本文關鍵詞：豬IL-12和IL-18對TGEV S基因核酸疫苗免疫效應的研究　出處：《東北農業(yè)大學》2011年碩士論文　論文類型：學位論文

  更多相關文章： 豬傳染性胃腸炎病毒 S基因 豬白細胞介素12 豬白細胞介素18 免疫佐劑

【摘要】：豬傳染性胃腸炎(Porcine transmissible gastroenteritis,TGE)是由豬傳染性胃腸炎病毒(Transmissible gastroenteritis virus,TGEV)引起的一種高度接觸性、急性消化道傳染病。預防該病主要是靠接種疫苗。本實驗分別將豬傳染性胃腸炎病毒S基因與豬IL-12基因亞克隆至真核表達載體pVAX1中,構建了兩個重組真核表達質粒pVAX1-TGEV S1和 pVAX1-pIL-12。通過脂質體轉染法將pVAX1-TGEV S1和pVAX1-pIL-12轉入BHK細胞中進行了表達,且表達的蛋白可以被多克隆抗體所檢測。以pVAX1-TGEV S1和pVAX1-pIL-12重組真核表達質粒免疫小鼠。共分為9組,分別是空白組、PBS組、pVAX1組、pIL-12組、pIL-12+pIL-18組、TGEV S組、TGEV S+pIL-12組、TGEV S+pIL-12+pIL-18組和疫苗組。利用淋巴細胞轉化試驗、酶聯免疫吸附試驗、流式細胞儀技術、檢測特異性CTL活性實驗、病毒中和試驗和細胞因子檢測試驗對不同處理組小鼠的各項免疫指標進行了檢測。試驗結果表明,各免疫組小鼠的各項免疫指標均比pVAX1組、PBS組和空白組高,加入佐劑聯合免疫組的結果尤為顯著。在淋巴細胞增殖試驗、酶聯免疫吸附試驗、病毒中和試驗、細胞因子檢測試驗、T細胞亞型CD4+、CD8+數量變化檢測以及特異性CTL活性的檢測 試驗中,加佐劑抗原組的效果明顯要優(yōu)于其他組別。病毒中和試驗和酶聯免疫吸附試驗檢測抗TGEV IgG的抗體結果顯示,疫苗組產生抗體的效果優(yōu)于其他各組。流式細胞術檢測和細胞因子檢測試驗中發(fā)現佐劑免疫組對機體免疫應答也有輔助的增強作用。 本實驗還構建了一個pGEX-6p-pIL-12(p40)原核表達質粒,質粒缺失pIL-12 N(p40)端22 aa信號肽序列。在大腸桿菌中獲得了高效表達的pIL-12(p40)蛋白。純化后蛋白免疫新西蘭兔獲得兔源多克隆抗體。酶聯免疫吸附試驗和免疫熒光結果表明pIL-15蛋白與多克隆抗體具有良好的特異性反應。 本研究首次將pIL-12基因、pIL-18基因與TGEV S基因質粒聯合免疫。實驗結果表明pIL-12基因以及pIL-12基因和pIL-18基因聯合作為TGEV S抗原基因的分子佐劑,不但能促進機體的細胞免疫應答,而且能夠提高機體體液免疫應答,進而提高了機體的免疫反應。為進一步研究細胞因子作為免疫佐劑的作用提供了依據,為新型核酸疫苗的研究奠定了理論基礎。
[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.
【學位授予單位】：東北農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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