【作者】 刘晔华；

【导师】 朱道银；

【作者基本信息】 重庆医科大学 ， 病原生物学， 2005， 硕士

【摘要】 目的: 本研究拟构建编码结核分枝杆菌 H37Rv Ag85B DNA 疫苗,一方面将该疫苗经小鼠鼻粘膜免疫,探讨其在小鼠体内诱导的免疫应答及机制;另一方面研究在 BCG初免后,将该疫苗滴鼻免疫进行增强免疫所诱导的免疫应答及机理。以上设计试图为结核病疫苗寻找最佳免疫途径和方案,为结核病的粘膜免疫防治提供理论和实验依据。 方法: 1、用 PCR 和基因克隆技术构建真核表达质粒 pcD85B,Ag85B 编码基因亚克隆 入原核表达质粒 pET32a 中, 纯化重组的 Ag85B 蛋白,制备 Ag85B 抗血清; pcD3.1(+)、pcD85B、转染 COS-7 细胞,用 RT-PCR、ELISA 和斑点印迹的方 法鉴定目的基因的表达;pcD3.1(+)、pcD85B 免疫 C57BL/6 小鼠,ELISA 法 测量免疫后相应的 PPD 特异性抗体和脾淋巴细胞 IL-4 以及 IFN-γ分泌水 平,MTT 法检测 PPD 特异性脾淋巴细胞增殖能力,评价 DNA 疫苗的免疫原性。 2、将 PBS、pcD3.1、pcD85B(滴鼻时溶于 PBS 或者以脂质体包裹)分别以肌 注和滴鼻免疫 C57BL/6 小鼠,采用 ELISA 法测量免疫后相应的 PPD 特异性 抗体和脾淋巴细胞 IL-4 以及 IFN-γ分泌水平; CTL 试验评估小鼠 CD8+T 细胞的杀伤活性。 3、C57BL/6 小鼠分三组,一组皮下接种 BCG,另外两组在 BCG 接种 14 周后, 分别以脂质体包裹的 pcD3.1、pcD85B 滴鼻进行增强免疫;采用 ELISA 法测 量免疫后相应的 PPD 特异性抗体和脾淋巴细胞 IL-4 以及 IFN-γ分泌水平; CTL 试验评估小鼠 CD8+T 细胞的杀伤活性。 结果:1、 经过限制性内切酶酶切及DNA双向测序证实pcD85B真核表达质粒构建成 功;用重组的 Ag85B 蛋白制备的鼠抗 Ag85B 血清效价为 1:64;重组质粒 4转染 COS-7 细胞后, Ag85B 多克隆抗体通过 ELISA 法检测到细胞培养上清液 Ag85B 的表达,对照组为为阴性反应;pcD85B 免疫 C57BL/6 小鼠, 末次免疫后 4 周各组 PPD 特异性抗体水平均有不同程度增高,脾淋巴细胞PPD 特异性 IFN-γ分泌水平以及脾淋巴细胞增殖能力均增高。2、 脂质体包裹的 pcD85B 滴鼻途径①可诱导局部粘膜免疫 BALF 中的 sIgA 抗体滴度最高(P<0.05),但血清中 IgA 的抗体滴度不高;②可诱导产生 Th1型细胞免疫。该组 IFN-γ水平较高,与同样用脂质体包裹的载体对照比,有显著差异(P<0.05),与肌注组无显著差异(P>0.05),低于 BCG 组(P<0.05)。溶于 PBS 的 DNA 疫苗组 IFN-γ水平非常低,低于载体对照组、肌注组和 BCG 组(P<0.05)。该组 IL-4 水平很低,与肌注组、阳性对照BCG 组、以及生理盐水 DNA 滴鼻组相比,都有显著差异(P<0.05)。该试验组 IgG2a 抗体滴度较高,和肌注组、阳性对照 BCG 组相比无显著差异(P>0.05);与生理盐水 DNA 滴鼻组有显著差异(P<0.05)。③体液免疫较弱 血清 IgG 抗体水平低于肌注组和 BCG 组。④CTL 试验 通过不同组别颗粒酯酶分泌百分率(%)来反映细胞毒性 T 细胞的功能。该指标在不同免疫途径区别不大,除与 PBS 对照有区别外,与载体对照差别甚微。3、 BCG 皮下初免,pcD85B 滴鼻增强的方案强于单纯 BCG 皮下免疫对照组,表现在①粘膜免疫 它的 BALF 中的 sIgA 抗体滴度高于 BCG 组(P<0.05)。②体液免疫 该组的特异性 IgG 抗体滴度略低于 BCG 组 ③Th1 型细胞免疫检测 PPD 刺激的脾淋巴细胞培养上清,其 IFN-γ 水平稍低于 BCG 组,但明显高于 pcD85B 肌注组;IL-4 水平明显低于 BCG 组和肌注组,特异性 IgG2a的抗体滴度高于 BCG 组(P<0.05)。④细胞杀伤活性 通过不同组别颗粒酯酶分泌百分率(%)来反映细胞毒性 T 细胞的功能。该组颗粒酯酶分泌百分率 86.3946%,低于 BCG 组的 93.3333%。结论:1、 pcD85B 疫苗构建成功, 能在 COS-7 细胞中分泌表达,可诱导小鼠产生特异性体液免疫和细胞免疫。2、 脂质体包裹的 DNA 疫苗组与同等剂量的肌注组产生的系统免疫差别不大,弱于 BCG 组;溶于 PBS 的 DNA 疫苗组弱于同等剂量的脂质体组和肌注组,也弱于 BCG 组;DNA 疫苗可以进行滴鼻尝试。更多还原

