登革2型病毒43株NS1基因重组质粒DNA的免疫原性及保护作用研究

DNA immunization with recombinant dengue 2 virus nonstructural protein NS1 elicits protective immunity in mice

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 。

Objective To assess the immunogenicity and protective efficacy of recombinant plasmid DNA encoding dengue 2 virus 43 strain(D2-43)NS1 protein. Methods Purified recombinant pcDNA-NS1 construct was injected i.m into 3-week BALB/c mice with immunization dosage of 100μg per time per animal. NS1-specific antibodies and specific cytotoxic T-cell responses were analyzed. After intracerebral challenge of 6-week BALB/c mice with neurovirulent D2-43 strain, protective efficacy of plasmid pcDNA-NS1 was defined. Results pcDNA-NS1 immunized BALB/c mice had an antibody titre up to 1∶800, 1∶10 diluted antisera from pcDNA-NS1 immunized mice was able to lyse D2-43 infected BHK-21 cells in the presence of complement. Specific cytotoxic T-cell responses were also elicited. When E/T ratio was 20∶1, the specific cytotoxicity was 22.6%. The primed BALB/c mice were challenged by intracerebral injection of 100 LD 50 of D2-43 strain virus, the results showed that survival rate in the pcDNA-NS1 immunized mice was 90.9%. The difference in survival between pcDNA-NS1 immunized group and pcDNA3.1 immunized control group was statistically significant (P<0.05). Conclusion pcDNA-NS1 plasmid DNA immunization can elicit anti-NS1 antibodies and specific cytotoxic T lymphocyte(CTL) activity. The results herein illustrate that immunization with DNA encoding NS1 protein is capable of protecting mice against a lethal D2-43 attack. The present result may be useful to the development of an effective dengue virus vaccine for humans.