Abstract:

Objective To construct the polyvalent recombinant nucleic acid vaccine of Toxoplasma gondii and measure its protective immune effect. Methods The gene of heat shock protein（HSP70）was amplified by PCR and inserted into the recom? binant plasmid of pcDNA3?ROP2?p30 to construct recombinant polyvalent nucleic vaccine（pcDNA3?ROP2?p30?Hsp70） . BALB/c mice were immunized with the constructed recombinant nucleic vaccine. CD4+ and CD8+ in the splenic lymphocytes and the lym? phocytes in anticoagulant whole blood，the immune indices such as antibodies（IgG，IgM and IgA）and IFN?γ，TNF，IL?2，IL?4， IL?12 in serum and splenic lymphocytes culture medium were detected，along with the challenge experiment. The protective im? mune responses that caused by the vaccine was measured by detecting the changes of immune indices of mice and the challenge ex? periment. Results 916 bp fragment of HSP70 gene was amplified by PCR. The recombinant polyvalent nucleic vaccine pcDNA3? ROP2?p30?HSP70 that included the whole open reading frame sequence of HSP gene was successfully constructed. The immuniza? tion results also showed this polyvalent nucleic vaccine could induce strong cellular and humoral responses by the detection of high? er antibody titer in the experimental mice group，the increasing proliferation of CD4+ and CD8+ cells with significant deviations among the groups（FCD4+ =45.00，FCD8+ =15.01， all P＜0.01）and the apparent up?regulated levels of several cytokines IFN?γ，IL?2 and IL?12 in serum and cultural supernatant of spleen cells，with more striking effect in serum. As a result of the challenge experi? ment，the immunized mice showed a longer survival time. Conclusion The recombinant nucleic acid vaccine pcDNA3?ROP2?p30 ?HSP70 possesses a strong immunogenicity and is able to induce an immune protection.

Key words: Toxoplasma gondii；Rhoptry protein 2（ROP2）；Surface antigen P30；Heat shock protein 70（HSP70）；Nucle

摘要：

目的 构建弓形虫多基因核酸疫苗， 评价其免疫保护性。方法 PCR扩增热休克蛋白 （HSP70） 基因片段， 克隆到 pcDNA3?ROP2?p30重组质粒中， 构建pcDNA3?ROP2?p30?HSP70多基因核酸疫苗。采用该疫苗免疫BALB/c小鼠， 检测小鼠脾淋巴细胞及全血CD4+ 、 CD8+ ， 血清、 脾淋巴细胞培养液中IgG、 IgM、 IgA和IFN?γ、 TNF、 IL?2、 IL?4、 IL?12等， 并进行弓形虫攻击感染实验。结果 应用PCR扩增出916 bp HSP70基因片段， 成功构建pcDNA3?ROP2?p30?HSP70重组体， 其中包含 HSP70 蛋白基因读码框内的完整序列。该弓形虫多基因核酸疫苗能诱发小鼠产生细胞免疫和体液免疫， 实验组小鼠血清抗体滴度高， CD4+ 和 CD8+ 均增殖明显， 组间差异有统计学意义（FCD4+ =45.00， FCD8+ =15.01， P均＜0.01）； 其血清及脾细胞培养液中IFN?γ、 IL?2和IL?12含量均明显升高， 尤以血清中升高显著。攻击实验结果显示， 实验组小鼠存活时间明显延长。结论 pcDNA3?ROP2?p30?HSP70多基因核酸疫苗具有较强的免疫原性， 能产生较好的免疫保护作用。

关键词: 刚地弓形虫； 棒状体蛋白2； 表面抗原P30； 热休克蛋白70； 核酸疫苗