectively expressed in Expi 293F cells,and retained the specific binding ability to OTA antigen.There was no cross-reaction with other mycotoxins DON,ZEN,AFB1 detection antigens.The OTA indirect competitive ELISA based on recombinant chimeric antibody showed that its IC50 was 1.624±0.129 ng/mL.The affinity constant Ka was 1.807×108 L/mol.