| Objective: Bovine tuberculosis is a zoonotic infectious disease that can seriously threaten the food healthy safety and economic security of human.In recent years,the epidemic spread rapidly,incidence has been increasing year by year.At present,the only available tuberculosis vaccine is still BCG,But because of the immune effect of the cattle is not ideal,As a result,the researchers are working hard to develop efficient bovine tuberculosis vaccines.MPB83,MPB70 is the early secretion protein of Mycobacterium bovis and the most studied protein.The MPB83 and MPB70 protein genes are fused together to improve the defense effect on bovine tuberculosis.As a diagnostic antigen,it has a high specificity and sensitivity.Therefore,the MPB83-70 fusion gene as the target gene of this experiment has far-reaching significance for the development of bovine tuberculosis vaccine.Lactococcus lactis pNZ8149/NZ3900 is known as food safety level microbes,it is also intestinal probiotics.It has weak antigenicity and does not produce extracellular enzymes.At the same time,antibiotic screening markers are not required in the screening process.Lactococcus lactis pNZ8149/NZ3900 can be used as natural delivery of exogenous antigen vector in vaccine research and development and has become hot spots in the vaccine research and development,bio-pharmaceutical and other areas.Therefore,this paper will use pNZ8149/NZ3900 as expression vector to express Bovine Mycobacterium bovis MPB83-70 fusion gene to construct recombinant Lactococcus lactis,and to evaluate the immune index of the recombinant bacterium to become theoretical reference for development of bovine tuberculosis vaccine.Method: The plasmid mpb83-70 x was used as a PCR template.The target gene mpb83-70 was amplified by PCR and connected with pMD18-T Simple Vector to constructcloning plasmid pMD18-T(S)-mpb83-70.KpnI and NcoI were used for enzyme digestion,the obtained target gene mpb83-70 was ligated with the expression vector pNZ8149/NZ3900.The ligation product was introduced into the lactic acid bacteria susceptibility by electroporation.Then it was coated on ELLKEI solid screening medium containing lactose and bromocresol violet,the yellow colonies were initially identified as positive bacteria pNZ8149-83-70/NZ3900.By By Nisin induction,SDS-PAGE and indirect immunofluorescence identification,Lactococcus lactis PNZ8149/NZ3900 express 37 kDa mpb83-70 target protein.Then the mice were immunized with recombinant pNZ8149-83-70/NZ3900 lactic acid bacteria,flow cytometry was used to detect the number of CD3+,CD4+,CD8+T lymphocytes and IL-2,TNF-? and IFN-? cytokines in immune mice,specific IgG in serumand the levels of sIgA in the feces supernatant were measured by indirect enzyme-linked immunosorbent assay(ELISA)and Mouse sIgA ELISA Kit kit.Finally,By the statistical analysis method i to analyze the data.Results:1.The target gene mpb83-70 was successfully amplified by PCR and ligated with Escherichia coli cloning vector pMD18-T Simple Vector.Mpb83-70 cloning gene was obtained by enzyme digestion;2.The target gene mpb83-70 was ligated with the expression vector pNZ8149/NZ3900 and identified by double digestion,the recombinant lactic acid bacteria pNZ8149-83-70/NZ3900 was successfullyconstructed.Recombinant lactic acid bacteria expressed 37 kDa exogenous protein by SDS-PAGE and indirect immunofluorescence to verify;3.The number of CD3+T lymphocytes in mice immunized with flow cytometry was higher than that no-load pNZ8149 group and 0.9% NaCl group,but lower than BCG group.There was no significant difference between the four groups(P>0.05);The number of CD4+T lymphocytes in the immunized mice was slightly lower than that in the BCG group,but the difference was not significant(P>0.05),compared with pNZ8149/NZ3900 group and PBS group,the number of CD4+T lymphocytes produced by recombinant lactic acid bacteria was higher and the difference was significant(P<0.05);The number of CD8+T lymphocytes was slightly lower than that of BCG group(P>0.05),but higher than that of pNZ8149/NZ3900 group and PBS group,the difference was significant(P<0.05);The number of IL-2 cells in mice immunized by flow cytometry was higher than that in BCG group and pNZ8149/NZ3900 group,but the difference was not significant(P>0.05),BCG group was higher than that of no-load pNZ8149/NZ3900 group and PBS group,but the difference was not significant(P>0.05);The number of IFN-? cells was less than that of BCG group and the difference was not significant(P>0.05),but compared with the other two groups,the numerical value was high and the difference was not significant(P<0.05);The number of TNF-? cells secreted by immunized mice was smaller than that of BCG group,higher than pNZ8149/NZ3900 group and PBS group,and the difference was not significant(P>0.05);ELISA was used to test the IgG antibody in the serum of immunized mice,the results showed that the recombinant lactic acid bacteria pNZ8149-83-70/NZ3900 was lower than that of BCG group,higher than the pNZ8149/NZ3900 group and PBS group,and the difference was not significant(P>0.05);Mouse sIgA ELISA kit was used to test the level of sIgA antibody in feces of mice immunized,the results show: the values of pNZ8149-83-70/NZ3900 group were significantly higher than those of BCG group,no-load pNZ8149/NZ3900 group and PBS group(P<0.05),it can be confirmed that pNZ8149-83-70/NZ3900 can cause local mucosal immune response,and can induce immune mice to produce better sIgA antibodies.In summary,the results of flow test and ELISA test showed that thepNZ8149-83-70/NZ3900 has immunogenicity,and it could provide a theoretical basis for the follow-up study of bovine tuberculosis vaccine. |
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