猪IL-12和IL-18对TGEV S基因核酸疫苗免疫效应的研究

发布时间：2018-01-15 18:32

本文关键词：猪IL-12和IL-18对TGEV S基因核酸疫苗免疫效应的研究　出处：《东北农业大学》2011年硕士论文　论文类型：学位论文

【摘要】：猪传染性胃肠炎(Porcine transmissible gastroenteritis,TGE)是由猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)引起的一种高度接触性、急性消化道传染病。预防该病主要是靠接种疫苗。本实验分别将猪传染性胃肠炎病毒S基因与猪IL-12基因亚克隆至真核表达载体pVAX1中,构建了两个重组真核表达质粒pVAX1-TGEV S1和 pVAX1-pIL-12。通过脂质体转染法将pVAX1-TGEV S1和pVAX1-pIL-12转入BHK细胞中进行了表达,且表达的蛋白可以被多克隆抗体所检测。以pVAX1-TGEV S1和pVAX1-pIL-12重组真核表达质粒免疫小鼠。共分为9组,分别是空白组、PBS组、pVAX1组、pIL-12组、pIL-12+pIL-18组、TGEV S组、TGEV S+pIL-12组、TGEV S+pIL-12+pIL-18组和疫苗组。利用淋巴细胞转化试验、酶联免疫吸附试验、流式细胞仪技术、检测特异性CTL活性实验、病毒中和试验和细胞因子检测试验对不同处理组小鼠的各项免疫指标进行了检测。试验结果表明,各免疫组小鼠的各项免疫指标均比pVAX1组、PBS组和空白组高,加入佐剂联合免疫组的结果尤为显著。在淋巴细胞增殖试验、酶联免疫吸附试验、病毒中和试验、细胞因子检测试验、T细胞亚型CD4+、CD8+数量变化检测以及特异性CTL活性的检测试验中,加佐剂抗原组的效果明显要优于其他组别。病毒中和试验和酶联免疫吸附试验检测抗TGEV IgG的抗体结果显示,疫苗组产生抗体的效果优于其他各组。流式细胞术检测和细胞因子检测试验中发现佐剂免疫组对机体免疫应答也有辅助的增强作用。本实验还构建了一个pGEX-6p-pIL-12(p40)原核表达质粒,质粒缺失pIL-12 N(p40)端22 aa信号肽序列。在大肠杆菌中获得了高效表达的pIL-12(p40)蛋白。纯化后蛋白免疫新西兰兔获得兔源多克隆抗体。酶联免疫吸附试验和免疫荧光结果表明pIL-15蛋白与多克隆抗体具有良好的特异性反应。本研究首次将pIL-12基因、pIL-18基因与TGEV S基因质粒联合免疫。实验结果表明pIL-12基因以及pIL-12基因和pIL-18基因联合作为TGEV S抗原基因的分子佐剂,不但能促进机体的细胞免疫应答,而且能够提高机体体液免疫应答,进而提高了机体的免疫反应。为进一步研究细胞因子作为免疫佐剂的作用提供了依据,为新型核酸疫苗的研究奠定了理论基础。
[Abstract]:Porcine transmissible gastroenteritis. TGEV is caused by transmissible gastroenteritis virus (TGEV) of swine transmissible gastroenteritis virus (TGEV). Acute gastroenteritis virus S gene and porcine IL-12 gene were subcloned into eukaryotic expression vector pVAX1. Two recombinant eukaryotic expression plasmids pVAX1-TGEV S1 and 2 were constructed. PVAX1-pIL-12. PVAX1-TGEV S1 and pVAX1-pIL-12 were transfected into BHK cells by liposome transfection. The expressed protein could be detected by polyclonal antibody. Mice were immunized with pVAX1-TGEV S1 and pVAX1-pIL-12 recombinant eukaryotic expression plasmids. The mice were divided into 9 groups. PVAX1 group, pIL-12 pIL-18 group and TGEV S pIL-12 group respectively. TGEV S pIL-12 pIL-18 group and vaccine group. Specific CTL activity was detected by lymphocyte transformation test, enzyme-linked immunosorbent assay and flow cytometry. Virus neutralization test and cytokine test were used to detect the immune indexes of mice treated with different treatments. The results showed that the immune indexes of each immunized group were higher than those of pVAX1 group. The PBS group and the blank group were high, especially in the adjuvant combined immunization group. In lymphocyte proliferation test, enzyme linked immunosorbent assay (Elisa), virus neutralization test, cytokine detection test. Quantitative changes of T cell subtype CD4 and CD8 and detection of specific CTL activity In the experiment, the effect of adjuvant antigen group was obviously better than other groups. Virus neutralization test and enzyme-linked immunosorbent assay (Elisa) showed that the antibody against TGEV IgG was detected by virus neutralization test and enzyme-linked immunosorbent assay (Elisa). The effect of antibody production in vaccine group was better than that in other groups. Flow cytometry and cytokine test showed that adjuvant immunized group also had auxiliary enhancement effect on immune response. A prokaryotic expression plasmid pGEX-6p-pIL-12p40) was constructed. Plasmid deletion of 22 AA signal peptide sequence at the end of pIL-12 nunp40. Highly expressed pIL-12 p40 was obtained in Escherichia coli. The purified protein was used to immunize New Zealand rabbits to obtain rabbit polyclonal antibodies. The results of enzyme-linked immunosorbent assay (Elisa) and immunofluorescence showed that pIL-15 protein reacted well with polyclonal antibodies. In this study, pIL-12 gene was used for the first time. PIL-18 gene and TGEV S gene plasmid were immunized. The results showed that pIL-12 gene, pIL-12 gene and pIL-18 gene were combined as TGEV. The molecular adjuvant of the S antigen gene. It can not only promote the cellular immune response, but also improve the humoral immune response of the body, and then improve the immune response of the body, which provides the basis for further study of the role of cytokines as immune adjuvants. It lays a theoretical foundation for the study of new nucleic acid vaccine.
【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

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