Construction Of Avian Influenza Viruses Oral Vaccine Based On Lactococcus Lactis Expression System And Analyses Of Its Immunogenicity

Objective:The oral vaccine of avian influenza virus was constructed based on L.lactis induced expression system,and its immunogenicity and immune protection efficacy were analyzed.Methods:The HA1 gene and M2 gene were amplified using pcDNA3.1-HA（Gen Bank:KY171732.1）and p GEM-M2（Gen Bank:AAT70528.1）as the templates,respectively.The Glycine-Serine gene complementary sequence（GS linker）was designed in the downstream primer of HA1 gene and upstream primer of M2 gene,and the target gene HA1-M2 was amplified by overlap PCR.The HA1-M2 gene and the expression plasmid p NZ8149 were digested with Nco I/Kpn I,and then ligated with T4 DNA ligase to obtain the recombinant plasmid p YD1-HA1-M2 which was electroporated into the competent L.lactis NZ3900,and the positive clone was obtained by lactose screening method,and further identified by PCR,double digestion and sequencing.The positive clone was named as the recombinant L.lactis/p NZ8149-HA1-M2.Nisin A was used as an inducer,the expression of recombinant L.lactis/p NZ814-HA1-M2 was qualitatively and quantitatively analyzed by Western blot,immunofluorescence labeling technique and ELISA in vitro.Further,Leghorn chicken was used as an animal model of oral immunization.The levels of humoral,mucosal and cellular immune responses induced by the recombinant L.lactis/p NZ8149-HA1-M2 were determined by ELSIA assay or ELISpot assay.The hemagglutination inhibition（HI）titers and neutralizing antibody titers of sera were measured by HI assay and microneutralization assay.Finally,the immune protection efficacy conferred by the recombinant L.lactis/p NZ8149-HA1-M2 was evaluated by H5N6 and H5N1 virus challenge experiments.Results:The target gene HA1-M2（1302bp）was amplified by overlap PCR.The HA1gene and M2 gene were connected by Glycine-Serine sequence（GS linker）.The recombinant L.lactis/p NZ8149-HA1-M2 was successfully constructed by using the competent L.lactis NZ3900 as a host strain and the L.lactis expression plasmid p NZ8149 as the skeleton.The antigen protein HA1-M2 was specifically expressed in L.lactis and detected by Western blot and immunofluorescence labeling technique,and its molecular weight was about 45 k Da.The expression of the recombinant L.lactis/p NZ8149-HA1-M2 was measured by ELISA,and 1×10⁸ CFU of the recombinant L.lactis/PNZ8149-HA1-M2 expressed about 7μg of antigen protein HA1-M2.Leghorn chickens orally vaccinated with the recombinant L.lactis/p NZ8149-HA1-M2 could produced meaningful HA1-or M2-specific sera Ig G antibodies,secretory Ig A antibodies and higher secreted level of IFN-γ.Furthermore,the HI titers of anti-H5N6 and anti-H5N1 induced by the recombinant L.lactis/p NZ8149-HA1-M2were 2^5.4±0.547and 2^5.2±0.836,respectively,and the neutralizing antibody titers were 45.4±8.41and 43±2.73,respectively.Most importantly,Leghorn chickens orally vaccinated with the recombinant L.lactis/p NZ8149-HA1-M2 could survive against H5N6 or H5N1 virus challenge.The results indicated that the recombinant L.lactis/p NZ8149-HA1-M2 could provide 100%immune protection efficacy.Conclusion:Oral vaccine of avian influenza virus was constructed based on L.lactis induction expression system,and showed the strong immunogenicity and high immune protection efficacy.This provides scientific support for effective preventing avian influenza virus infection in poultry industry,also broadens the research insight of L.lactis as an oral vaccine delivery vector,and further provides the reliable reference for the development of oral vaccines against other viruses or bacteria.