【Abstract】 Objective We aimed to construct a DNA vaccine encoding Mtb Ag85B, then appliedto the C57BL/6 murine model by two different strategies. One is in aplasmid-lipid complex delivered by the intranasal route; the other is aBCG/plasmid-lipid complex prime/boost regime. It would lay the groundworkfor a new tuberculosis vaccine and provide data for a further study atmucosal immunogenicity. Methods 1. The eukaryotic expression plasmid pcD85B was constructed. The recombinantplsmid was transfected into COS-7 cells and the expressed target protein was tested byRT-PCR, ELISA and dot blotting. The C57BL/6 mice were immunized with pcD85B,then the total serum IgG, the proliferation of splenocytes and IFN-γlevel were detectedby MTT and ELISA to analyze the specific immune responses induced by DNAvaccination. 2. C57BL/6 mice were intranasally immunized with pcD85B, then the total serumIgG、IgA、IgG2a,the BALF sIgA and IFN-γ level 、IL-4 level and cytoxicity ofCD8+ T cells were tested by ELISA to analyze the specific immune responsesinduced by DNA vaccination. 3. C57BL/6 mice were vaccined s.c. with 4×105 CFU of BCG, after 14 wk,micewere boosted i.n. with pcD85B-lipid complex. Ten days later, serum was detectedfor the total IgG and IgA and IgG2a isotype, BALF for the sIgA; supernants of spleencells were assayed for the IFN-γ、IL-4 level and cytoxicity response. All of thesementioned were done by ELISA. Results 1. pcD85B was constructed and the inserted target gene was confirmed byrestriction enzyme analysis as well as DNA sequencing. The supernatant of COS-7 cellcultures transfected with pcD85B, showed positive reaction to rAg85B antibodies byELISA. The antibody titer against PPD, the proliferation of antigen-specific lymphocyteand IFN-γ production in DNA vaccination group were higher than those of the controlgroups(P<0.05). 2.Among the intranasally immunized C57BL/6 mice ,group of plasmid-lipidcomplex ①induced local mucosal immunity, the level of BALF antibody sIgA is thehighest(P<0.05).②induced Th1 type cell immunity, IFN-γ production was similar toi.m. group,while was lower than the positive control(P<0.05); IL-4 level under thelower detection limitation(P<0.05) ; the level of serum antibody IgG2a is thehighest(P<0.05).③ total serum antibody IgG level lower than i.m. group and thepositive control(P<0.05). ④ a controversial cytotoxicity responses. Perhaps themethod for this test is unmature,no variance was observed between the vector controland the plasmid groups,only the cytotoxicity responses in the positive control wasvaluable . 3. The BCG/ plasmid-lipid complex prime/boost group was induced a higherantibody titer of BALF sIgA and serum antibody isotype IgA、IgG2a than the BCGcontrol(P<0.05); while the serum antibody IgG was lower; IFN-γ production waslower than the BCG control(P<0.05); IL-4 level under the lower detectionlimitation(P<0.05) ; spleen cells’cytotoxicity responses was a little lower than theBCG control.Conclusions1. Recombinant pcD85B was constructed correctly. Target protein wasexpressed in COS-7 cells. The plasmid could induce specific humoral andcellular immune responses in mice after DNA vaccination.2. pcD85B -lipid complex delivered by the intranasal route could inducemucosal immunity, which is an advantage compared to the i.m. group.but itstotal serum IgG and Th1 type cell immunity couldn’t transcend the BCGgroup .3. BCG/ plasmid-lipid complex prime/boost strategy was superior to s.c.BCG、 i.m. and i.n. pcD85B regimes;it has a promising future.更多还原