CD40L对单纯疱疹病毒2型DNA疫苗的免疫增强作用研究

Plasmid CD40L Enhances Immune Response of HSV- 2 DNA Vaccine

目的研究重组质粒pc DNA3-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pc DNA3-Kan/CD40L。(2)体外细胞实验:检测重组质粒p CD40L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p K组、pg D组、pc CD40L+pg D组和p K+pg D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 Ig G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pc DNA3-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pc DNA3-Kan(P<0.05)。(2)体内实验结果:小鼠血清抗HSV-2 Ig G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)。pc DNA3-Kan/CD40L+pg D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pc DNA3-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)pc DNA3-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。

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 .