中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (10): 688-692.

• 论著 • 上一篇    下一篇

白介素2与梅毒螺旋体膜蛋白Gpd双价核酸疫苗的免疫活性及保护作用研究

余坚1,2,赵飞骏3,张晓红1,4,顾伟鸣1,5,刘双全6,曾铁兵1,7,张跃军1,7,吴移谋8   

  1. 1.
    2. 南华大学病原生物学研究所
    3. 南华大学医学院病原生物学研究所
    4. 南华大学医学院组织学与胚胎学教研室
    5. 上海皮肤病性病医院检验科
    6. 南华大学第一附属医院
    7. 湖南省南华大学病原生物研究所
    8. 衡阳南华大学医学院病原微生物学研究所
  • 收稿日期:2010-12-29 修回日期:2011-05-12 出版日期:2011-10-15 发布日期:2011-09-30
  • 通讯作者: 吴移谋 E-mail:yimouwu@sina.com
  • 基金资助:

    国家自然科学基金;湖南省科技厅重点课题;湖南省卫生厅课题

Analysis of immunogenicity and protective effect of a bivalent DNA vaccine expressing interleukin-2 and an outer membrane protein of Treponema pallidum （Gpd）

  • Received:2010-12-29 Revised:2011-05-12 Online:2011-10-15 Published:2011-09-30

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摘要：

目的 探讨白介素2（IL-2）基因与梅毒螺旋体（Treponema pallidum, Tp）Gpd抗原编码基因融合构建双价核酸疫苗免疫新西兰兔后的免疫应答效果及感染Tp后的保护作用。方法 定向克隆构建真核表达重组体pcDNA3.1（+）/Gpd-IL-2，与先期构建的pcDNA3.1（+）/Gpd真核表达重组体分别设为两个疫苗实验组，同时设pcDNA3.1（+）空质粒对照组及PBS对照组，共4组，每组18只雄性新西兰兔，肌肉多点注射初次免疫，于初次免疫后第10周各实验组兔皮下接种Tp 标准株进行感染实验，酶联免疫吸附试验（ELISA）检测不同时间点免疫兔特异性抗体产生水平和脾细胞IL-2及干扰素γ（IFN-γ）诱导水平，噻唑蓝法检测兔脾淋巴细胞增殖水平。结果 用pcDNA3.1（+）/Gpd-IL-2融合双价疫苗和pcDNA3.1（+）/Gpd单基因疫苗在免疫期间和感染期间均能检测到高滴度的IgG特异性抗体，最高滴度分别可达1 ∶ 4096和1 ∶ 1024（P值均 < 0.01）；两疫苗组之间在免疫及感染期间不同时间点比较差异也有统计学意义（P值均 < 0.01）；免疫后第8周双价融合核酸疫苗组及单基因核酸疫苗组兔脾细胞培养上清中IFN-γ分别为（447 ± 22.4） μg/L、（225 ± 17.6） μg/L，IL-2分别为（167 ± 15.7） μg/L、（110 ± 12.6） μg/L，均高于空质粒对照组和空白对照组（P值均 < 0.01）；感染期间不同时间点兔脾细胞受相应蛋白刺激均有明显增殖反应，检测指标均显著高于空质粒对照组和空白对照组（P值均 < 0.01）。早期感染皮损观察显示双价融合核酸疫苗组较之单基因疫苗组有着更低的皮损Tp检测阳性率（17.5%）、溃疡病灶发生率（15%）以及更短的皮损愈合时间。结论 用pcDNA3.1（+）/Gpd-IL-2双基因融合疫苗在兔体内能更有效地诱导保护性体液免疫和细胞免疫应答。

关键词: 免疫应答

Abstract:

Objective To investigate the immune response to and protective effect of a bivalent DNA vaccine expressing interleukin-2 （IL-2） and Gpd proteins in New Zealand rabbits. Methods Seventy-two male New Zealand white rabbits were equally and randomly divided into 4 groups to be immunized with recombinant plasmids pcDNA3.1（+）/Gpd-IL-2 （pcD/Gpd-IL-2）, pcDNA3.1（+）/Gpd （pcD/Gpd）, empty plasmid pcDNA3.1 （+） （pcD） and phosphate buffered saline （PBS）, respectively. Immunization was carried out by intramuscular injection at multiple sites with a 2-week interval for 3 times. On week 10 after the initial immunization, the rabbits were challenged intradermally with T. pallidum （Nichols strain）. Enzyme-linked immunosorbent assay （ELISA） was used to quantify the serum level of anti-Gpd antibodies in the rabbits and the level of IL-2 and interferon （IFN-γ） in the supernatant of Gpd protein-stimulated spleen cells from the rabbits at different time pionts. MTT assay was conducted to detect the proliferation response of spleen cells collected from the rabbits on day 0, 14, 28, 140 and 168 after the challenge. Results Compared with pcD and PBS, both the vaccines pcD/Gpd and pcD/Gpd-IL-2 elicited significantly higher levels of anti-Gpd IgG antibodies in rabbits at different time points during the vaccination and infection period, with the titers peaking at 1 ∶ 1024 and 1 ∶ 4096, respectively （both P < 0.01）. There were also significant differences in the serum levels of anti-Gpd IgG antibodies between the pcD/Gpd- and pcD/Gpd-IL-2-immunized rabbits at different time points （all P < 0.01）. The levels of IL-2 in the supernatant of spleen cells from pcD/Gpd- and pcD/Gpd-IL-2-immunized rabbits on week 8 after the immunization were 110 ± 12.6 and 167 ± 15.7 μg/L respectively, and those of IFN-γ were 225 ± 17.6 and 447 ± 22.4 μg/L respectively, significantly higher than those in that from the other two groups of rabbits （all P < 0.01）. Furthermore, an apparent proliferation response was observed in spleen cells from pcD/Gpd- and pcD/Gpd-IL-2-immunized rabbits with a higher stimulation index compared with pcD- and PBS-immunized rabbits （all P < 0.01）. Dark-field microscopic examination of early-stage infected lesions revealed that pcD/Gpd-IL-2-immunized rabbits had a lower detection rate （17.5%） of Tp from lesions, occurrence of ulcerative lesions （15%） and shorter curing time compared with pcD/Gpd-immunized rabbits. Conclusion The recombinant plasmid pcDNA3.1（+）/Gpd-IL-2 could induce protective humoral and cellular immune response more efficiently in rabbits.

Key words: immune-modulatory