中国寄生虫学与寄生虫病杂志 ›› 2008, Vol. 26 ›› Issue (6): 3-416.

• 论著 • 上一篇    下一篇

血吸虫多基因非融合性膜锚定表达疫苗的研究

唐成武,刘朔婕,马彦彬,梁靓,郭萍,王淑玉,邹镇,高红,段秋红,程继忠,戴五星*   

  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2008-12-30 发布日期:2008-12-30
  • 通讯作者: 戴五星

A Multi-gene DNA Vaccine Encoding Non-fusion Membrane-anchoring Antigen against Schistosoma japonicum

TANG Cheng-wu,LIU Shuo-jie,MA Yan-bin,LIANG liang,GUO Ping,WANG Shu-yu,GAO Hong,DUAN Qiu-hong,CHENG Ji-zhong,DAI Wu-xing*   

  • Received:1900-01-01 Revised:1900-01-01 Online:2008-12-30 Published:2008-12-30

摘要: 目的 构建日本血吸虫多基因非融合性膜锚定表达疫苗pIRES-Sj97-Sj14-Sj26,并研究其免疫原性及免疫保护作用。 方法 构建日本血吸虫脂肪酸结合蛋白(Sj14)、谷胱甘肽-S-转移酶(Sj26)和副肌球蛋白(Sj97)的跨膜共表达质粒pIRES?鄄Sj97-Sj14-Sj26,转染HeLa细胞。通过RT?鄄PCR法检测Sj14、Sj26和Sj97 mRNA的表达,免疫荧光(IFA)检测Sj14、Sj26和Sj97蛋白的表达。用纯化的该质粒免疫BALB/c小鼠(100 μg/只),设生理盐水和空载体对照组,20只/组,每隔2周加强免疫1次,共免疫3次。末次免疫2周后,每组取10只小鼠,眼球取血并断颈处死,取脾淋巴细胞及腹腔巨噬细胞。分别检测免疫小鼠血清中总IgG抗体及脾淋巴细胞培养上清干扰素-γ(IFN-γ)水平,淋巴细胞刺激指数(SI)及 NO释放量,并分析脾淋巴细胞亚群。各组中余下小鼠每只经腹部感染40±1条尾蚴,45 d后剖杀,计数成虫数及肝卵数,计算减虫率和减卵率。结果 构建的质粒可在体外表达。疫苗组、生理盐水组和空白质粒组血清总IgG抗体含量分别为(5.62±0.64)、 (1.22±0.20)及(1.48±0.36) mg/ml,疫苗组与各对照组差异均具有统计学意义(P<0.01, P<0.05)。疫苗组、生理盐水组和空白质粒组小鼠腹腔巨噬细胞NO含量分别为(321.19±18.03)、 (184.12±11.05)及(213.51±15.93) nmol/ml,疫苗组与生理盐水组间差异有统计学意义(P<0.05)。疫苗组、生理盐水组、空白质粒组小鼠脾淋巴细胞刺激指数分别为(2.25±0.29)、(1.18±0.07)及(1.22±0.09),疫苗组显著升高(P<0.01)。疫苗组INF-γ水平与各对照组相比,差异具有统计学意义(P<0.01);CD4+和CD8+T细胞百分比明显升高。疫苗组减虫率和肝减卵率分别为39.9%和43.94%。 结论 pIRES-Sj97-Sj14-Sj26疫苗具有较强的免疫原性,可对日本血吸虫感染的小鼠产生一定保护作用。

关键词: 日本血吸虫, 多基因DNA疫苗, 免疫保护作用

Abstract: Objective To construct DNA vaccine (pIRES-Sj97-Sj14-Sj26) and study its immunogenicity and protective immunity against Schistosoma japonicum. Methods The plasmid pIRES-Sj97-Sj14-Sj26 containing fatty binding protein (Sj14), GST (Sj26) and paramyosin (Sj97) was constructed and expressed on the membrane. RT-PCR was used to detect the expression of Sj14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, and IFA for detecting the ex-pression of trans-membrane Sj14, Sj26 and Sj97. Sixty BALB/c mice were randomly divided into 3 groups. Mice in each group respectively received normal saline, pIRES blank vector and pIRES?-Sj97-Sj14-Sj26 by intramuscular injection. Two weeks after the 3rd immunization, 10 mice from each group were sacrificed and total IgG in serum and the level of IFN-γ were detected by ELISA and lymphocyte stimulating index(SI) by MTT. FCM was used to analyze the subgroups of splenocytes. The level of NO secreted by peritoneal macrophages was determined by nitrate reductase approaches. The left 10 mice in each group were challenged with (40±1) cercariae of S. japonicum by abdominal skin penetration. Forty-five days after challenge, mice were sacrificed,and numbers of recovered worms and hepatic eggs were counted. Results RT-PCR showed the expression of Sj14 mRNA, Sj26 mRNA and Sj97 mRNA. IFA proved the expression of Sj26, Sj14 and Sj97 protein. Level of total IgG in the vaccination group, saline group and pIRES blank vector group was (5.62±0.64)、 (1.22±0.20) and (1.48±0.36) mg/ml respectively, showing a statistical significance (P<0.01, P<0.05). The NO level in macrophages was (321.19±18.03)、(184.12±11.05) and(213.51±15.93) nmol/ml in the 3 groups respectively (P<0.05), and the lymphocyte stimulating index in the 3 groups was (2.25±0.29)、(1.18±0.07) and(1.22±0.09) respectively(P<0.01). The INF-γ level was higher in the vaccination group than others (P<0.01). The percentage of CD4+ and CD8+ T cells increased considerably (P<0.01). The vaccination group showed a worm reduction rate of 39.9% (P<0.01) and an egg reduction rate of 43.9% (P<0.01). Conclusion The vaccine candidate pIRES-Sj97-Sj14-Sj26 induces an immune protection in BALB/c mice against Schistosoma japonicum.

Key words: Schistosoma japonicum, Multi-gene DNA vaccine, Protective immunity