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目的 研究以pcDNA3.1为载体的登革 2型病毒 43株 (D2 43)NS1基因重组DNA的免疫原性及对登革病毒感染所致小鼠神经毒的免疫保护作用。方法 将纯化的pcDNA NS1重组质粒DNA采用肌肉多点注射途径免疫 3周龄BALB/c小鼠 ,剂量为每只 10 0 μg/次 ,检测了免疫鼠血清抗体滴度及特异性细胞毒作用。并以D2 43病毒脑内攻击 6周龄BALB/c小鼠产生的神经毒症状为实验模型 ,对pcDNA NS1的免疫保护作用进行了初步探讨。结果 用间接ELISA测得pcDNA NS1免疫后抗体滴度为 1∶80 0 ,在补体存在下 ,对D2 43病毒感染的BHK 2 1细胞特异性杀伤率可达到 6 1.6 %。由免疫的BALB/c小鼠脾制备的效应细胞在体外可特异性地杀伤D2 43感染的P 815细胞 (H 2 d)。当效靶比(E/T)为 2 0∶1时 ,pcDNA NS1质粒免疫后的特异性CTL杀伤百分率为 2 2 .6 %。将 10 0LD50 的D2 43病毒经脑内攻击BALB/c小鼠 ,结果表明免疫pcDNA NS1组小鼠存活率最高 (90 .9% ) ;与免疫pcDNA3.1对照组比较 ,P值 <0 .0 5。结论 pcDNA NS1质粒免疫BALB/c小鼠不仅可诱导体液免疫 ,还可诱导特异性细胞免疫。初步结果还显示 ,用含NS1基因的重组质粒DNA免疫的小鼠能免受致死剂量登革病毒的攻击 ,为登革热新型疫苗的研究奠定了基础
Objective To study the immunogenicity of recombinant DNA of NS1 gene of Dengue virus type 2 (43) (43 43) with pcDNA3.1 and the protective effect against dengue virus-induced neurotoxicity in mice. Methods Three weeks old BALB / c mice were immunized with the purified recombinant plasmid DNA of pcDNA NS1 at the dose of 10 μg / mouse. The antibody titer and the specific cytotoxicity of the immunized rat serum were detected. The neuroprotective effects of pcDNA3.1 NS1 were also investigated by intracerebral challenge with D2 43 virus in 6-week-old BALB / c mice. Results The titer of pcDNA NS1 after immunization was 1: 80 0 by indirect ELISA. In the presence of complement, the specific killing rate of BHK 2 1 cells infected by D2 43 virus reached 61.6%. Effector cells prepared from the spleens of immunized BALB / c mice specifically kill D2 43-infected P 815 cells (H 2 d) in vitro. When the effective target ratio (E / T) was 20: 1, the specific CTL killing rate of pcDNA NS1 plasmid after immunization was 22.6%. The results showed that the survival rate of mice immunized with pcDNA NS1 was the highest (90.9%). Compared with pcDNA3.1-immunized mice, P <0. 0 5. Conclusion Immunization of BALB / c mice with pcDNA NS1 plasmid not only induced humoral immunity but also induced specific cellular immunity. Preliminary results also showed that mice immunized with the recombinant plasmid DNA containing the NS1 gene were protected from the lethal dose of dengue virus and laid the foundation for the study of a new dengue vaccine