| Shigellosis is an intestine contagious diarrhea by Shigella bacteria,which is one of the most common intestinal infectious diseases in summer and autumn.This disease is widespread,serious harm,can infect humans and a variety of animals,cause disease even death,harm to human heath,and cause havoc for the aquaculture industry in China.Therefore,the study of disease prevention and biological vaccine is not only important for veterinary public health significance,but also has significant medical public health significance.This study use chicken Shigella Ipa C protein 、 chicken Shigella inactivated bacterial vaccine and a mixture of both immunized mice,to get more efficient primary immune by determined mice humoral and cellular immunity and challenge protection test.To properly evaluate the effect of the vaccine,provides a reliable means of detection and methods of effective prevention and control of disease.The results are as follows:1、Cloning and expression of chicken Shigella Ipa C geneAmplified Ipa C gene by PCR from recombinant plasmid p MD18-T-Ipa C,and cloned into the expression vector p ET32 a to construct the recombinant plasmid p ET32a-Ipa C,and transformed into E.coli BL21(DE3),taking IPTG as the inducer for inducing expression.SDS-PAGE results showed successfully expressed Ipa C protein,expressed both in supernatant and sediment,the molecular weight of the fusion protein is 63 k D.The supernatant was purified to electrophoretic homogeneity by using Ni-Agarose His tag,measured and adjusted the concentrate to 1mg/m L.Western-blot analysis showed that the protein could be recognized by monoclonal antibody,and has a good responsed immunity.2、The humoral immune function of chicken Shigella Ipa C protein and bacterial inactivated vaccine and the mixture of the two vaccinesDetermination of humoral immunity,prepared three kinds of immunogen primary:the purified Ipa C protein(concentration 1mg / m L),chicken Shigella ZMD01 strains inactivated vaccine(concentration 1010cfu/m L),and 1:1 of the mixture.Three kinds of immunogen are divided into two batches,one of the batch add Freund’s complete adjuvant for first immunization,another add Freund’s incomplete adjuvantfor the second strengthen immunization.Strengthen immunization 21 d after the first immunization.Both first and second immunization groups are according to the0.2m L/only immunized by subcutaneous injection,and set the PBS control group.Determination different time antibody of collected blood,both first and second immunization are tail blood after 7d and 14 d,separate serum,testing serum antibody titers by indirect ELISA method of this laboratory established.The results showed that each immunized group mice geometric mean titer(GMT) was significantly higher than the control group(P<0.05),reached a peak at second immunized 14 d,Ipa C protein group、inactivated bacterial group and mixed group GMT were 1:15241、1:16591、1:18093,PBS group was 1:20,the three immunized groups were not significant(P>0.05),Ipa C protein group GMT > inactivated bacterial group GMT > mixed group GMT.3 、 The cell-mediated immune function of chicken Shigella Ipa C protein and bacterial inactivated vaccine and the mixture of the two vaccinesDetermination of cellular immunity,types of vaccines、animal grouping、immune method and time of blood sampling as the humoral immune assay.After the second immunize 21 d,each group of mice was measured spleen lymphocytes stimulation index(SI) by four methyl thiazolyl tetrazolium chromatometry(MTT) assay、use flow cytometry to detect peripheral blood T lymphocyte subsets proportion 、 Goat anti mouse ELISA kit to detect level of IL-2 and IFN-γ. The results showed that,①after the second immunize 21 d,each immunized group lymphocyte stimulation index were significantly higher than the PBS control group(P<0.05),the difference of each immune between groups was not significant(P>0.05),mixed group(2.241±0.243) is slightly higher than Ipa C protein group(2.120±0.210),Ipa C protein group was slightly higher than inactivated bacterial vaccine group(2.085±0.185).②T lymphocyte subsets analysis shows,CD3+,CD4+percentage of each immunized group were higher than the PBS control group,while CD8+ ratio was less than PBS group,CD4+/CD8+ratio of Ipa C protein group,inactivated bacterial vaccine group,mixed group were higher than the PBS control group 17.92%,26.27%,28.11%.③From the beginning of the first immunize 7d,in addition to the PBS control group,IL-2 and IFN-γ content of the other groups are gradually increased,has a very significant difference(P<0.01) with the PBS control group after the second immunize 21 d,but the difference between immunized groups was not significant(P>0.05).4、Poison attack immune protectionFirst determination LD50 of the Shigella strain,with six different levels of Shigella ZMD01 strains infection dose challenged on mice,observe the death number of mice in each group after challenged 7d,use the Bliss method to calculate Shigella ZMD01 strains on mice median lethal dose(LD50).Challenged on second immunize21 d with dose of LD50,observed and recorded incidence of each group mice.The results showed that the incidence mice were depression、deceased appetite、feces abnormal.After 7d of infection the survival rate of mice in each group were:Ipa C protein group was 5/10,inactivated bacterial group was 5/10,mixed group was6/10,PBS group 3/10.Necropsy lesions were mainly for liver enlargement, reddish brown, bleeding; bleeding and swelling of the spleen; swelling of the intestinal contents, wall thinning, a small amount of bleeding.In the liver, spleen and intestinal contents of the mice were recovered pathogenic bacteria.Conclusion:Chicken shigella Ipa C gene can expression in prokaryotic effectively,the expression protein had a good response of immunity.Chicken Shigella Ipa C protein and bacteria inactivated vaccine and combined vaccine can improve the effect of humoral immunity,and enhance cellular immunity,and the mixed group vaccine were higher than the other two groups, mixed vaccine is expected to be a candidate vaccine. |
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