CAJ | 학술논문

目的研究重组质粒pcDNA3-Kan/CD40L辅佐HSV-2 DNA疫苗增强小鼠体液免疫和细胞免疫的作用效果,探讨其作为HSV-2 DNA疫苗佐剂的潜力. 方法 (1)构建鼠CD40L基因的重组真核表达质粒pcDNA3-Kan/CD40L. (2)体外细胞实验:检测重组质粒pCD40L刺激小鼠外周血淋巴细胞增殖情况和脾细胞分泌IFN-γ的能力. (3)体内动物实验:48只雌性BALB/c小鼠随机分为4个免疫组pK组、pgD组、pcCD40L+pgD组和pK+pgD组. 小鼠后腿肌内注射,共免疫2次,间隔3周. 末次免疫3周后,每组随机取4只小鼠进行致死剂量攻毒实验验证疫苗的保护作用.ELISA检测小鼠血清抗HSV-2 IgG抗体水平和趋化因子RANTES;流式细胞术检测全血中CD4 +和CD8 +T细胞百分率以及分泌IFN-γ和IL-4的T细胞的百分率;MTS法检测小鼠脾脏T细胞的增殖能力. 结果 (1)体外实验结果:重组质粒pcDNA3-Kan/CD40L对小鼠外周血淋巴细胞的增殖能力和刺激脾细胞分泌IFN-γ的能力均显著大于空质粒pcDNA3-Kan(P<0.05). (2)体内实验结果:小鼠血清抗HSV-2 IgG水平、趋化因子RANTES、脾淋巴细胞刺激指数和外周血CD4 +T细胞数和分泌IFN-γ的Th1细胞数均高于其他免疫组(P<0.05). pcDNA3-Kan/CD40L+pgD组预防小鼠感染HSV-2效果好于其他免疫组. 结论 (1)重组质粒pcDNA3-Kan/CD40L能够诱导外周血淋巴细胞增殖并刺激脾细胞分泌IFN-γ具有作为疫苗佐剂的潜力. (2) pcDNA3-Kan/CD40L可以辅助HSV-2 DNA疫苗诱导BALB/c小鼠产生特异性抗HSV-2的体液免疫和细胞免疫,具备作为HSV -2 DNA疫苗免疫佐剂的能力.
목적 연구중조질립pcDNA3-Kan/CD40L보좌HSV-2 DNA역묘증강소서체액면역화세포면역적작용효과,탐토기작위HSV-2 DNA역묘좌제적잠력. 방법 (1)구건서CD40L기인적중조진핵표체질립pcDNA3-Kan/CD40L. (2)체외세포실험:검측중조질립pCD40L자격소서외주혈림파세포증식정황화비세포분비IFN-γ적능력. (3)체내동물실험:48지자성BALB/c소서수궤분위4개면역조pK조、pgD조、pcCD40L+pgD조화pK+pgD조. 소서후퇴기내주사,공면역2차,간격3주. 말차면역3주후,매조수궤취4지소서진행치사제량공독실험험증역묘적보호작용.ELISA검측소서혈청항HSV-2 IgG항체수평화추화인자RANTES;류식세포술검측전혈중CD4 +화CD8 +T세포백분솔이급분비IFN-γ화IL-4적T세포적백분솔;MTS법검측소서비장T세포적증식능력. 결과 (1)체외실험결과:중조질립pcDNA3-Kan/CD40L대소서외주혈림파세포적증식능력화자격비세포분비IFN-γ적능력균현저대우공질립pcDNA3-Kan(P<0.05). (2)체내실험결과:소서혈청항HSV-2 IgG수평、추화인자RANTES、비림파세포자격지수화외주혈CD4 +T세포수화분비IFN-γ적Th1세포수균고우기타면역조(P<0.05). pcDNA3-Kan/CD40L+pgD조예방소서감염HSV-2효과호우기타면역조. 결론 (1)중조질립pcDNA3-Kan/CD40L능구유도외주혈림파세포증식병자격비세포분비IFN-γ구유작위역묘좌제적잠력. (2) pcDNA3-Kan/CD40L가이보조HSV-2 DNA역묘유도BALB/c소서산생특이성항HSV-2적체액면역화세포면역,구비작위HSV -2 DNA역묘면역좌제적능력.
Objective To detect HSV-2-specific humoral immunological response and cellular immunological response in BALB /c mice which were induced by plasmid CD40L-adjuvanted HSV-2 DNA vaccine.Methods ①The murine CD40L gene transcript was inserted into the pcDNA3 vector to obtain the recombinant plasmid pcDNA 3-Kan/CD40L.②In vitro study: MTS colorimetric method was employed in the detection of the rat peripheral blood lymphocytes proliferation and SYBRgreen qPCR assay was used to test the IFN -γsecretion ability of spleen cells .③In vivo study:Forty eight female BALB/c mice were randomly divided into four groups:pKan, pgD, pCD40L+pgD and pKan+pgD, and inoculated through intramuscular immunization at the weeks 0 and 3.After 6 weeks the protection given to the mice was assayed by a fatal dose of HSV -2.The humoral immunological response and the cellular immunological response were detected by enzyme linked immunosorbent assay (ELISA), MTS colorimetric assay and flow cytometry (FCM).Results ①The a-bility of stimulation lymphocytes proliferation of rat PBMC and IFN -γlevel in spleen cells of cDNA3-Kan/CD40L group were signifi-cantly better than that of pK group (P<0.05).②The level of anti -HSV-2 IgG, RANTES, stimulation index (SI), CD4 +and IFN-γin pCD40L+pgD group were significantly higher than anther groups .Furthermore,mice of pCD40L+pgD group were prophylactically protected from challenge with a high dose of HSV -2.Conclusion ①The potential of pcDNA3-Kan/CD40L could be used as an adju-vant in vaccines.②The potential of pCD40L-adjuvantd HSV-2 DNA vaccine could induce systemic humoral immune responses and cel-lular immune responses intramuscular vaccinated mice .