CAJ | 학술논문

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.
목적 탐토백개소2(IL-2)기인여매독라선체(Treponema pallidum,Tp)Gpd항원편마기인융합구건쌍개핵산역묘면역신서란토후적면역응답효과급감염Tp후적보호작용.방법 정향극륭구건진핵표체중조체pcDNA3.1(+)/Gpd-IL-2,여선기구건적pcDNA3.1(+)/Gpd진핵표체중조체분별설위량개역묘실험조,동시설pcDNA3.1(+)공질립대조조급PBS대조조,공4조,매조18지웅성신서란토,기육다점주사초차면역,우초차면역후제10주각실험조토피하접충Tp표준주진행감염실험,매련면역흡부시험(ELISA)검측불동시간점면역토특이성항체산생수평화비세포IL-2급간우소γ(IFN-γ)유도수평,새서람법검측토비림파세포증식수평.결과 용pcDNA3.1(+)/Gpd-IL-2융합쌍개역묘화pcDNA3.1(+)/Gpd단기인역묘재면역기간화감염기간균능검측도고적도적IgG특이성항체,최고적도분별가체1∶4096화1∶1024(P치균＜0.01)；량역묘조지간재면역급감염기간불동시간점비교차이야유통계학의의(P치균＜ 0.01)；면역후제8주쌍개융합핵산역묘조급단기인핵산역묘조토비세포배양상청중IFN-γ분별위(447±22.4)μg/L、(225±17.6)μg/L,IL-2분별위(167±15.7)μg/L、(110±12.6)μg/L,균고우공질립대조조화공백대조조(P치균＜ 0.01)；감염기간불동시간점토비세포수상응단백자격균유명현증식반응,검측지표균현저고우공질립대조조화공백대조조(P치균＜ 0.01).조기감염피손관찰현시쌍개융합핵산역묘조교지단기인역묘조유착경저적피손Tp검측양성솔(17.5％)、궤양병조발생솔(15％)이급경단적피손유합시간.결론 용pcDNA3.1(+)/Gpd-IL-2쌍기인융합역묘재토체내능경유효지유도보호성체액면역화세포면역응답.
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 supematant 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-γwere 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.