| In recent years,The H9N2 subtype of low pathogenic avian influenza has become one of the most widespread subtypes among poultry in China.Infected poultry usually do not develop disease and mainly show reduced production performance,but when mixed with H5,H7 or other pathogens(such as Escherichia coli,infectious bronchitis),it will significantly increase the morbidity and mortality of poultry,which seriously hinders the healthy development of China’s poultry farming industry.Probiotics has the advantages of green,safe and pollutionfree,and has become ideal alternative to antibiotics in livestock and poultry farming.Live recombinant Lactobacillus vaccine can be directly administered orally,convenient to administer,safe and non-toxic,etc.so it has been widely researched and applied.Lactobacillus constitutive expression system can express exogenous genes while the bacterium is alive without additional induction agent,which simplifies the operation and is relatively safe,so it is more suitable to be applied to the actual industrial production.In the study,we constructed a live recombinant Lactobacillus vaccine by introducing the HA2 gene into a constitutive Lactobacillus expression vector,and the HA2 protein is located on the surface of recombinant Lactobacillus.The study is divided into two main parts as follows.1.Screening for probiotic LactobacillusIn the study,we compared the acid resistance(p H = 2.5),bile salt resistance(0.125% and0.25%),pepsin resistance(3 mg/ml)and Trypsin resistance(1 mg/ml)and antimicrobial activity(Escherichia coli,Salmonella and Staphylococcus aureus)of L.casei 393,L.casei 0331,L.plantarum NC8,L.plantarum 3356,L.plantarum 3335 and L.rhamnosus 8945 to screen for probiotic Lactobacillus.This study further studied the effects of selected Lactobacillus on growth performance,digestive function,immune function,vaccine immune titer,serum antioxidant activity and cecal flora of Huang chickens.Three strains of L.casei 0331,L.plantarum NC8 and L.plantarum 3356 with good stress resistance were screened by in vitro simulation test and bacteriostatic test.Animal test showed that the three strains of Lactobacillus had different promoting effects on the growth performance,digestive function and immune function of Huang chickens,but the strength of probiotic effect was closely related to the strains.L.plantarum NC8 has a good effect on improving the immune function of Huang chickens,and can improve the levels of Ig A,Ig G and IFN-γ and NDV antibody,which were better than those of control group and other treatment groups;L.plantarum 3356 had a good effect on improving the production performance and digestive performance of Huang chickens,and the effect was equivalent to of aureomycin;The probiotic effect of L.casei 0331 was weaker than that of aureomycin and the other two strains of Lactobacillus.2.Construction of recombinant Lactobacillus expressing H9N2 AIV HA2 proteinBecause L.plantarum NC8 has the best effect on improving the immune performance of Huang chickens,it was selected as the host strain for the construction of recombinant Lactobacillus.Using enhanced green fluorescent protein(EGFP)as the reporter gene,which was fused with the truncated anchor gene pgs A(poly-r glutamate synthase A),and compared the promoter activities of HCE,Slp A and p32 constitutive promoters in L.plantarum NC8.Compared with the three constitutive promoters of HCE,Slp A and p32,the results showed that p32 constitutive promoter had strong promoter activity in L.plantarum NC8 and the truncated pgs A’ protein could anchor EGFP on the cell surface.From 2019 to 2020,our laboratory isolated 9 strains of H9N2 AIV from Shandong,Henan,Jiangsu and Sichuan Province.The genetic evolution analysis of HA gene showed that the 9 strains belonged to Y280 like branch of Eurasian branch.The results of homologous evolution analysis of the isolated strains showed that the homology of HA gene was close,and the homology of nucleotide and amino acid was more than 92.8%.The HA gene homology between the isolated strains and vaccine strains SS,F and 6 was slightly far,with nucleotide homology less than 89.5% and amino acid homology less than 91.8%.The cleavage sites of HA protein of 9 strains of H9N2 AIV were RSSR↓GLF,which accorded with the characteristics of typical low pathogenic avian influenza virus.Among the 9 isolated strains,the strain SD isolated from chicken in Shandong Province had close homology with the other 8 strains,and the homology of nucleotide and amino acid reaches more than 94.5%.So,polyclonal antibody against the HA2 protein of the strain was prepared,the results of indirect ELISA showed that the titer of the polyclonal antibody reached 1:25600,and the polyclonal antibody can specifically bind to the protein in Western blot test.Therefore,polyclonal antibody against the HA2 protein of the strain was prepared,the titer of the polyclonal antibody measured test was 1:25600 by indirect ELISA test,and the polyclonal antibody can specifically bind to the protein by Western blot test.Finally,the truncated anchor gene pgs A’ gene was fused with the HA2 gene of SD to construct recombinant L.plantarum p32-pgs A’-HA2/NC8.Western Blot showed that the recombinant Lactobacillus successfully expressed fusion protein and IFA showed that the recombinant Lactobacillus could display HA2 protein on the surface.This study showed that L.plantarum NC8 had a good effect on the immune performance of Huang Chickens through in vitro simulated gastrointestinal environment test,bacteriostatic test and animal test,so it was selected as the host strain for the construction of recombinant Lactobacillus.The HA genes of 9 H9N2 AIV strains isolated in our laboratory from 2019 to2020 were analyzed,the strain SD isolated from chicken in Shandong Province had close homology with the other 8 strains,and successfully constructed recombinant Lactobacillus p32-pgs A’-HA2/NC8 with HA2 protein on the surface,which provided a new vaccine candidate strain for the development of oral microecologics of Lactobacillus to prevent H9N2 subtype avian influenza. |
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