The Effect Analysis Of Six BVDV-E2Truncated Gene Recombination BCG Vaccine In Cattle After Immunication

Bovine Viral Diarrhea-Mucosal Disease Virus(BVD-MDV), is one of the members ofFlaviviridae. The virus is a single strand RNA and infectious, with membrane structure.Thedisease is the spread of worldwide infectious disease,with the key features of gastrointestinalmucous membrane with inflammation, erosion, necrosis and diarrhea,and can cause chronic,acute and mucosal infection, influence seriously the cattle reproduction and productionperformance, and also can cause inordinately infection to cattle, sheep, pigs, horses etc andseriously hinder the development of aquaculther in our country even all over the world.The BVDV E2genehas low conservation, the strongest variability, its various glycosylationdegree, and lower immune escape rate.E2genecan not only decide the main part of BVDVantigenicity, but also participating the combination of anti-BVDV antibody,mediated immunereaction and with host cell discriminating and adsorbing. So far, scholars at home and abroaddevelop and research new BVDV vaccine from various ways, the BVDV E2protein is the firsttarget protein of people researching new vaccine, as immune dominant protein.Recombination BCG(rBCG) vaccine focus on all kinds of advantages to produce strongand lasting immune with various specificity. It has been become the competing research hottopic for current scholar at home and abroad to contrast rBCG vaccine. The aim of research isto study the six sections BVDV-E2truncated gene rBCG vaccine（E11、E12、E13、E21、E22、E23）, and test the antibody level, cell factor, lymphocyte cell breeding, and CD4+, CD8+Tlymphocyte subsets to the immunity cattle to make useful attempt to evaluating to choose thenew rBCG vaccine which can prevent tuberculosis and BVDV.The research made bacteria liquid PCR reaction with reserved6groups of BVDV-E2truncated gene recombinant BCG which had been diluted in the lab, tested gene fragment sizeby1%agarose gel electrophoresis, then amplified preservation with7H9cultivated method,counted rBCG with7H9solid medium plate method and calculated colony forming units,which can be rBCG to prepare for immunity cattle. The six sections rBCG had no other bacteriawith gram staining and eosin methylene blue plate detection, then made intraperitonealinjection to6-8week-old male cavies, and all experiment cavies had healthy living and noobvious symptoms after observing, the results indicated that rBCG were no pollution and nopathogenicity to experimental animals. Sixty healthy cattle about24month-old were selected randomly, and tested their maternal antibody levels in serum with BVDV antibody detection kit.There were2positive serum samples and51negative serum samples, so the rate of negative is85.00%. The18negative cattle from the detection of serum were chosen for dividing into6groups randomly, each group had three cattle, and made subcutaneous injection respectivelywith control group of physiological saline and5groups of rBCG vaccine which dose were105CFU/mL,106CFU/mL,107CFU/mL,108CFU/mL,109CFU/mL, then determined the bestimmune does of rBCG by testing humoral immunity levels. The results showed that the groupinjected108CFU/ml was the best one, so the best immune does was108CFU/mL. Then the27negative cattle were chosen for dividing into9groups randomly to make subcutaneous injection,each group had three cattle,6groups of BVDV-E2truncated gene recombinant BCG which hadbeen diluted were rBCG, the rest were BCG control group, BVDV inactivated seedlings groupand normal saline control group. The does of rBCG vaccine group and BCG control group were108CFU/mL, the does of BVDV inactivated seedlings was106.0TCID50/mL, and the does ofphysiological saline control group was1mL. The cattle were immunized in0week, the forthweek and the eighth week, then collected serum before each immune, collected anticoagulantblood and serum after twelfth week. The lymphocytes were separated from anticoagulant bloodwithin48hours for flow cytometry detection and lymphocyte proliferation experiment. Theresults showed that the lymphocyte stimulation indices of E11and E23rBCG were highest, andthe contents of CD4+, CD8+T lymphocyte subsets of E22and E11rBCG were highest. The fourtimes collected serum were used for testing BCG, BVDV antibody levels and IL-4, IL-10, IL-12and IFN-γcell factor levels. The results showed that the BCG antibody levels of E11and E12rBCG were higher, the BVDV antibody levels of E11and E13rBCG were higher, the IL-4levels of E11and E13rBCG were higher, the IL-10levels of E21and E13rBCG were higher,the IL-12levels of E11,E21and E13rBCG were higher, the IFN-γ levels of E11and E21rBCGwere higher. Comprehensive the above indicators showed that the immune effect of E11of6groups of BVDV-E2truncated gene recombinant BCG which had been diluted was obviouslybetter than E12、E13、E21、E22、E23.