弓形虫GRA4基因真核表达重组质粒DNA免疫小鼠的研究  

STUDY ON IMMUNORESPONSE OF RECOMBINANT PLASMID pVAC CONTAINING GRA4 GENE FROM TOXOPLASMA GONDII IN MICE

作　　者：林绮萍[1] 吴少庭[2] 翁亚彪[1] 高世同[2] 张仁利[2] 黄达娜[2] 袁仕善[2] 雷明军[3] 温见翔[4] 潘晖榕[3] 秦莉[3] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]深圳市疾病预防控制中心,广东深圳518020 [3]华中科技大学同济医学院,湖北武汉430030 [4]广东医学院微生物学教研室,广东湛江524023

出　　处：《中国寄生虫病防治杂志》2005年第3期180-183,共4页Chinese Journal of Parasitic Disease Control

摘　　要：目的用pVACGRA4真核表达重组质粒免疫小鼠,观察诱导的免疫应答及对弓形虫感染的保护作用。方法大量制备pVACGRA4真核表达重组质粒,经基因枪腹部皮内注射免疫BALB/c小鼠3次,以pVAC空质粒及不经任何处理的空白组为对照。于末次免疫4周后作免疫指标测定(包括MTT法测定小鼠脾脏T淋巴细胞增殖活性、间接免疫荧光法测定T淋巴细胞亚群数目、双夹心ELISA法测定细胞因子IFNγ及IL4含量、间接ELISA法测定IgG抗体滴度),并观察攻毒试验后小鼠存活情况。结果脾淋巴细胞增殖各组间差异无显著性(P>0.05);pVACGRA4组CD4+T细胞百分率无明显变化(P>0.05),CD8+T细胞百分率明显增加(与pVAC对照组比较P<0.05,与空白对照组比较P<0.01),CD4+/CD8+比值也较空白对照组明显降低(P<0.01);pVACGRA4免疫组IFNγA值比对照组略高,但差异无显著性(P>0.05),IL4A值各组间无明显变化(P>0.05);pVACGRA4组可诱导产生特异性IgG抗体,但滴度不高;pVACGRA4免疫组小鼠存活率显著高于两对照组,死亡小鼠的平均存活时间延长(与空白对照组比较P<0.05)。结论用pVACGRA4重组质粒DNA免疫小鼠,可诱导产生以细胞免疫为主的免疫应答及对弓形虫攻击感染的部分保护作用。Objective To observe the immune responses of constructed recombinant plasmid, pVAC, containing GRA4 gene from Toxoplasma gondii and its protective effect against T.gondii in mice. Methods Plasmid DNA, pVAC and pVACGRA4, was prepared in large quantity BALB/c mice were inoculated with plamid DNA by gene gun injection. Four weeks after the third immunization, the following tests were conducted: spleens were taken out from mice, the splenocytes were isolated and counted. Those splenocytes were used to detect the proliferative activity of T lymphcytes by means of MTT assay and the number of CD4+, CD8+ T cells with direct immunofluorescent assay. The splenocytes′ culture fluid was collected, and was used to detect the cytokines, IFNγ and IL4 by means of sandwich ELISA. Sera were isolated from the experimental mice, and were used to detect the titers of IgG antibody by ELISA. The rest of the experimental mice were challenged by tachyzoits of T. gondii RH isolate through intraperitonea injection four weeks after immunization. The living status of mice was observed. Results The proliferative activity of spleen T lymph cells were generated when stimulated by ConA, but there was no remarkable difference between immunized mice and control ones. The number of CD4+ T cells showed no obvious increase compared with that of the control groups but CD8+T cells were obviously increased (pVAC group, P<0.05; nontreated group, P<0.01). The value of CD4+/CD8+ was decreased compared with that of nontreated group (P<0.01). The A value of IFNγ was higher in the vaccinated group than that of the control groups, and the A value of IL4 was not significantly different among these groups. The specific IgG could be detected, but the titer was low. After challenged with the virulent RH T. gondii isolate, the survival rate and the living time of mice in the immunized group were also prolonged obviously (compared with nontreated group, P<0.05). Conclusi

关 键 词：弓形虫 GRA4 DNA免疫 小鼠 

分 类 号：R382.5[医药卫生—医学寄生虫学]