Study On The Infection Mechanism Of Infectious Bursal Disease Virus To Chicken Embryo Fibroblast

Infectious bursal disease virus(IBDV),a member of genus Avibirnavirus of the Birnaviridae family,causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry.Virus receptor plays an important role in virus binding and cell recognition.Viral attachment to a specific receptor on the surface of susceptible host cells is the first step in virus infection.To study the virus infection at the levels of virus binding,receptor identification is critical for understanding the virus-host cell interactions and pathogenesis of the viral disease.Chicken embryo fibroblast(CEF) cells,susceptible host cells of IBDV,commonly are used for the propagation of IBDV. Constructed a cDNA library of a virus susceptible host cell and used a monoclonal antibody(mAb) that can blocks virus infection to screen the library is commonly used in virus receptor identification research.To identify the IBDV receptor or the infection fator on the surface of CEF,we have done series of works as follows.First,a CEF cDNA expression library displayed on phage T7 was constructed via phage display technique,we also constructed a cDNA eukaryotic expression library of CEF and established a simple method to prepare high efficient electrocompetent cells of E.coli strain DH10B.Then on the basis of immunocytochemistry we set up a method in which the biotin-streptavidin systerm is applied.It can be used to screen the monoclonal antibody(mAb) that can blocks IBDV infection.With the mathod we selected three strains of mAb that can block IBDV infection.And we identified some charactors of the mAb.What we have done have settled the basis to clone the gene of IBDV receptor on the surface of CEF or to reveal the mechanism of IBDV infection.1.Construction and quality identification of chicken embryo fibroblast cells(CEF) cDNA expression library displayed on Phage T7To construct the CEF cell eDNA expression library displayed on phage T7 and to identify its quality,mRNA was prepared from CEF by oligo(dT)-cellulose affinity chromatography and reverse transcripted into cDNA.The CEF cell cDNA expression library was constructed after cloning cDNA into T7 EcoRI/Hindâ…¢vector arms and in Vitro packaging.Titer and recombinant rate of the prime library were determined.The size of the inserts was identified by PCR after a round of library amplification.The titer of the prime constructed library and amplified library was 2.2×10⁷ pfu/mL and 3.5×10¹⁰ pfu/mL.The inserts size largely ranged from 0.4 to 3kb with the average length of 1.3kb.The CEF cell cDNA expression library displayed on phage T7 has high quality.2.Construction and identification of a cDNA eukaryotic expression library of CEF.The mRNA was extracted from CEF and the cDNA was made by reverse transcription. The cDNA was ligated with the EcoR I adaptor and then digested with Not I.Fragments smaller than 500 bp were removed by a spin column.And then the large fragments were ligated into the eukaryotic expression plasmid pAP3neo predigested by EcoR I and Not I. Competent cells DH10B were transformed with the ligated product by electroporation.The size of the cDNA library and that of inserts were also identified through colony PCR.As a result a cDNA eukaryotic expression library of CEF was constructed.The total original bacterial colonies is 8.8×10⁵ in the library.The inserts of the recombinant plasmids larger than 1.0 kb is 85.7%.It indicates that the cDNA library is probably sufficient for cloning of low abundance cDNAs.3.Optimize the protocol of preparing high efficient electrocompetent cellsTo construct a cDNA library,a high efficient electro-competent cell is vital.We optimized several aspects which have a great influence on the transformative efficiency of DH10B electro -competent cell such as OD₆₀₀ value,the final resuspend volume and so on. At last we find out a good method to prepare the electro-competent cell DH10B with the high efficiency of up to 2～6×10⁹cfu/μgDNA.It is satisfied with the harsh requirement of constructing cDNA library,even in gene expression and clone under some special conditions.4.Establish a method to screen the mAb of anti-IBDV receptor on the CEF surfaceAn efficient method must be esbablished which is used for screenning the mAb that can block IBDV infection.In this study based on the method of immunocytochemistry,we employ the Biotin-Streptavidin system to establish an efficient method of the number of infection positive cell reduction test which is used for screenning the mAb that can block IBDV infection.It is a simple and sensitive and practical method for screenning the mAb of blocking virus infection.The method has solve the key problem of screenig the anti-virus receptor monoclonal antibody,which make it possible to reveal the IBDV receptor.It is of great important in methodology because this method can also be used to study to other virus receptor.5.Prepare the mAb of anti-IBDV receptor on the CEF surface and identify some charactors of the mAbThat to prepare the anti-virus receptor monoclonal antibody(mAb) is an efficient method to identify the virus receptor or to clone the receptor gene.Chicken embryo fibroblast(CEF) is a kind of the susceptible cell for infectious bursal disease virus(IBDV)and there are some molecular serves as receptor to mediate the virus infection. Four immunization program was adopted to immunize the BALB/c mouse.With the established mathod we selected three strains of mAb that can block IBDV infection.Some charactors of the mAb were identified.The infection inhibitive ratio of mAb is up to 84.5%. It also has the character of does dependent in infection inhibation.By flow cytometry we definated that the molecular that the mAb against is on the surface of CEF.It indicated that the molecular plays an important role in the course of IBDV infection.We speculate that it is one of the possible IBDV receptors or at least it is a relative element in IBDV infection. Because the mAb can not block IBDV infection completely,we speculate that another receptors or molculars also exsist that play an important role in IBDV infecion.It is consistant with what have been reported that there are at least three receptors existing on CEF.What we have done have settled the basis to reveal the mechanism of IBDV infection.