| Cow mastitis is an infectious disease that is caused by pathogenic microorganisms. The pathogens that cause cow mastitis can be classified into four categories, namely bacteria, virus, mycoplasma and fungi according to category. Among them, Staphylococcus aureus is a major pathogen. S.aureus, which is a gram positive (G+) contagious non-specific mammary gland parasite, has the capacity of adherence and anchorage to mammary gland epithelium and the characteristic of easy to produce antibiotic-resistant strains. That makes the infection become chronic. Therefore cellular immune state has an important effect on development and outcome of cow mastitis infected by S.aureus.Recognizing and specifically anchoring of S.aureus adhesins to definite molecules on the cell is one of the key steps in the initiation of an infection. Blocking bacterial adherence to cell and the colonization of the mucosal surface may be the most effective strategy to prevent infection. Clumping factor A (ClfA) and Fibronectin binding protein (Fnbps) are important adhesion factors involved in the pathogenesis of S.aureus that causes bovine mastitis. Based on the work done before, in this paper, colony-stimulating factor (GM-CSF) gene of bovine, region D of FnBPB gene and region A of ClfA gene of S.aureus were obtained by the method of PCR. And three fused DNA fragments, GM-CSF-FnBPB, FnBPB-ClfA and GM-CSF-FnBPB-ClfA, amplified by overlap extension PCR were subcloned into the expression vector pET-32a (+) respectively. Three fusion proteins expected were properly expressed. SDS-PAGE showed that the molecular weights of them were 39kDa,51kDa,64kDa respectively. The fusion proteins were immunogenic by Western blot analysis. Furthermore, mice were immunized three times with purified fusion proteins, adhesion molecules and cell factors through singly immunization and combined immunization Strategy. The serum antibody titers of vaccinated mice were tested by the methods of ELISA (Eenzyme Linked Immunosorbent Aassay) and SAT (Standard Tube Agglutination Test). The results showed that antibodies were all detected in the serum of mice immunized three times. The serum antibody titers of fusion proteins immunization group and combined immunization group were highly significantly different from that of the control group (P<0.01); The serum antibody titers of singly adhesion immunization group was significantly different from that of the control group (P<0.05); The serum antibody titers of fusion proteins immunization group and combined immunization group were significantly different from that of singly adhesion immunization group (P<0.05).The antiserum was used for opsonophagocytic assay and anti-binding of S. aureus experiments. The humoral immune protection efficacy of vaccines constructed from fusion proteins, mixed proteins and singly expressed adhesion was assessed. The results of opsonophagocytic assay proved that the bactericidal capability of whole blood of mice immunized by fusion proteins and mixed proteins were highly significantly different from that of the control group (P<0.01); the bactericidal capability of whole blood of mice immunized by adhesion was significantly different from that of the control group(P<0.05); the bactericidal capability of whole blood of mice immunized by fusion proteins and mixed proteins were significantly different from that of the adhesion immunization group(P <0.05).The results of anti-binding of S. aureus experiments demonstrated that the adhesion ability of S. aureus cultured by serum of mice to fibrinogen and fibronectin,which was immunized by fusion proteins and mixed proteins, was highly significantly different from that of the control group(P<0.01); the adhesion ability of S. aureus cultured by serum of mice to fibrinogen and fibronectin, which was immunized by adhesin, was significantly different from that of the control group(P<0.05); the adhesion ability of S. aureus cultured by serum of mice to fibrinogen and fibronectin, which was immunized by fusion proteins and mixed proteins, was significantly different from that of the adhesin immunization group(P<0.05).The cell immune protection efficacy was assessed through detection of Th1/Th2 Types Cytokines level and T lymphocyte Proliferation assay.The results of detection of Th1/Th2 Types Cytokines level showed that the contents of Th1type Cytokines IL-2 and IFN-γand Th2 type Cytokines IL-4 and IL-10 in the serum of mice immunized by fusion proteins and mixed proteins were also highly significantly different from that of the control group(P<0.01); the contents of those in the serum of mice immunized by adhesin was significantly different from that of the control group(P<0.05); the contents of those in the serum of mice immunized by fusion proteins and mixed proteins were significantly different from that of the adhesin immunization group(P<0.05). Draw a conclusion, immune adjuvant can not only increase the level of cell immune, but also enhance the humoral immune reactivity. The results of T lymphocyte Proliferation assay revealed that T lymphocyte Proliferation ability of fusion proteins immunization group and mixed proteins immunization group were also highly significantly different from that of the control group(P<0.01);T lymphocyte Proliferation ability of adhesion immunization group was significantly different from that of the control group(P<0.05);T lymphocyte Proliferation ability of fusion proteins immunization group and mixed proteins immunization group were also significantly different from that of the adhesin immunization group(P<0.05).In conclusion, immune adjuvant can enhance the specific cellular immune response induced by the vaccine.This study provided theoretical basis and experimental evidence for developing genetically-engineered subunit vaccine against cow mastitis infected by S. aureus.The innovation of this research also covers two aspects:(1)Genetically-engineered subunit vaccine was constructed from genes encoding adhesion ClfA, FnBPB and cell factor GM-CSF for the first time. And it was proved that the fusion protein has better protective immunity. The high level of antibody may improve anti-adherence to Cells of S.aureus and strengthen the phagocytosis and bactericidal capability of macrophage PMN.(2)Tandem genes were linked by linker sequences of lower molecular weight and good flexibility. The sequence data of them proved that the tandem order of gene fragments was correct and the drift of open reading frame did not arise. The results of animal immune experiment indicated that inserting linker sequences did not impact on expression and conformation of protein, and assured proper translation of protein.
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