中国寄生虫学与寄生虫病杂志 ›› 2011, Vol. 29 ›› Issue (2): 8-122-125.

• 论著 • 上一篇    下一篇

杜氏利什曼原虫无鞭毛体蛋白基因重组质粒的免疫原性研究

李金福1,陈建平2 *,田玉2,杨志伟2,马莹2,胡孝素2   

  1. 1 贵阳医学院寄生虫学教研室,贵阳 550004;2 四川大学华西基础医学与法医学院寄生虫学教研室,成都 610041
  • 出版日期:2011-04-30 发布日期:2012-09-27

Immunogenicity of the Recombinant Plasmid of Leishmania donovani Amastin Gene

LI Jin-fu1,CHEN Jian-ping2 *, TIAN Yu2,YANG Zhi-wei2,MA Ying2,HU Xiao-su2   

  1. 1 Department of Parasitology,Guiyang Medical College,Guiyang 550004,China;2 Department of Parasitology,School of Preclinical and Forensic Medicine,Sichuan University,Chengdu 610041,China
  • Online:2011-04-30 Published:2012-09-27

摘要: 目的  研究杜氏利什曼原虫无鞭毛体蛋白基因重组质粒pcDNA3.1-amastin的免疫原性。 方法  将18只雌性BALB/c小鼠随机分为实验组和对照组,每组9只。两组分别肌肉注射重组质粒pcDNA3.1-amastin和空质粒pcDNA3.1(+) (50 μg/只),2周后同法加强免疫1次。加强免疫后第7、14和21天每组各取小鼠3只,内眦采血,分离血清,间接ELISA法测定血清中抗原特异性抗体水平。随后脱颈处死小鼠,无菌取脾,分离脾细胞,用刀豆球蛋白A刺激培养,3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测脾淋巴细胞增殖活性和细胞毒性T淋巴细胞(CTL)杀伤活性。双抗体夹心ELISA法检测脾淋巴细胞培养上清中γ干扰素(IFN-γ)、白细胞介素-2(IL-2)和IL-4的水平。 结果  加强免疫后第7、14和21天,实验组均检测到特异性IgG抗体,效价在1 ∶ 640以上,而对照组未检测到IgG抗体(P<0.01);实验组脾淋巴细胞增殖活性刺激指数分别为4.28±0.51、5.01±0.60和4.39±0.50,均高于对照组(P<0.01);实验组脾淋巴细胞培养上清中IFN-γ含量分别为(42.06±4.26)、(66.02±6.02)和(58.29±3.75) pg/ml,IL-2含量分别为(38.21±5.11)、(64.79±8.67)和(52.69±7.15) pg/ml,均高于对照组(P<0.01),两组均未检测到IL-4;实验组脾淋巴细胞CTL杀伤活性分别为(42.20±5.96)%、(63.66±5.44)%和(52.24±4.56)%,均高于对照组(P<0.01)。 结论  杜氏利什曼原虫无鞭毛体蛋白基因重组质粒pcDNA3.1-amastin免疫小鼠后可诱导其产生特异的体液免疫应答和Th1型细胞免疫应答。

关键词: 杜氏利什曼原虫, 无鞭毛体蛋白, 基因疫苗, 免疫原性

Abstract: Objective   To investigate the immunogenicity of recombinant plasmid pcDNA3.1-amastin with Leishmania donovani amastin gene.  Methods   Eighteen female BALB/c mice were randomly divided into experimental group and control group. Mice in experimental group and control group were intramuscularly injected with 50 μg recombinant plasmid pcDNA3.1-amastin and blank plasmid vector pcDNA3.1(+), respectively, and then received equivalent dose of plamid after 2 weeks. On days 7, 14, and 21 after the second immunization, serum samples were collected from 3 mice each group. The mice were then sacrificed, spleens were removed and splenocytes were collected. Serum antibody level was determined by indirect ELISA. Splenocyte proliferation responses and cytotoxicity of spleen-derived lymphocytes were analyzed by MTT colorimetry after stimulation with ConA. Level of IFN-γ, IL-2 and IL-4 in the splenocyte culture supernatants was determined by double antibody sandwich ELISA.   Results   On days 7, 14, and 21 after the second immunization, specific IgG antibody (more than 1 ∶ 640) was found in experimental group, but not in the control(P<0.01); stimulation index (SI) of spleen cells in experimental group (4.28±0.51, 5.01±0.60, and 4.39±0.50) was higher than that of control group (P<0.01); the level of IFN-γ[(42.06±4.26), (66.02±6.02), and (58.29±3.75) pg/ml] and IL-2 [(38.21±5.11), (64.79±8.67), and (52.69±7.15) pg/ml] in splenocyte culture supernatants of experimental group was higher than that of control group (P<0.01); IL-4 was not found in the two groups; cytotoxicity of spleen-derived lymphocytes in experimental group [(42.20±5.96)%, (63.66±5.44)%, and (52.24±4.56)%]was stronger than that of control (P<0.01).   Conclusion   The recombinant plasmid pcDNA3.1-amastin can induce specific humoral and Th1 type cellular immune responses in mice.

Key words: Leishmania donovani, Amastin, DNA vaccine, Immunogenicity