HBcAg融合蛋白DNA疫苗的免疫保护力在小鼠模型上的研究

发布时间：2018-03-20 12:35

本文选题：乙型肝炎病毒　切入点：DNA疫苗　出处：《华中科技大学》2009年博士论文　论文类型：学位论文

【摘要】： 目的 1.构建3种免疫细胞表面分子的胞外段与乙型肝炎病毒核心抗原(HBcAg)融合蛋白的真核表达质粒：pwCTLA-4-HBc、pmCD27-HBc和pmCD40L-HBc,并检测其编码的融合蛋白在真核细胞中的表达和分泌情况。 2.了解以上构建的3种质粒：pwCTLA-4-HBc、pmCD27-HBc和pmCD40L-HBc以及2个对照质粒：单纯表达HBcAg的质粒pHcex和单纯表达HBeAg的质粒pHeex电击免疫诱导小鼠体液免疫应答和细胞免疫应答的情况。 3.了解质粒DNA疫苗pwCTLA-4-HBc和pHcex在小鼠尾静脉高压水注射感染性克隆模型上的免疫保护效力。方法 1.利用分子克隆技术分别构建真核表达质粒pwCTLA-4-HBc、pmCD27-HBc和pmCD40L-HBc。经酶切鉴定、间接免疫荧光方法、转染上清和细胞裂解液酶联免疫吸附实验(ELISA)检测融合蛋白表达,证实其构建成功。 2.将质粒pwCTLA-4-HBc、pmCD27-HBc、pmCD40L-HBc和对照质粒pHcex和pHeex采用肌肉注射联合电击免疫小鼠；采集系列血清间接ELISA有限稀释法检测小鼠产生的抗HBV核心抗原的总IgG和IgG2a/IgG1亚型抗体的滴度,了解其体液免疫应答情况和Th1/Th2型细胞免疫应答的情况；3次免疫后4周处死小鼠,用IFN-γ的酶联免疫斑点实验(ELISPOT)检测核心抗原CTL表位肽刺激下产生IFN-γ的小鼠脾脏淋巴细胞的相对数量,了解其特异性CTL细胞免疫应答的情况。 3.选取2种质粒pwCTLA-4-HBc和pHcex同样方案免疫小鼠,完成免疫周期后2周,尾静脉高压水注射pAAV/HBV1.pAAV/HBV 1.2;采集系列血清：ELISA定性检测小鼠产生HBsAg的情况,了解HBV表达情况；荧光实时定量PCR(realtime PCR)检测血清中HBV DNA的拷贝数(copy number),了解HBV复制情况。尾静脉高压水注射后29和33天分别放血和处死各组的半数小鼠,分离血清间接ELISA有限稀释法检测小鼠产生的抗HBV核心抗原和抗HBV表面抗原的总IgG和IgG2a/IgG1亚型抗体的滴度,了解其体液免疫应答情况和Th1/Th2型细胞免疫应答的情况；用IFN-γ的酶联免疫斑点实验(ELISPOT)检测核心抗原和表面抗原CTL表位肽刺激下产生IFN-γ的小鼠脾脏淋巴细胞的相对数量,了解其特异性CTL细胞免疫应答的情况。结果 1.构建质粒pwCTLA-4-HBc、pmCD27-HBc、pmCD40L-HBc和对照质粒pHcex、pHeex转染的BHK细胞可检测到HBc抗原特异性免疫荧光,转染Hela细胞后细胞裂解液和培养上清中针对HBeAg的ELISA反应强弱不等,但模式与预期吻合。 2.构建质粒pwCTLA-4-HBc、pmCD27-HBc、pmCD40L-HBc和对照质粒pHcex、pHeex免疫小鼠,3次免疫周期结束,小鼠均能产生强弱不等的针对HBV核心抗原的抗体应答,其产生抗体亚型的模式和动力学略有不同,pwCTLA-4-HBc能够引起Th1/Th2相对平衡的应答,并且在再次加强免疫后能保持这一平衡状态,而且能够较快诱导出较强的抗体应答；5种质粒免疫后小鼠脾淋巴细胞经核心抗原CTL表位肽刺激的IFN-γ分泌细胞形成的斑点数显著多于Mock免疫的对照组,其中pwCTLA-4-HBc和pHcex免疫组显著多于其它3组。 3.尾静脉高压水注射体内转染pAAV/HBV1.2,经pwCTLA-4-HBc和pHcex免疫的小鼠,能较快清除循环中的表面抗原,在体内转染后第5天外周血中的HBV DNA水平较对照组显著降低。经核心抗原CTL表位肽刺激的IFN-γ分泌细胞形成的斑点数DNA免疫组显著多于Mock组；针对核心抗原的Th1/Th2型、总IgG抗体反应DNA免疫组比Mock组高；pwCTLA4-HBc免疫组Th2型抗体反应高于pHcex免疫组,但Th1型抗体反应前者低于后者；针对表面抗原的Th1型和总IgG抗体反应：无统计学差异,Th2型抗体反应：2个DNA免疫组都比Mock组抗体滴度高，pwCTLA4-HBc免疫组高于pHcex免疫组。结论 1.成功构建真核表达质粒pwCTLA-4-HBc、pmCD27-HBc和pmCD40L-HBc。 2.以上构建的3种质粒与对照质粒pHcex和pHeex电击免疫小鼠,能够在小鼠体内诱导体液和细胞免疫应答,其中pwCTLA-4-HBc和pHcex的应答最强。 3. pwCTLA-4-HBc和pHcex电击免疫能够部分抑制体内转染的pAAV/HBV1.2在小鼠体内的复制,较快清除抗原,细胞免疫和体液免疫可能都参与了这一过程。
[Abstract]:objective
1., we constructed 3 eukaryotic expression plasmids pwCTLA-4-HBc, pmCD27-HBc and pmCD40L-HBc of the fusion protein of the outer segment of the immune cell surface molecule and the hepatitis B virus core antigen (HBcAg), and detected the expression and secretion of the encoded fusion protein in eukaryotic cells.
2. understand the above 3 plasmids: pwCTLA-4-HBc, pmCD27-HBc and pmCD40L-HBc, as well as 2 control plasmids: plasmid pHcex expressing HBcAg only and plasmid pHeex expressing HBeAg only. The immune response to humoral immune response and cellular immune response in mice is induced by electric shock.
3. the protective effect of plasmid DNA vaccine pwCTLA-4-HBc and pHcex on the infected clone model of mouse tail vein high pressure water injection was studied.
Method
1., molecular cloning technology was used to construct eukaryotic expression plasmid pwCTLA-4-HBc. PmCD27-HBc and pmCD40L-HBc. were identified by enzyme digestion. Indirect immunofluorescence assay was used to detect the fusion protein expression by supernatant and cell lysate enzyme linked immunosorbent assay (ELISA).
2. the plasmid pwCTLA-4-HBc, pmCD27-HBc by intramuscular injection combined with immune shock control mice pmCD40L-HBc and plasmid pHcex and pHeex were collected; anti HBV core antigen and produces a series of indirect ELISA detection method of limited dilution of serum of mice IgG and IgG2a/IgG1 subtype antibody, the solution of the humoral immune response and cellular immune response of Th1/Th2 type the mice were sacrificed; 4 weeks after the 3 immunization, IFN- gamma enzyme-linked immunospot (ELISPOT) assay the relative number of CTL core antigen table generation IFN- gamma peptide stimulated mouse spleen lymphocytes, the solution of the specific CTL cell immune response.
