【摘要】 为研究结核分枝杆菌Ag85B、MPT64和MPT83抗原DNA疫苗在奶牛体内诱导的免疫应答,以PJW4303为载体,构建了上述3种抗原基因的三价DNA疫苗。采用阴离子交换色谱方法提取3种质粒,混合后添加佐剂DDA免疫3月龄荷斯坦奶牛3次,采用ELISA法检测免疫奶牛的特异性抗体效价。利用双抗夹心ELISA定量测定免疫奶牛各月的IFN-γ浓度。结果显示,纯化后的DNA疫苗经质量检验达到动物免疫试验的要求;免疫前奶牛体内特异性抗体为阴性;三免后第30天MPT83、Ag85B和MPT64特异性抗体效价达到最高,均值分别为1∶3 200、1∶2 560和1∶2 880;同时免疫奶牛MPT83、Ag85B、MPT64三种蛋白诱导产生的IFN-γ在三免后第30天达到最高,分别为(630.25±94.45)、(341.65±76.75)、(51.38±9.80)pg/mL,阴性对照组PBS的IFN-γ浓度为(6.48±20.88)pg/mL。每个月的阴性对照与3种抗原相比,差异均极显著(P<0.01)。证实该三价核酸疫苗能同时诱导机体产生细胞免疫应答和体液免疫应答,可能有利于增强疫苗对结核病的抗性。更多还原

【Abstract】 In order to evaluate the immune response in vivo induced by the DNA vaccine based on Mycobacterium tuberculosis Ag85B,MPT83 and MPT64 antigen in cow, three plasmids were extracted by anion exchange chromatography method and three-month-old Holstein cows were immunized with the eukaryotic expression vector PJW4303 and dime-thyldioctyldeeyl ammonium bromide. Enzyme-linked immunosorbent assay(ELISA) was used to detect anti-Ag85B,MPT83 and MPT64 antibody titers in sera of the vaccinated cow,and double antibody sandwich ELISA was used to quantitatively determine the concentration of IFN-γ. Results showed that the purified DNA vaccines met the require of animal immune test. The specific antibody titers of cattle were negative before the pre-immunization,and MPT83,Ag85B and MPT64 can induce to the highest antibody titers on day 30 after third immunization with average titers of 1∶3200、1∶2560 and 1∶2880. Antigen-specific IFN-γ level of MPT83,Ag85B,MPT64 and PBS were(630.25±94.45)pg/mL,(341.65±76.75)pg/mL,(51.38±9.80)pg/mL and(6.48±20.88)pg/mL, respectively. The negative control group(PBS) showed significant difference(P<0.01) with the three antigen groups in each month.In conclusion,the trivalent DNA vaccines can simulate humoral and cellular immune responses simultaneously,which suggested that the trivalent DNA vaccines might be able to enhance protective response of cow against tuberculosis.更多还原