The Construction Of Recombinant Baculovirus Containing S And N Proteins Of Infectious Bronchitis Virus Strain GX-YL5

Infectious bronchitis(IB)is a highly contagious and infectious disease caused by infectious bronchitis virus(IBV)and results in huge economic losses to the world poultry industry.IBV genome is proned to mutations and recombination,resulting in multiple IBV serotypes and variants.The attenuated and inactivated vaccines commonly used for the prevention of IB are deficient in the safety,efficacy and economic costs.Therefore,the development of safe,efficient and inexpensive new vaccines is very important for the control of IB.IBV structural proteins S1 and N are the main immunogen of IBV,playing an important role in humoral and cellular immunity,respectively.Thus,the development of new vaccines including S1 and N proteins has good prospects.Baculovirus expression system is a mature expression system and plays an important role in the expression of processing recombinant exogenous proteins,because it has good capacity of processing post-translation,cutting of signal peptide of the recombinant exogenous proteins,glycosylation modification and so on.Previous researches showed that the introduction of honeybee melittin(HBM)signal peptide recognized by insect cells can mediate heterologous protein expression in the form of secretion in the culture supernatant,enhance recombinant protein expression and glycosylation,which makes the recombinant protein is closer to the native conformation.Baculovirus expression system was applied only in the expression of single IBV proteins.There is no reports about the co-expression of S1 and N proteins by using baculovirus expression system at present,and the expression products are flawed in molecular weight,biological activity and secretory expression.So it is very important to express secreted structural protein and function closer to the natural S1 and N protein.In the present study,the baculovirus expression system was used for the expression or co-expression of S and N protein of IBV strain GX-YL5,which was the representative and dominant serotype strain in Guangxi.The introduction of the HBM signal peptide identified by insect cells,the C-terminal fusing with 6 histidine tag and TEV protease cleavage sites to facilitate purified protein were carried out.The details are as followings:Firstly,the N gene of IBV and HBM signal peptide were amplified by PCR,then the fusion gene HBM-N was obtained by the method of fusion PCR.The cloning vector contained HBM-N was identified by PCR,restriction endonuclease digestion and sequencing,and then was subcloned into pFastBacDual plasmid,transformed to DH10Bac for transposition.The purified recombinant bacmid rHBM-N contained HBM-N gene was obtained after several times of screen.Secondly,S1,S genes and the genes S1,S removed their signal peptides were amplified.HBM was amplified and fused with the genes S1,S removed their original signal peptides,and then the fused gene HBM-S1 and HBM-S were obtained.S and S1 genes contained their own signal peptides and the HBM-S,HBM-S1 genes were cloned and identified correctly.Then HBM-S1,S1,HBM-S and S genes were subcloned into pFastBacDual,identified by PCR,restriction endonuclease digestion and sequencing.The recombinant transposon vectors containing HBM-S 1,S1,HBM-S and S genes were obtained and transformed into the DH10Bac respectively.Recombinant bacmids rHBM-S,rHBM-S1 carrying HBM and recombinant bacmids r-S and r-S1 containing its own signal peptide were obtained.Thirdly,HBM-S1 gene was subcloned into pFast-HBM-N recombinant transposition vector and identified by PCR,restriction endonuclease digestion and sequencing.The recombinant binary transposition vector pFast-HBM-Sl-N was constructed successfully,and then transformed into DH10Bac.The purified recombinant expression bacmid rHBM-S1-N containing S1 and N gene was obtained after several times of identification by PCR.The six recombinant baculovirus expression vectors constructed in the present study will lay a good foundation for the expression or co-expression of S and N proteins,for the study of biological function of S and N proteins,and also for the mutation,pathogenesis diagnostic antigen and new vaccine of IBV.The present study also will provide evidence for the impact of HBM on the expression of IBV structural proteins.The innovations of this study are as followings.Firstly,it is the first time to research co-express IBV major protective antigen protein S1 and the conservative N protein playing an important role in cell-mediated immunity using the baculovirus expression system.Secondly,the introduction of HBM signal peptide that can be recognized by insect cells was carried out in the present study in order to obtain efficiently the secretory expression proteins with native activities.The present study will lay good foundations for the development of diagnostic antigens and genetic engineering vaccines.