| Avian influenza(AI), which is found worldwide and causes one of the most prevalent infectious diseases, it is caused significant economic loss for breeding industry,But also a serious threat to human health in our country.Currently, vaccination is the most effective means for controlling Avian Influenza.Traditional vaccines are inactivated vaccine, but immunological effectiveness is not ideal.Therefore,there is a broad application prospect of oral mucosal immune universal vaccine.In this study, we use a foreign protein expression vector—Lactobacillus(Lactobacillus plantarum, NC8) to connect the gene of protective antigen protein M2 e with hemagglutinin gene HA2 of H9N2 subtype Avian influenza virus,combined with exogenous gene probiotic lactic acid bacteria developing recombinant lactobacillus oral vaccine of M2 e gene fusion the HA2 hemagglutinin gene and make a preliminary evaluation of the immune function.As the research contents and results followed:(1)The preparation and immunogenicity analysis of lactobacillus p SIP409-pgs A-3M2e-HA2 and p SIP409-pgs A-HA2.We compound two groups of recombinant plasmid, including plasmid p SIP409-pgs A-3M2e-HA2,plasmid p SIP409-pgs A-HA2 by means of enzyme digestion,connection,transformation etc. By means of the target recombinant plasmid electroporated into Lactobacillus plantarum,which obtained the target protein by Spp IP peptide inducing expression in lactic acid bacteria.Then analysing by Western blotting and to further detect exogenous gene,two fusion proteins of p SIP409-pgs A-3M2e-HA2 and p SIP409-pgs A-HA2 presented reactionogenicity as the polyclonal antibody of AIV. The results showed that the proteins expressed by the lactobacillus p SIP409-pgs A-3M2e-HA2 and p SIP409-pgs A-HA2 are provided good immunogenicity,lay the foundations for the next experiment.(2)The comparation and evaluation of immune protection effect of lactobacillus p SIP409-pgs A-3 m2e-HA2 and p SIP409-pgs A-HA2.The experimental chicks in six groups, each group of 20.preparation and recovery the lactobacillus that can expresse the proteins of p SIP409-pgs A-3M2e-HA2 and p SIP409-pgs A-HA2 and two new functional lactobacillus which can obtained the target protein by inducing expression p SIP409-pgs A-3M2 e and p SIP409-pgs A(the laboratory have been constructed before).Use the method of oral administration and performed prime and boost immunization to the chicks with these four new functional lactobacillus. At the same time designed blank control group and H9N2 subtype avian influenza inactivated vaccine group.The primer immunization from 21 days to23 days, interval of 14 days we began to booster immunization, for three days. Then kill the immunized chicks for taking samples after 3days.Use flow cytometry to detect the activation of T lymphocytes in spleen of chicks after booster,thereby to determine the immunity effects of new functional lactobacillus.In addition,separated collection each group chicks serum and tracheal lavage,detected the expression levels of Specific Ig G and Specific secretory immunoglobulin A(SIg A) in chicks.Infected of two kinds of H9N2 avian influenza virus after booster by intranasal.Observe the mental state and record the weight change of chicks after attack drug.10 days after challenge,killed the chicks and remove the lungs and throats,determination of lung virus titers of each group of the chicks,made pathological section by HE staining for lungs and throats.Comparison of virus titers and pathological changes in the lung and throat, Thereby further validate the immunological effectiveness to chicks by immunization of new functional lactobacillus.The results show that,.T lymphocytes were improved significantly in spleen of chicks after immunization by new functional lactobacillus p SIP409-pgs A-3M2e-HA2 and p SIP409-pgs A-HA2 and p SIP409-pgs A-3M2 e compared with blank control group, in which the p SIP409-pgs A-3M2e-HA2 and p SIP409-pgs A-HA2 group showed significant increases.The expression levels of specific antibodies was significantly increased in trachea lavage and serum of chicks.A good protective effect to against H9N2 subtype of avian influenza virus infected is confirmed with the final results of observation of mental state Etc.But the immunological effectiveness of p SIP409-pgs A-3M2e-HA2 and p SIP409-pgs A-HA2 is better.Description of the conservative antigen gene HA2 of avian influenza virus hemagglutinin have a certain immunological effectiveness against the different H9N2 avian influenza virus. it has also indicate that the recombinant gene fusion lactic acid bacteria of M2 e have the ability to resist the AIV. The research provided dara for further study to against AIV by the new functional probiotics. |
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