The Stable Expression And Activity Detection Of Newcastle Disease Virus F Protein In 293T Cell Line

Newcastle disease(ND)is a highly contagious,acute and potent infectious avian viral disease caused by Newcastle disease virus(NDV),characterized by infections of the respiratory,enteric,nervous and reproductive systems,causing nervous impact on poultry industry because of its substantial morbidity and mortality.The present effective poultry disease control program demands professional vaccination regimen with strict biosecurity measures in the world,and this program will lasts long in the future.At present,commercial ND vaccines can provide a robust pvevention against ND,however most vaccines' production of these are based on the chick embryo,which can influence the bio-safety and environmental protection.Advances in recombinant DNA technology have made it possible to rapidly formulate subunit vaccines producted by mammalian cell lines,which has many advantages such as high security and easily mass production,is the main development direction of new animal vaccines.The F protein is the neutralizing and protective antigen of NDV which is highly related of the virulence of NDV,so it is always considered as the focus of ND subunit vaccine.Lentivirus vector is a eukaryotic expression system which can express foreign gene based on lentivirus,the vector has the same bio-characteristics as lentivirus that can insert genes into the host cell genome and it has been one of the most widely used eukaryotic expression systems.In this research,we established a F-293 T cell line expressing the F protein of NDV by lentivirus vector,providing the foundation for ND subunit vaccine which is a new route for ND vaccines.In order to establish the F-293 T cell lines that can stably express the F protein.Firstly,we constructed the transfer vector pHB-F carrying GFP and F gene of NDV LaSota strain,and transfected pHB-F?pX and pG into 293 T cells by liposome-mediated way.Secondly,we obtained the lentivirus by ultracentrifugation and then used the virus to infect the 293 T cells.Thirdly,we selected the appropriate F-293 T cells for subculture by means of gradual dilution to screen hig-purity cells.At last,we established the cell lines called F-293 T cells.The F gene was detected in different generations of cells by PCR,and the fluorescence intensity has no significant difference.Then,we detected the target band in SDS-PAGE and Western Blot using the medium supernatant of F-293 T cells.All the consequences indicate we successfully constructed the F-293 T cell lines that could express F protein steadily,and the protein has a good reactogenicty.we also detected the immunogenicity of the F protein expressed by F-293 T cells.Before detecting the immunogenicity,we need to concentrate the F protein and emulsify the protein with oil adjuvant to prepare the subunit vaccine.Then we vaccinated the chicken with the subunit vaccine,and collected the serum samples after 14 days and vaccinated them again.We collected the serum samples after 21 days and 35 days again.Chicken embryo neutralization experiment was performed to detect the immunogenicity,and the results showed the F protein expressed by F-293 T cells has a satisfying immunogenicity.All the consequences indicates that the F-293 T cell line could be used to produce the ND subunit vaccines.