本文選題：日本血吸蟲　切入點：DNA疫苗　出處：《中國農(nóng)業(yè)科學(xué)院》2012年碩士論文

【摘要】：【目的】日本血吸蟲病是危害嚴(yán)重的熱帶亞熱帶消耗性疾病，分布廣泛。研制安全、有效的疫苗是重要的研究方向之一。本文以血吸蟲重要的體被蛋白—日本血吸蟲四跨膜蛋白2（SjTSP2）和硫氧還蛋白谷胱甘肽還原酶（SjTGR）為抗原候選分子，制備DNA疫苗，比較不同免疫途徑誘導(dǎo)的保護效果，研究其免疫保護機制。 【方法】利用分子克隆方法構(gòu)建DNA疫苗，應(yīng)用脂質(zhì)體轉(zhuǎn)染293T細胞，間接免疫熒光法檢測目的蛋白在細胞中的表達，分別采取基因槍轟擊法和肌肉注射法免疫接種BABL/c小鼠，進行動物免疫保護試驗，計算減蟲、減卵率。用ELISA法檢測抗體水平，用流式細胞術(shù)檢測細胞因子，用硫氧還蛋白還原酶試劑盒檢測蟲體SjTGR的酶活力。 【結(jié)果】成功構(gòu)建了DNA疫苗（pVAX1/SjTSP2和pVAX1/SjTGR），，間接免疫熒光法檢測表明兩種蛋白均在293T細胞中表達良好。采用基因槍轟擊法免疫pVAX1/SjTSP2，ELISA檢測結(jié)果表明免疫組小鼠產(chǎn)生了較高水平的特異性抗體，用流式細胞術(shù)檢測發(fā)現(xiàn)免疫組小鼠產(chǎn)生了較高水平的IL-4、IFN-γ，然而，免疫組小鼠平均成蟲數(shù)較對照組增加12.04%，每克肝臟蟲卵數(shù)增加10.01%，差異不顯著。大量純化pVAX1/SjTGR，分別采用小鼠腿部肌肉注射法和腹部皮膚基因槍轟擊法免疫小鼠，進行小鼠日本血吸蟲病免疫保護效果評估。結(jié)果基因槍免疫小鼠獲得了27.83%（P0.05）的減蟲率和40.38%（P0.05）的減卵率，而肌肉注射組沒有顯著的保護效果。ELISA檢測結(jié)果顯示免疫組小鼠均產(chǎn)生了較高水平的特異性抗體，且基因槍免疫組產(chǎn)生的抗體水平高于肌肉注射法免疫。用基因槍轟擊免疫法進行免疫保護重復(fù)試驗，結(jié)果誘導(dǎo)了38.83%（P0.05）的減蟲率和44.51%（P0.05）的減卵率。流式細胞術(shù)結(jié)果顯示基因槍免疫pVAX1/SjTGR，致使小鼠產(chǎn)生了較高水平的IL-4、IFN-γ，體外TGR酶活力測定表明，免疫組蟲體的酶活力顯著低于對照組，而且高濃度特異性抗SjTGR抗體可抑制SjTGR酶活力。 【結(jié)論】用基因槍轟擊法免疫小鼠pVAX1/SjTSP2DNA疫苗，未誘導(dǎo)理想的免疫保護效果。而基因槍法免疫pVAX1/SjTGR誘導(dǎo)了顯著的保護效果，誘導(dǎo)的免疫保護作用可能和高水平的特異性抗體和較強的細胞免疫反應(yīng)有關(guān)。SjTGR具有作為抗日本血吸蟲病疫苗候選抗原分子的潛力。本文為血吸蟲疫苗研究提供了有益的數(shù)據(jù)資料。
[Abstract]:[objective] Schistosomiasis japonicum is a serious tropical, subtropical and expendable disease, which is widely distributed. The effective vaccine is one of the important research directions. In this paper, DNA vaccine was prepared by using Schistosoma japonicum four-transmembrane protein (SjTSP2) and thioredoxin glutathione reductase (SjTGR) as antigen candidate molecules. The protective effects of different immune pathways were compared and the mechanism of immune protection was studied. [methods] DNA vaccine was constructed by molecular cloning, and 293T cells were transfected with liposome. Indirect immunofluorescence assay was used to detect the expression of target protein in the cells. BABL/c mice were immunized with gene gun bombardment and intramuscular injection respectively. ELISA method was used to detect antibody level, flow cytometry was used to detect cytokines, and thioredoxin reductase kit was used to detect the enzyme activity of SjTGR. [results] DNA vaccine pVAX1 / SjTSP2 and pVAX1 / SjTGR2 were successfully constructed. Indirect immunofluorescence assay showed that both proteins expressed well in 293T cells. Flow cytometry showed that the immunized mice produced a high level of IL-4 and IFN- 緯, however, Compared with the control group, the average adult number of mice in the immunized group increased 12.04 and 10.01 per gram of liver eggs, but the difference was not significant. A large number of purified pVAX1 / SjTGRwere used to immunize mice with leg intramuscular injection and abdominal skin gene gun bombardment, respectively. Results the rate of worm reduction and egg reduction of mice immunized with gene gun were 27.83% (P0.05) and 40.38% (P0.05), respectively. The results of Elisa showed that the immunized mice produced higher levels of specific antibodies. The antibody level of the gene gun immunized group was higher than that of the intramuscular injection method. Results the worm reduction rate and egg reduction rate of 44.51% and 44.51% were induced. The results of flow cytometry showed that the gene gun immunized pVAX1 / SjTGRR resulted in a higher level of IL-4 and IFN- 緯 in mice. The activity of TGR in vitro was significantly lower than that of the control group, the results of flow cytometry showed that the enzyme activity of the immunized group was significantly lower than that of the control group, and the results of flow cytometry showed that the expression of IL-4 and IFN- 緯 in the immunized group was significantly lower than that in the control group. And high concentration of specific anti SjTGR antibody can inhibit the activity of SjTGR enzyme. [conclusion] immunizing mice with pVAX1/SjTSP2DNA vaccine with gene gun bombardment did not induce the ideal immune protective effect, but pVAX1/SjTGR immunized with gene gun had a significant protective effect. The immune protective effect induced by SjTGR may be related to the high level of specific antibodies and the strong cellular immune response. SjTGR has the potential as a candidate antigen molecule for the vaccine against schistosomiasis japonicum. Useful data.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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