HSV-2多表位复合DNA疫苗的构建与诱导小鼠免疫效果的研究

发布时间：2018-08-12 11:14

【摘要】： 目的构建单纯疱疹病毒2型(HSV-2)糖蛋白gD、gB、gC和早期表达蛋白ICP 27的多表位复合DNA疫苗和免疫佐剂IL2质粒，以及在复合疫苗质粒基础上修饰的信号肽质粒与泛素化质粒，并探讨其诱导小鼠机体免疫应答的能力。方法国内HSV-2野生株经PCR鉴定后利用PCR技术从其基因组中扩增出HSV-2 ICP27 377-459、gD146-179、gD223-306、gB529-606、gC247-282、gD1-77六个基因片段，PCR鉴定后按先后顺序，采用多基因片段一步酶切连接法获得HV融合基因片段，经pGEMT载体克隆后定向插入真核表达质粒pcDNA3.1载体中，构建出重组真核表达质粒HV-pcDNA3.1，并对其进行酶切分析及测序鉴定。 PCR扩增gD146-179信号肽片段，进一步酶切连接构建重组质粒gDs-HV-his-pcDNA3.1，并对其进行酶切分析及测序鉴定。 PCR扩增人基因组DNA和HSV-2基因组中泛素全长基因和HSV-2 ICP27 377-459，酶切连接获得融合基因片段，插入真核表达质粒pcDNA3.1载体中，构建出重组质粒Ub-ICP-pcDNA3.1，并对其进行酶切分析及测序鉴定。 PCR扩增人IL-2信号肽基因和人IL-2尾段基因，取上述两段PCR扩增产物作为融合PCR的模板，扩增IL-2 cDNA全长基因，连入pcDNA3.1质粒中，构建出重组质粒IL-2-pcDNA3.1，并对其进行酶切分析及测序鉴定。 C57／BL6雌性6周龄小鼠40只，随机分为8组，每组均5只，根据免疫的质粒类型和免疫方式的不同，各组依次为pcDNA3.1(空质粒对照)、HV-pcDNA3.1、HV-pcDNA3.1+IL2-pcDNA3.1、gDs-HV-his-pcDNA3.1、gDs-HV-his-pcDNA3.1+IL2-pcDNA3.1、Ub-ICP-pcDNA3.1和HV-pcDNA3.1、HV-pcDNA3.1+IL2-pcDNA3.1。免疫前先用特异性抗原刺激，前6组小鼠每只用5ug的相应质粒(与IL2共表达的组用5+5ug的量)和1ug的脂质体充分混合，，20min后转染到同批次C57／BL6雌性鼠脾淋巴细胞中，37℃、5％CO_2培养4h后注射入小鼠皮下；后2组小鼠也用5ug的相应质粒(与IL2共表达的组用5+5ug的量)直接注射到双侧胫骨前肌中。14天后正式免疫，前6组小鼠每只用100ug的相应质粒(与IL2共表达的组用100+100ug的量)和10ug的脂质体充分混合，20min后注入小鼠双侧胫骨前肌中；后2组小鼠也用100ug的相应质粒(与IL2共表达的组用100+100ug的量)直接注入小鼠双侧胫骨前肌中。14天后再加强免疫一次，方法和剂量与正式免疫完全一致。加强免疫后3周小鼠断尾取血，分离血清。断颈处死小鼠取脾，分离淋巴细胞。ELISA检测小鼠血清HSV-2特异性IgG、IL-2和IFN-γ，MTT法检测小鼠脾T淋巴细胞特异性增殖，乳酸脱氢酶法检测杀伤性T淋巴细胞(CTL)功能和流式细胞仪检测CD4+／CD8+T细胞亚群分类。结果构建的HV-pcDNA3.1质粒、IL2-pcDNA3.1质粒、Ub-ICP-pcDNA3.1质粒、gDs-HV-his-pcDNA3.1质粒经酶切鉴定并进行DNA测序，测序结果正确，表明已成功构建出HSV-2多表位复合DNA疫苗。免疫小鼠后实验结果显示：gDs-HV-his-pcDNA3.1与IL2-pcDNA3.1联合免疫后体液免疫最强，特异性IgG效价达到400倍左右；T细胞表面信号分子CD4的阳性率与空载体pcDNA3.1组相比有显著差异(P＜0.05)。而HV-pcDNA3.1与IL2-pcDNA3.1联合免疫后细胞免疫水平最强，特异性T淋巴细胞的刺激指数SI达到2.75左右，而空质粒对照组为0.7左右；淋巴细胞杀伤实验CTL反应的杀伤率(效靶比为50：1时)接近50％，而空质粒对照组为8％左右；T细胞表面信号分子CD8的阳性率与空载体pcDNA3.1组相比也有显著差异(P＜0.05)，但CD4~+／CD8~+的值无显著差异(P＞0.05)；血清中细胞因子IL2和IFN-γ含量检测结果显示：IL2水平(ng／L)为1421.16±220.98，而空质粒对照组为685.21±104.20；IFN-γ水平(ng／L)为1956.19±219.60，而空质粒对照组为547.71±189.33。另一组有关不同免疫方式比较的实验总体上看则说明了脂质体包裹的DNA疫苗比单纯的裸DNA注射法能显著提高免疫活性，若用脂质体包裹起来同时转染IL2质粒和复合疫苗质粒则更强。结论 1．成功构建了HSV-2多表位复合DNA疫苗，包括质粒HV-pcDNA3.1、信号肽质粒gDs-HV-his-pcDNA3.1、泛素化质粒Ub-ICP-pcDNA3.1和佐剂质粒IL2-pcDNA3.1。 2．小鼠动物实验表明，我们构建的疫苗质粒HV-pcDNA3.1和信号肽质粒gDs-HV-his-pcDNA3.1能有效诱导小鼠体液免疫和细胞免疫应答，与佐剂质粒IL2-pcDNA3.1共同免疫效果更强。信号肽质粒gDs-HV-his-pcDNA3.1与佐剂质粒IL2-pcDNA3.1共注射诱导的体液免疫反应最强，而质粒HV-pcDNA3.1与佐剂质粒IL2-pcDNA3.1共注射诱导的细胞免疫反应最强。 3．质粒经脂质体包裹并在免疫前通过淋巴细胞体外转染小鼠皮下预注射方法比单纯裸质粒注射方法能明显提高体液免疫和细胞免疫反应。 4．泛素化质粒Ub-ICP-pcDNA3.1不能诱导有效的体液免疫和细胞免疫应答。
