SARS冠状病毒免疫增强型核酸疫苗构建及免疫效果评价

发布时间：2018-03-16 20:24

本文选题：SARS　切入点：S1基因　出处：《东北农业大学》2011年硕士论文　论文类型：学位论文

【摘要】：本研究首先利用原核系统表达SARS-CoV S1蛋白和霍乱毒素B亚单位CTB蛋白制备了相应的多克隆抗体。然后,成功构建了真核表达质粒pVAX-S1、pVAX-CTB;并利用SOE-PCR技术将S1基因和CTB基因进行连接,得到融合基因真核表达质粒pVAX-S1-CTB。利用脂质体2000将各种真核表达质粒转染BHK-21细胞。间接免疫荧光的结果表明,各种真核表达质粒均能在哺乳动物细胞中表达,为后续动物免疫实验提供了理论基础。利用6周龄昆明鼠进行DNA疫苗免疫效果研究。在动物免疫实验中设计6个处理组,即PBS组(免疫灭菌PBS)、pVAX载体对照组(免疫真核载体pVAX1)、病毒对照S1组(免疫真核表达质粒pVAX-S1)、佐剂对照CTB组(免疫真核表达质粒pVAX-CTB)、联合免疫S1~+CTB组(联合免疫pVAX-S1和pVAX-CTB)和融合免疫S1-CTB组(免疫融合基因真核表达质粒pVAX-S1-CTB)。检测小鼠脾脏和外周血特异性T淋巴细胞增值效果、CD4~+和CD8~+T淋巴细胞数量变化、特异性CTL活性、IL-4和IFN-γ产量以及血清中特异性IgG抗体效价。瞬时转染实验结果表明,重组真核表达质粒pVAX-S1、pVAX-CTB和pVAX-S1-CTB均可在哺乳动物细胞中表达。免疫后42d,两疫苗组T淋巴细胞增殖效果明显高于各对照组,差异极显著(P0.01)。联合组脾脏和外周血CD4~+T细胞均在免疫后42d达到最大值;联合组脾脏和外周血CD8~+T细胞均在免疫后42d达到最大值。特异性细胞毒性作用结果表明,免疫后42d联合组及融合组细胞毒性效果明显,融合组效果尤佳。小鼠血清抗S1蛋白IgG含量检测结果表明,两疫苗组IgG含量均呈一定的增长趋势;免疫后42d,融合组IgG含量明显高于其他各组,差异极显著(p0.01)。外周血IFN-γ和IL-4含量检测结果表明,免疫后42d联合组及融合组以上两种细胞因子含量明显高于其他各对照组,且融合组刺激产生更多的细胞因子,二者差异显著(p0.05)。综上所述,通过本试验研究表明,CTB基因在配合SARS-CoV S1抗原基因免疫过程中,不但能够促进机体的细胞免疫应答能力,而且能够提高机体体液免疫应答的能力。本实验结果为进一步研究CTB的生物活性提供了实验依据,同时为探讨开发研制新型免疫增强型抗SARS病毒核酸疫苗提供了理论基础。
[Abstract]:In this study, SARS-CoV S1 protein and cholera toxin B subunit CTB protein were first expressed in prokaryotic system. Then the eukaryotic expression plasmid pVAX-S1 pVAX-CTB was successfully constructed, and the S1 gene and CTB gene were ligated by SOE-PCR technique. The eukaryotic expression plasmid pVAX-S1-CTB was obtained. Various eukaryotic expression plasmids were transfected into BHK-21 cells by liposome 2000. The results of indirect immunofluorescence showed that all eukaryotic expression plasmids could be expressed in mammalian cells. It provides a theoretical basis for the follow-up animal immune experiment. The immune effect of DNA vaccine in 6-week-old Kunming mice was studied. Those in PBS group (immunized PBSX pVAX vector control group), virus control S1 group (immune eukaryotic expression plasmid pVAX-S1), adjuvant control CTB group (immunized eukaryotic expression plasmid pVAX-CTBN), combined immunized S1 ~ CTB group (combined immunized pVAX-S1 and pVAX-CTB), In fusion immunized S1-CTB group (eukaryotic expression plasmid pVAX-S1-CTB), the changes of CD4 ~ and CD8 ~ T lymphocytes in spleen and peripheral blood of mice were detected. The specific CTL activity and the production of IL-4 and IFN- 纬, as well as the titer of specific IgG antibody in serum. The results of transient transfection showed that the recombinant eukaryotic expression plasmids pVAX-S1pVAX-CTB and pVAX-S1-CTB could be expressed in mammalian cells. After 42 days of immunization, the proliferation effect of T lymphocytes in the two vaccine groups was significantly higher than that in the control group. The difference was very significant (P 0.01). The CD4T cells in spleen and peripheral blood reached the maximum at 42 days after immunization in the combined group, and the CD8 ~ T cells in the spleen and peripheral blood of the combined group reached the maximum value at 42 days after immunization. The results of specific cytotoxicity showed that CD4T cells in the spleen and peripheral blood of the combined group reached the maximum value at 42 days after immunization. The cytotoxic effect of the combined group and the fusion group was obvious 42 days after immunization, especially in the fusion group. The detection of serum anti-S1 protein IgG content in mice showed that the IgG content of both vaccine groups showed a certain increasing trend. At 42 days after immunization, the IgG content in fusion group was significantly higher than that in other groups, and the difference was very significant (p 0.01). The results of detection of IFN- 纬 and IL-4 in peripheral blood showed that the levels of above two cytokines in combination group and fusion group were significantly higher than those in other control groups at 42 days after immunization. In the fusion group, more cytokines were produced by stimulation, and the difference between the two groups was significant (p 0.05). To sum up, this study shows that CTB gene can not only promote the cellular immune response ability in the process of immunization with SARS-CoV S1 antigen gene, The results provide a theoretical basis for the further study of the biological activity of CTB and the development of a novel immune-enhanced nucleic acid vaccine against SARS virus.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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