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Expression And Regulation Mechanism Of Chlorothalonil-specific Monoclonal Antibody Genes From Hybridoma Cell

Posted on:2021-12-28Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y LiuFull Text:PDF
GTID:1480306509999289Subject:Food Science
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At present,the pesticide residue is a prominent food safety problem and there is an urgent need to establish fast and reliable residue analysis methods.The immunoassay method occupies a huge market in the field of rapid diagnosis due to its high sensitivity,rapid detection,and easy operation.Hybridoma cells,as the main engineering cell for the preparation of monoclonal antibodies which are the core element of the immunoassay method,have the advantages of mature preparation technology,high antibody expression and high antibody sensitivity.This study explored the expression of pesticide-specific monoclonal antibodies and their regulatory mechanisms in hybridoma cells under in vitro culture conditions,and provided a theoretical basis for improving the stability of hybridoma cells and antibody expression.Research content and results:(1)Chlorothalonil-specific hybridoma cells were taken as the research objects,some of them did not secrete specific antibodies during the culture process.The “negative” cells had accelerated proliferation rate and the antibody light chain and/or heavy chain proteins were not secreted.It was found that the negative cells lost BJQ-specific antibody gene sequences or not lost them but the expression level of antibody was significantly down-regulated by comparing and analyzing the antibody genes and their transcriptional expression.(2)Transcriptome comparison analysis was performed between the positive cell line E2 and the negative cell line D4 with significantly reduced expression of BJQ-specific antibody gene in their logarithmic growth phase.It was found that oncogene Pet2 was up-regulated and the tumor suppressor gene Lrig1 was down-regulated in D4 cell line;RNAi revealed that the Vapa gene which was related to vesicle transport,the Prdm1 gene which was the key factor of plasma cell differentiation,and the Foxb1 gene which was the transcription factor significantly correlated with antibody expression.(3)The CRISPR library was used to perform genome-wide gene editing of chlorothalonil hybridoma cells.The CRISPR knockout cell library should be screened by pesticide pressure screening and FACS in the full growth cycle according to the increase and decrease of antibody expression.To analyze the enriched functional genes of the screened hybridoma cells: The main "throttle" genes that regulate the expression of antibodies in hybridoma cells were the tumor suppressor genes Hivep3?Lrig1,MAPK pathway genes Grap?Kras,transcriptional regulator Cebpb?Foxb1?Prdm1?Prdm2?Pax5,vesicle transport-related proteins Rab12?Vapa?Vapb;the main "brake" genes are the cancer-promoting genes Pet2?Rad51?Rad50?Wnt2?Relb;apoptosis-inhibiting genes Ucp2?Perp,genes that promote cycle progression cdc25b?Id1?Plk2?Cdkl2and cyclin kinase in the cell cycle process gene related to intercellular signaling Rabgef1,MAPK and its upstream receptor pathways genes Rgs4?Gapt?Ralb?Rasef,PI3k/Akt pathway gene Akt3.Among them,Foxb1?Prdm1 and Vapa genes were significantly positively correlated with antibody expression,which were verified by si RNA;Pet2 and Ccnd2 genes were significantly negatively correlated with antibody expression,which were verified by sh RNA.The cell cycle and antibody gene expression were regulated by the Erk/MAPK signaling pathway.(4)Transcriptome analysis of spleen cells from various stages immunizated by chlorothalonil.After stimulated by chlorothalonil pesticide antigen,the process of B cells activation,proliferation and differentiation into plasma cells were mainly through the BCR receptor cascading the Ras-Erk/MAPK signaling pathway,which initiates cell growth and antibody gene expression.That was consistent with the main pathway of hybridoma cells.(5)Antibody expression and regulation mechanism of hybridoma cells were summarized: hybridoma cells fuse B cells with the function of specific antibodies expression and the SP2/0 cells with the function of continuously proliferate and divide,both in vitro.These two functions were synchronized and restricted with each other.The cell growth and antibody expression were regulated through the Erk/MAPK pathway and related regulatory factors from its upstream and downstream.Inhibition of cell growth in the platform growth phase could promote the expression of antibody,and abnormal cell proliferation due to gene mutations could inhibit antibody expression.This study characterized the diversity of antibody genes and their expression from hybridoma cells,discussed the expression and regulation mechanism of chlorothalonilspecific monoclonal antibodies,and clarified the main key regulatory genes and signaling pathways.This mechanism provides a certain theoretical basis to improve the stability and the output of the specific antibody expressed by hybridoma cells in vitro culture systems.
Keywords/Search Tags:chlorothalonil, hybridoma cell, monoclonal antibody, CRISPR library, gene expression regulation
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