[Abstract]:Objective To construct a multi-epitope compound DNA vaccine and immune adjuvant IL-2 plasmid of herpes simplex virus-2 (HSV-2) glycoprotein gD, gB, gC and early expression protein ICP-27, as well as a signal peptide plasmid and a ubiquitination plasmid modified on the basis of the composite vaccine plasmid, and to investigate the ability of the plasmid to induce immune response in mice.
Methods Six gene fragments of HSV-2 ICP27 377-459, gD146-179, gD223-306, gB529-606, gC247-282 and gD1-77 were amplified from the genome of HSV-2 wild strain in China by PCR. The fragments of HV fusion gene were cloned by pGEMT vector and inserted directionally. The recombinant eukaryotic expression plasmid HV-pcDNA3.1 was constructed and identified by enzyme digestion and sequencing.
The gD146-179 signal peptide fragment was amplified by PCR, and the recombinant plasmid gDs-HV-his-pcDNA3.1 was constructed by enzyme digestion and ligation.
Full-length ubiquitin gene and HSV-2 ICP27 377-459 in human genome DNA and HSV-2 genome were amplified by PCR. Fusion gene fragments were obtained by enzyme digestion and ligation. The recombinant plasmid Ub-ICP-pcDNA3.1 was constructed by inserting into eukaryotic expression plasmid pcDNA3.1 vector. The recombinant plasmid Ub-ICP-pcDNA3.1 was analyzed by enzyme digestion and sequenced.
The full-length gene of IL-2 cDNA was amplified by PCR. The recombinant plasmid IL-2-pcDNA3.1 was constructed and identified by restriction enzyme digestion and sequencing.
C57/BL6 female mice aged 6 weeks were randomly divided into 8 groups. Each group consistof 5 mice randomly divided into 8 groups. According to the different immunplasmid types and immunimmunmodes, each group was pcDNA3.1 (blankplasmid control), HV-pcDNA3.1, HV-pcDNA3.1, HV-pcDNA3.1+IL2-pcDNA3.1, HV-pcDNA3.1+IL2-pcDNA3.1, HV-pcDNA3.1+IL2-pcDNA3.1, gDs-HV-HV-his-pcDNA3.1, gDs-HV-HV-pcDNA3.1+his-pcDNA3.1, gDDs-HDs-HV-HV-pcDNA3.1+IL2-ppcDNA 3.1.
