The Construction And Expression Of Infectious Bursal Disease VP2 Gene Transfer Vector In 293t Cells

Infectious bursal disease(IBD)is an acute and highly contagious disease in young chickens caused by IBD virus(IBDV).IBDV is found worldwide and has been an economically significant disease in the production of poultry since it was first recognized in 1962.The major choice for controlling IBD currently is vaccination which can provide chickens with active or passive immune protection,and that is why developing vaccines is critical to controlling IBD.Though there are some high quality vaccines available in many countries,the antigenicity of commercial vaccines still has no good match with epidemic strain because of the variation of antigen and the arise of vvIBDV.Vaccination failure always happens in the clinical conditions,so we need urgent advancement in vaccine to keep pace with the evolution of IBDV so that we can control IBD more efficiently.Molecular biology and recombinant DNA technique have made it possible to rapidly formulate subunit vaccines,which is the main development direction on IBDV.Compared with inactivated vaccine,subunit vaccine has a low production cost,a quick good match with epidemic strain and a relatively low risk.VP2 is main host protective antigen of IBDV which can induce neutralizing antibodies and it is always considered as the focus of research because of its favourable immunogenicity and stability.VP2 protein expressed in eukaryotic expression system can be modified correctly and so it may offer a much better immunogenicity.In addition,there is still no available such subunit vaccines in the market.Firstly,we constructed the eukaryotic expression vector by inserting the VP2 gene of IBDV HB96 strain into the pCAGGs vector,and transfected the recombinant plasmid into the 293 T cells using the high efficient transfection reagent.We use indirect immunofluorescent detection to identify the expression of the VP2 and prove the successful transient expression.Secondly,We constructed a transfer vector encoding GFP and VP2 by inserting the VP2 gene into the lentiviral vector pHB carrying GFP gene,and transfected pHB-VP2 into 293 T cells with pX and pG by liposome-mediated.During this period,we grouped for the best proportion for gene expression and selected the appropriate 293 T cells for subculture by means of gradual dilution to screen high-purity cells.The cells were selected by the fluorescence signal and preserved to expand culture.At last,we obtain a monoclonal cell named as 293T-VP2.The VP2 gene was detected in different generations of cells by PCR,and the fluorescence intensity has no significant difference.Therefore we successfully constructed the 293 T cells that could express VP2 gene steadily by lentivirus packaging system.In this study,We constructed a transient expression system expressing VP2,and obtain a 293T-VP2 cell stably expressing IBDV-VP2.This study laid the foundation for the further of the structure and function of VP2,IBD subunit vaccine and DNA vaccine.