摘要
Early etiological diagnosis is very important for the control of sudden viral infections,and requires antibodies with both high sensitivity and high specificity.Traditional antibody preparation methods have limitations,such as a long and arduous cycle,complicated operation,and high expenses.A chicken lymphoma cell line,DT40,is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture.Here,the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro.Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase(AID)gene,AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected.In this study,we generated a novel AID-inducible DT40 cell line(DT40-H7),in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system,and an inducible AID gene,based on the Tet-Off expression system,was stably transfected.AID expression was controlled in DT40-H7 cells in a simple and efficient manner;gene conversion and point mutations were observed only when AID was expressed.Using the antibody library generated from this cell line,we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus.The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.
Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce Ig M-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase(AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line(DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus.The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.
基金
sponsored by the Project of the National Defense Science and Technology Innovation Special Zone (to HH, 17-163-12-ZT-005-013-01)
the CAMS Innovation Fund for Medical Sciences (to HH, CIFMS 2016-12 M-1-013 and to ZZ, CIFMS 2016-12 M-1-014 and 2016-12 M-3-020)
the National Key Research and Development Program (to ZZ, 2016YFD0500300)
the Fundamental Research Funds for the Central Universities (to HH, 2018PT31032)