The mice in the first six groups were stimulated with specific antigens before immunization. Each mouse in the first six groups was adequately mixed with the corresponding plasmids of 5+5 UG (the amount of 5+5 UG in the group co-expressed with IL2) and the liposome of 1 ug. The spleen lymphocytes of the same batch of C57/BL6 female mice were transfected 20 minutes later. The spleen lymphocytes were cultured at 37 C for 4 hours with 5% CO_2 and injected subcutaneously into the mice in the second two groups with the corresponding plasmids of 5 UG (the L2 co-expression group was injected directly into bilateral anterior tibial muscles with the dose of 5+5ug. 14 days later, the mice in the first six groups were immunized with 100 UG of corresponding plasmids (100+100ug of the group co-expressed with IL2) and 10 UG of liposome. After 20 minutes, the mice in the latter two groups were injected into bilateral anterior tibial muscles with 100 UG of corresponding plasmids (with IL2). The expression group was injected directly into the bilateral anterior tibial muscles of mice with the dose of 100 + 100 ug. The method and dose of the immunotherapy were completely consistent with the formal immunization.
Three weeks after the immunization, the mice were killed by neck-cutting and the spleens were taken out for lymphocyte isolation. The specific IgG, IL-2 and IFN-gamma of HSV-2 in serum were detected by ELISA, the specific proliferation of splenic T lymphocyte was detected by MTT, the killer T lymphocyte (CTL) function was detected by lactate dehydrogenase assay and the CD4 + / CD8 + T cell was detected by flow cytometry. Classification of subgroups.
Results The constructed HV-pcDNA3.1 plasmid, IL2-pcDNA3.1 plasmid, Ub-ICP-pcDNA3.1 plasmid and gDs-HV-his-pcDNA3.1 plasmid were identified by enzyme digestion and DNA sequencing. The results showed that the HSV-2 multi-epitope compound DNA vaccine was successfully constructed.
The results showed that the combined immunization of gDs-HV-his-pcDNA3.1 and IL2-pcDNA3.1 had the strongest humoral immunity, and the specific IgG titer was about 400 times. The positive rate of CD4 on T cell surface was significantly different from that of pcDNA3.1 group (P < 0.05). The stimulus index SI of specific T lymphocyte reached about 2.75, while that of blank plasmid control group was about 0.7; the killing rate of CTL reaction in lymphocyte killing experiment (the effective target ratio was 50:1) was close to 50%, while that of blank plasmid control group was about 8%; the positive rate of CD 8 on T cell surface signal molecule was also obvious compared with blank vector pcDNA3.1 group. There was significant difference (P < 0.05), but there was no significant difference (P > 0.05) in CD4 + / CD8 +; the levels of IL 2 and IFN - gamma in serum were 1421.16 + 220.98, 685.21 + 104.20 in blank plasmid control group, 1956.19 + 219.60 in ng / L, 547.71 + 189.33 in empty plasmid control group.
Another group of experiments comparing different immune modes showed that liposome-encapsulated DNA vaccines could significantly improve the immune activity compared with bare DNA vaccines, and the liposome-encapsulated DNA vaccines could transfect IL2 plasmids and composite vaccine plasmids simultaneously.
conclusion
1. HSV-2 multi-epitope DNA vaccine was successfully constructed, including plasmid HV-pcDNA3.1, signal peptide plasmid gDs-HV-his-pcDNA3.1, ubiquitinated plasmid Ub-ICP-pcDNA3.1 and adjuvant plasmid IL2-pcDNA3.1.
2. Animal experiments in mice showed that the Vaccine Plasmid HV-pcDNA3.1 and the signal peptide plasmid gDs-HV-his-pcDNA3.1 could effectively induce humoral and cellular immune responses in mice, and the co-immunization effect was stronger with the adjuvant plasmid IL2-pcDNA3.1. The signal peptide plasmid gDs-HV-his-pcDNA3.1 and the adjuvant plasmid IL2-pcDNA3.1 co-injected to induce humoral immunity. The strongest immune response was induced by co-injection of plasmid HV-pcDNA3.1 and adjuvant plasmid IL2-pcDNA3.1.
3. Subcutaneous pre-injection of plasmid encapsulated in liposome and transfected with lymphocytes in vitro before immunization can significantly improve humoral and cellular immune responses compared with bare plasmid injection.
4. ubiquitin plasmid Ub-ICP-pcDNA3.1 can not induce effective humoral and cellular immune responses.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392;R752.1

【相似文献】