Study On The DNA Vaccine Against Bovine Tuberculosis

Bovine tuberculosis (BT) is a chronic wasting disease primary caused by Mycobacterium bovis thatis prevalent throughout the world, particularly in developing countries. This disease not only causestremendous economic hardship to the livestock industry, but it is also a serious human health threat.Hence, the development of strategies to control the disease is tremendously important to preventinghuman tuberculosis. Currently, no clinical vaccine against BT is available.In the present study, the DNA fragments of ag85b, esat-6, hsp65, mpb64 and ag85b-esat-6,hsp65-esat-6, mpb64-esat-6 were amplified by PCR and spliced by overlapping extension (SOE) fromthe genome DNA of Mycobacterium bovis Vallee HI.These seven fragments were inserted intopCDNA3.1(+) vector to construct recombinant plasmids pCA, pCE6, pCH, pCM, pCAE, pCHE andpCME. The seven plasmids were transfected into SP2/0 cell in vitro to detect the expression of targetgenes. BALB/c mice were intramuscularly vaccinated with the seven plasmids and the control vectorpCDNA3.1(+) and PBS respectively. The animals in each group were immunized 3 times with 2-wkintervals.The serum antibody titers were determined using indirect enzyme-linked immunosorbent assay(iELISA) coated with with M. bovis PPD every week. Two weeks after each immunization, spleniclymphocytes proliferation (SLP) assay were detected by MTT method using M. bovis PPD and ConA asstimulator. Two weeks after the third immunization, secreted IFN～γ, of spleen were tested. The results ofindirect ELISA showed the levels of antibodies in all recombinant plasmids groups were significantlyhigher than the two control groups(p＜0.05). SLP experiments indicated the SI value in the groups offusion gene were higher than the single gene groups (p＜0.05). The IFN～γexperiments showed thelevels of IFN～γ, induced with PPD in the groups of fusion gene were significantly higher than the singlegene (p＜0.05). The levels of IFN～γinduced with ConA were higher than with PPD, and the levelswere higher in groups pCAE and pCME than in other groups. So the results showed fusion gene DNAvaccines surpass single gene DNA vaccines in the induction of cellullar immunologic response.The fusion DNA fragments of ag85b-mpb64 and ag85b-mpb64-esat-6 were obtained by PCR andSOE technique. Various DNA vaccines were constructed with the pcDNA3.1(+): fusion of two genes(pCMA), and of three genes (pCMAE), bivalent combinations (pCA+pCM) and trivalent combinations(pCA+pCM+pCE6). BALB/c mice were vaccinated with this DNA vaccines.The mice injected withBCG were positive control and the mice injected with pCDNA3.1(+) and PBS were negative control.The mice were immunized 3 times with 2-wk intervals. The animals in group BCG were only inoculatedsubcutaneously with 1×10~6 CFU BCG at initial vaccination. The serum IgG titers and IgG isotype weredetermined using iELISA coated with M. bovis PPD and rMAE protein expressed and depurated inprokaryotic expression system every week. Two weeks after each immunization, SLP and IFN～γ, IL-2assay were detected using rMAE protein and M. bovis PPD as stimulator. Three weeks after the 3rdDNA immunization, mice were challenged with 1×10~6 CFU of BCG. Three weeks after the challengethe protective efficacy were evaluated based on immunofluorescence assay, lung tissue bacterial loadsand pathohistologic changes. The data demonstrate that immunization with fusion (of two or three genes) DNA vaccines results in significantly higher serum antibody levels, lymphocyte proliferation (SI value), IFN-γ, and IL-2 levels than immunization with polyvalent DNA vaccines (p＜0.05).These index in group BCG were significantly lower than the groups of fusion DNA vaccines and equivalent to groups of polyvalent DNA vaccines when rMAE protein was custodite antigen and stimulator. However, these index in group BCG were significantly higher than other groups when M. bovis PPD was custodite antigen and stimulator. The protective efficacy of the fusion DNA vaccines was surpass the polyvalent DNA vaccines and equivalent to that of the BCG vaccine, suggesting that fusion DNA vaccines represent a promising approach for the prevention of BT. So our experiment may be useful for the development of Bovis tuberculosis vaccine.