Studies On Chicken Oviduct Bioreactor Expressing HA Protein Of Avian Influenza Virus

Transgenic chicken bioreactor has become one of the hotspots in the field of biological science because its outstanding advantages such as short generation interval,high protein production and high glycosylation degree.Transgenes in birds has always lagged behind the advances made in mammals because of the special reproductive system structure of poultry,which limits the research process of transgenic chicken bioreactors.With the extensive application of lentivirus vectors and the development of chicken primordial germ cell culture technology,it has been reported that foreign protein is highly efficient and specific expression in poultry oviduct.Hemagglutinin(HA)protein is the most important protective antigen of avian influenza virus,and also has strong immunogenicity.The genetic engineering vaccine based on HA protein has good immune efficacy.Using EGFP as reporter gene,the eukaryotic expression vector containing the OV promoter was constructed and effective promoter was screened by analyzing the expression of GFP in the primary cell cultures of chicken oviduct and CHO cell,OV promoter of 1.1 kb was identified which is used for further experiments.A recombinant vector named p OV_{1.1 k}-HA to express HA protein of H5N1 subtype influenza virus was constructed.It was subsequently transfected into CHO cells,PCR and RT-PCR analysis of HA gene suggested that the vector could be delivered into CHO cells and then get them transcribed.The immunoreactivity and hemagglutination activity of HA protein were determined by Western blot and HA test.The4-week-old SPF chickens were vaccinated with purified HA protein and boosts were conducted with the same dosage after two weeks.The HI antibody level was 6.3log2 of three weeks after the boost.All chickens were challenged with 10⁶ EID50 of H5N1 viruses(A/Goose/Guangdong/1/96).The survival rate of all vaccinated chickens was 100%and that of control group was 0,also no detoxification in the vaccine group.It was indicated that complete protection was provided.Results show that the screened 1.1 kb OV promoter could effectively drive the expression of HA protein and the expression of HA protein immunizing SPF chicken provided complete protection against avian influenza virus attack.In order to establish the oviduct bioreactor expressing HA protein,we analyzed the expression activity of OV promoter in chicken oviduct transient expression model.Recombinant vector p OV_1.1k-HA was mixed with polyethylenimine and injected into wing vein of laying hens,the eggs were collected and egg whites were isolated from the eggs,the western blot of egg whites denoted 70 KD band,which reveal that HA has been expressed in egg white.The results of hemagglutination test showed that HA protein had hemagglutination activity.The results indicated that 1.1 kb OV promoter could effectively drive the expression of HA protein in the oviduct of laying hens,which lays the foundation for further obtaining transgenic chicken oviduct bioreactor.On the basis of the successful establishment of the transient expression model,the lentiviral vector that 1.1 kb OV promoter to drive HA gene expression was constructed.The lentivirus was packaged and the virus titer was 9.8×10⁷ Tu/m L.The standard of virus titer was determined and the lentivirus was injected into developing embryos(stage X)through the equatorial window method.219 fertilized eggs were injected,39 chickens were hatched and the hatch rate was 17.8%.14 G0 chickens were identified as positive for transgene by PCR,the positive rate of transgene was 35.8%.The results showed that transgenic chicken with integrated HA gene was successfully obtained by the method of gene transfer by lentivirus mediated.In this study,we screened 1.1 kb OV promoter and expressed HA protein at the cellular level.The purified HA protein provided 100%protection against avian influenza virus infection.On the basis of the successful establishment of the transient expression model,the transgenic chicken with integrated HA gene was obtained through the gene transfer technique mediated by lentivirus,which laid the foundation for the study of the expression of protective antigen and precious drug protein in the chicken oviduct bioreactor.