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Effects of Surface IgM Lamda Light Chain Gene Silencing on the Binding of IBDV to DT40 Cells

Author: LiPeiPei
Tutor: WangAiPing
School: Zhengzhou University
Course: Biological Engineering
Keywords: IBDV λlight chain of SIgM RNAi H1 promoter
CLC: S858.31
Type: Master's thesis
Year: 2011
Downloads: 17
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Abstract


Infectious Bursal Disease (IBD) is a contagious immunosuppressive disease caused by Infectious Bursal Disease virus (IBDV). Bursa of Fabricius is the target organ for IBDV, and the surface immunoglobulin M (SlgM)-positive B cells in the organ are liable to get infected. Recent study has proved thatλlight chain of SIgM is the primary binding site for the virus to attach host cells. However, the mechanism ofλlight chain of SIgM, which functions in infecting cells for IBDV, has not well described.In order to reveal the function ofλlight chain of SIgM in the infection of IBDV to cells, an expression vector pAnGHl-shCRL that interfered gene expression ofλlight chain of SIgM had been constructed by using RNAi method in this study. The vector was introduced into DF-1 and DT40 cells. The expression ofλ. light chain of SIgM was identified by flow cytometry (FCM) and RT-PCR. DT40 cells, which expressed gene sequence of pAnGHl-shCRL and then were infected IBDV, were tested by virus infection assay.To obtain RNAi vector that could specifically suppress the expression ofλlight chain of SIgM in cells, the gene sequence of SIgM was analyzed. SV40 promoter on the pCDNA3.1 (+) vector was replaced by chickenβ-actin promoter in order to drive anti-Neomycin gene expression. At the same time, a gene express frame involving enhancing green fluorescence protein (EGFP) and H1 promoter starting shRNA transcription were also added to the pCDNA3.1 (+) vector, respectively. The vector could not only be used to label and track positive cells, but screened stable cell line that was continuous transcription shRNA gene silence.FCM and RT-PCR results showed the binding fraction of DT40 cells to monoclonal antibody L1, which were introduced pAnGHl-shCRL vector, was obvious lower than control groups, the expression ofλlight chain of SIgM was inhibited and percentage of cells to bind IBDV was also reduced. Such results suggested that the expression ofλlight chain of SIgM was specifically disorder by expression vector pAnGHl-shCRL in DT40 cells. The cells were introduced into expression vector pAnGHl-shCRL and screened in anti-G418 medium. Monoclonal antibodies were chosen using chessboard method and then were cultured massively and were identified. The results indicated pAnGHl-shCRL vector could inhibit the expression ofλlight chain of SIgM all the time when the monoclonal antibodies were sub-cultured; revealing that RNAi of pAnGHl-shCRL vector we designed was effective.In this study, DT40 cells of specifically IBDV-binding gene silence were obtained through RNAi method. The result suggestedλlight chain of SIgM was an important site on DT 40 cells to bind IBDV and whether it expressed could also influence the binding effect of DT40 to IBDV, which provided theoretical basis to further study the molecular mechanism and infecting process of IBDV.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Chicken
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