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

Immunogenicity of the Recombinant Plasmid of Leishmania donovani Amastin Gene

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(+)(50μg/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。

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（P0.01）;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（P0.01）;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（P0.01）;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（P0.01）.Conclusion The recombinant plasmid pcDNA3.1-amastin can induce specific humoral and Th1 type cellular immune responses in mice.