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Study on the Role of SIgMλ Light Chain Playing in the Infection of IBDV to DT40 Cells

Author: YouLeiMing
Tutor: WangAiPing
School: Zhengzhou University
Course: Cell Biology
Keywords: Infectious bursal disease virus SIgMλ light chain VOPBA β-actin promoter Knockout vector
CLC: S858.31
Type: Master's thesis
Year: 2009
Downloads: 20
Quote: 0
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Abstract


Infectious bursal disease virus (infectious bursal disease virus, IBDV) is the causative pathogens cause infectious bursal disease (infectious bursal disease, IBD) , mainly against chick central immune organs - bursal B lymphocytes damage causing severe immunosuppression . B cells are the of IBDV most susceptible cells having a surface immunoglobulin M (surfaceimmunoglobulin M, SIgM) . lambda light chain is not only the main types of the B cells in the light chain , and participates in the formation of the SIgM not clear , but its role in the infection process of IBDV . Light chains in order to reveal SIgMλ IBDV infection the B lymphocyte DT40 process , before the chicken Bursal In this study, analysis of the sequence of the λ light chain of the B cell , and the λ light chain gene from the DT40 cell clone . Construction of prokaryotic expression vector pET28a-λ expression was induced in E.coli BL21 (DE3) , E. coli . Induced expression of the λ light chain proteins separated by SDS-PAGE transferred to PVDF (polyvinylidene fluoride) film , using a virus cover the western blot technique (Virus overlay protein blot assay, VOPBA) the combination of the identification of λ light chain and of IBDV . Build targeted DT40 cells SIgMλ light chain gene gene targeting vector into DT40 cells establish SIgMλ light chain missing cell model to further explore the lambda light chain , this study β-actin promoter cloned from chicken DT40 cells replace pCDNA3 .1 ( ) vector with the SV40 promoter, construct the β-actin promoter driven Neomycin resistance gene expression cassette . In the chromosomal localization after of SIgMλ light chain gene , cloning its gene seating on both sides of each of the flanking sequence of about 2kb and respectively inserted into both sides of the resistance expression cassette as homology arms build the targeting SIgMλ light chain gene targeting carrier and targeting vector PCR, enzyme digestion and sequencing . VOPBA results show λ light chain can be combined with different virulence IBDV strain - specific , lambda light chain in the DT40 cell IBDV infection plays a binding role of the virus , may be the receptor of IBDV infection . Targeting vector sequencing results show that properly to meet the requirements of the design of the vector sequence , targeted SIgMλ light chain gene targeting vector was successfully constructed , which transformed DT40 cells of establishing SIgMλ light chain gene knockout cell model to examine SIgMλ light chain IBDV laid the foundation infected DT40 cells .

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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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