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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。
In order to study the immune response of Mycobacterium tuberculosis Ag85B, MPT64 and MPT83 antigen DNA vaccines induced in dairy cows, a trivalent DNA vaccine of the above three antigen genes was constructed using PJW4303 as a carrier. Three kinds of plasmids were extracted by anion exchange chromatography. After being mixed, adjuvant DDA was used to immunize 3-month-old Holstein cows for 3 times. The specific antibody titers of immunized cows were detected by ELISA. The concentration of IFN-γ in each month of immunized dairy cows was determined by double-antibody sandwich ELISA. The results showed that the purity of the purified DNA vaccine reached the requirement of animal immunization test. The specific antibody of the cow before immunization was negative. The titer of the specific antibody of MPT83, Ag85B and MPT64 reached the highest at 30d 1: 3 200,1:2 560 and 1: 2 880 respectively. IFN-γ induced by three kinds of cow milk protein MPT83, Ag85B and MPT64 reached the highest on the 30th day after immunization (630.25 ± 94.45), (341.65 ± 76.75) and (51.38 ± 9.80) pg / mL, respectively. The negative control group had an IFN-γ concentration of (6.48 ± 20.88) pg / mL. The negative control of each month compared with the three antigens, the difference was extremely significant (P <0.01). Confirmed that the trivalent nucleic acid vaccine can simultaneously induce the body to produce cellular and humoral immune responses, may help to enhance the vaccine against tuberculosis resistance.