Construction And Immunization Of Recombinant Lactic Acid Bacteria Expressing HA2 Protein Of H9N2 Avian Influenza Virus

Avian Influenza（AI）,one of the acute and highly fatal infectious diseases of poultry,is widely spread throughout the country.H9N2 has replaced H5N6 and H7N9 as the main subtype threatening the poultry industry in China.In general,H9N2 AIV infection does not cause death of poultry,which is characterized by respiratory tract and intestinal inflammation and decreased egg production.However,when mixed with other pathogens,H9N2 AIV infection will lead to a significant increase in poultry mortality and cause serious economic losses to the poultry industry.At present,vaccination is still the most common way to prevent and control avian influenza,but the traditional vaccine has the risk of short immune time and strong virulence.Probiotics using lactic acid bacteria as the carrier to deliver immune antigens through the mucosal route have a wide application prospect.They are safe and non-toxic,can directly stimulate the body to produce mucosal immunity,and have the probiotic effect of lactic acid bacteria.In this study,HA2,the main protective antigen of H9N2 AIV,was introduced into the constitutive expression system and inducible expression system of lactic acid bacteria,respectively,and two recombinant lactic acid bacteria were developed,aiming to develop new agents that can prevent and control H9N2 AIV.The study content and results are as follows.1.Construction of recombinant Lactobacillus and Lactococcus lactis expressing H9N2AIV HA2 protein:RNA was extracted from the H9N2 AIV epidemic strain and reverse transcribed into c DNA.The HA2 gene was amplified,ligated into the p MD18-T plasmid and heat-stimulated into DH5αcompetent cells to construct the cloned plasmid p MD18-T-HA2.The p32-p SIP409plasmid was digested by NcoⅠand HindⅢdouble enzymes,and the linearization vector p32-p SIP409 and target gene fragment HA2 were connected by homologous recombination.Heat-stimulated transformation to TOP10 competent cells,extraction of plasmids,electroporation into Lactobacillus plantarum NC8 competent cells,the positive strains were screened by erythromycin resistance,and the plasmid was extracted.After sequencing verification,the strain containing the correct recombinant plasmid was named p32-p SIP409-HA2/NC8.The recombinant strain was spontaneously expressed overnight at 30°C.SDS-PAGE and Western blot were used to detect the specificity of the target protein.The 27 k Da HA2 protein was successfully expressed in the recombinant L.lactis p32-p SIP409-HA2/NC8.The HA2 gene fragment and plasmid p NZ8149 were digested by NcoⅠand KpnΙ.they were ligated by T₄ DNA ligase and electroporated into L.lactis NZ3900 competent cells.Elliker medium was used to screen positive strains,and the plasmid was extracted.After sequencing verification,the strain with the correct recombinant plasmid was named p NZ8149-HA2/NZ3900.The recombinant Lactococcus lactis was induced by Nisin.SDS-PAGE and Western blot analysis showed that the 27 k Da HA2 protein was successfully expressed in the recombinant L.lactis p NZ8149-HA2/NZ3900.2.The immune protection of recombinant lactic acid bacteria expressing H9N2 AIV HA2protein on chicks:In order to avoid the interference of maternal antibodies in the eggs,one-day-old SPF chickens were selected as the immune test animals,and the chicks were randomly divided into5 groups,with 20 chicks in each group.Chicks were immunized three times with the successfully prepared recombinant lactic acid bacteria p NZ8149-HA2/NZ3900 and p32-p SIP409-HA2/NC8 and empty vector lactic acid bacteria p32-p SIP409/NC8 and p NZ8149/NZ3900,respectively.The mice were immunized every 7 days for 3 consecutive days,and the oral inoculation dose was 1×10¹¹ CFU/mouse At the end of the three immunization,serum,tracheal mucosa and small intestinal mucosa were collected on the 21^st day of age,and HA and s Ig A antibodies were detected.The results showed that chickens could produce high levels of HA antibodies after immunization with two groups of recombinant lactic acid bacteria,and the induced recombinant lactic acid bacteria p NZ8149-HA2/NZ3900 had better immune effect than the synthetic recombinant lactic acid bacteria p32-p SIP409-HA2/NC8.It has a serum antibody potency of 7log₂.Both groups of recombinant lactobacilli preparations could induce the production of s Ig A antibody in the tracheal and small intestinal mucosa,and the overall level of s Ig A antibody in the small intestinal mucosa was higher than that in the tracheal mucosa.On the 22^nd day of age,10 SPF chickens in each group were artificially infected with H9N2 AIV by subcutaneous injection at a dose of 10⁶ EID₅₀/chicken.Throat swabs and cloacal swabs were collected at 3,5,7 and 9 dpi to detect the positive rate of H9N2 AIV.The results showed that none of the chickens in each group died after infection with H9N2 AIV,and the amount of virus excreted by oropharyngeal swabs was significantly higher than that by cloacal swabs in each group.In the PBS blank group,virus shedding reached the peak from day 3 to day 5,and the amount of virus shedding gradually decreased after day 5.Compared with the PBS control group,the two groups of recombinant lactic acid bacteria could significantly reduce the amount of H9N2 AIV shedding in chicks,and the induced recombinant lactic acid bacteria p NZ8149-HA2/NZ3900 had a slightly better effect on reducing the amount of H9N2 AIV shedding in chicks than the constitutively recombinant lactic acid bacteria p32-p SIP409-HA2/NC8.In conclusion,two recombinant L.lactis strains,p NZ8149-HA2/NZ3900 and p32-p SIP409-HA2/NC8,expressing HA2 protein of H9N2 AIV were successfully constructed.local mucosal immune response and humoral immune response could be induced after immunization with SPF chickens,and can significantly reduce the amount of H9N2 AIV shedding.The recombinant Lactobacillus p NZ8149-HA2/NZ3900 is more immunocompetent with a high serum antibody potency of 7log₂,which is expected to be a new preparation for the prevention and control of H9N2 AIV.