3. selected 2 kinds of plasmid pwCTLA-4-HBc and pHcex the same scheme of immunized mice 2 weeks after immunization period, intravenous injection of pAAV/HBV1.pAAV/HBV 1.2 high pressure water; serial serum: detection of ELISA mice produce qualitatively HBsAg, understand the expression of HBV; real-time fluorescence quantitative PCR (realtime PCR) HBV DNA in serum to detect copy number (copy number), understand the replication of HBV. Tail vein hydrodynamic injection after 29 and 33 days respectively, and half of the mice of each group were sacrificed by bloodletting, titer detection of mice serum separation of indirect ELISA method of limited dilution of anti HBV and anti HBV core antigen surface antigen IgG and IgG2a/IgG1 subtype antibody, the humoral immune responses and Th1/Th2 cell immune response; using IFN- gamma enzyme-linked immunospot (ELISPOT) detection of core antigen and surface antigen CTL IFN- gamma peptide stimulation in mice The relative number of lymphocytes in the spleen to understand the immune response of its specific CTL cells.
Result
1. plasmid pwCTLA-4-HBc, pmCD27-HBc, pmCD40L-HBc and control plasmid pHcex pHeex transfected BHK cells were detected HBc antigen specific immunofluorescence, Hela cells transfected with cell lysates and culture supernatants in response to ELISA or HBeAg range, but the mode and is expected to coincide.
2. plasmid pwCTLA-4-HBc, pmCD27-HBc, pmCD40L-HBc and control plasmid pHcex, pHeex mice after 3 times immunization period, the mice can produce different strength for HBV core antigen antibody response, the antibody subtype pattern and dynamics is slightly different, pwCTLA-4-HBc can cause the relative balance of Th1/Th2 responses, and keep this the equilibrium state after second immunization, and can rapidly induce strong immune responses; the control group were 5 plasmids of immunized mice spleen lymphocytes by IFN- gamma CTL core antigen epitope peptide stimulated the secretion of cell formation was significantly higher than Mock immunity, which pwCTLA-4-HBc and pHcex were significantly more than the other 3 groups.
3. high pressure water injection intravenous in vivo transfection of pAAV/HBV1.2, the pwCTLA-4-HBc and pHcex immunized mice, can quickly remove surface antigen in circulation in vivo, fifth days after transfection of HBV in peripheral blood of DNA was significantly lower than the control group. The number of spots DNA immunized by IFN- gamma CTL core antigen epitope peptide stimulated secretion the cell formation was significantly higher than in group Mock; for Th1/Th2 core antigen, IgG antibody response in DNA immunized group were higher than those of group Mock; pwCTLA4-HBc immune group Th2 antibody was higher than that of pHcex group, but Th1 antibody response to the former than the latter; for Th1 surface antigen and antibody response: Total IgG had no significant difference. Th2 antibody reaction: 2 DNA immune group than the Mock group with high antibody titer, pwCTLA4-HBc immune group was higher than that of pHcex immune group.
conclusion
1. the eukaryotic expression plasmids pwCTLA-4-HBc, pmCD27-HBc and pmCD40L-HBc. were successfully constructed
More than 2. of the 3 plasmids were immunized with the control plasmid pHcex and pHeex, which could induce humoral and cellular immune responses in mice, of which pwCTLA-4-HBc and pHcex had the strongest response.
3., pwCTLA-4-HBc and pHcex can partially inhibit the replication of pAAV/HBV1.2 transfected in vivo, and quickly eliminate antigen, cellular immunity and humoral immunity may be involved in this process.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

【相似文献】