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  本文選題：日本血吸蟲　切入點：重組抗原　出處：《安徽醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:利用rSjLAP和rSjFBPA加佐劑分組免疫BALB/c小鼠,探討基于2-DE和血清蛋白質(zhì)組篩選的上述二種重組抗原的免疫保護效果;通過福氏佐劑和納米C60佐劑在增強免疫效果方面的比較,觀察納米C60佐劑在血吸蟲分子疫苗研發(fā)中的價值。方法:從重組質(zhì)粒中誘導(dǎo)表達rSjLAP和rSjFBPA,分別用親和層析法(His-Bind Purification Kit)純化,并用Bradford法分別檢測二者的含量。將6周齡BALB/c小鼠隨機分為7組,每組6只。除對照組外,每組均給予rSjLAP或rSjFBPA 100μg/次/鼠,腹部皮下多點注射,共免疫3次,每次間隔2周。按以下分組免疫:①對照組:皮下注射等體積的生理鹽水;②rSjLAP+福氏佐劑組;③rSjFBPA+福氏佐劑組;④rSjLAP+ rSjFBPA+福氏佐劑組;⑤rSjLAP+納米C60佐劑組;⑥r(nóng)SjFBPA+納米C60佐劑組;⑦rSjLAP+ rSjFBPA+納米C60佐劑組。免疫5周后,收集小鼠血清,用ELISA法測定小鼠血清IFN-γ和IL-4的含量(pg/ml)。用尾蚴感染BALB/c小鼠,30±2條/鼠,尾蚴感染6周后,處死小鼠,收集成蟲,計算減蟲率;收集肝組織蟲卵,計算減卵率。結(jié)果:不同實驗組BALB/c小鼠,測得2、3及4組重組蛋白加福氏佐劑組誘導(dǎo)的減蟲率分別為:35.5%(P0.05); 33.0%(P0.05)、38.1%(P0.05);5、6及7組重組蛋白加納米C60佐劑誘導(dǎo)的減蟲率分別為:43.1%(P0.05)、39.1%(P0.05)、45.2%(P0.05); 2、3及4組誘導(dǎo)的減卵率分別為:40.2%(P0.05),42.6%(P0.05),48.9%(P0.05),5、6、及7組誘導(dǎo)的減卵率分別為41.6%(P0.05),45.4%(P0.05),46.9%(P0.05)。ELISA法測定對照組、rSjLAP+福氏佐劑組、rSjFBPA+福氏佐劑組各組IFN-γ水平(pg/ml)分別為:20.4,20.1,19.6;IL-4水平(pg/ml)分別為:192.0,382.0,518.0。結(jié)果表明:rSjLAP和rSjFBPA加佐劑均能夠明顯提高蛋白對BALB/c小鼠抗血吸蟲感染的保護性免疫作用。結(jié)論:rSjLAP和rSjFBPA,加福氏佐劑或納米C60佐劑免疫BALB/c小鼠,獲得了一定的免疫保護作用;福氏佐劑和納米C60佐劑均增強了重組蛋白的免疫效果,實驗結(jié)果表明福氏佐劑的免疫保護效果和納米C60佐劑差別不大,但低劑量的納米C60佐劑對細胞不造成損傷,危害較小。以上結(jié)果表明,rSjLAP和rSjFBPA有可能作為日本血吸蟲病的候選疫苗分子,為其開發(fā)應(yīng)用研究打下了基礎(chǔ)。各實驗組小鼠IFN-γ水平無顯著變化,IL-4水平變化明顯,表明日本血吸蟲感染BALB/c小鼠伴隨蛋白抗原免疫,主要表現(xiàn)為Th2細胞應(yīng)答反應(yīng)。
[Abstract]:Objective: to immunize BALB/c mice with rSjLAP and rSjFBPA plus adjuvant to investigate the immunoprotective effects of these two recombinant antigens screened based on 2-DE and serum proteome, and to compare the effects of Freund's adjuvant and nano-C60 adjuvant in enhancing the immune response. To observe the value of nano-C60 adjuvant in the development of Schistosoma japonicum molecular vaccine. Methods: rSjLAP and rSjFBPA were induced from recombinant plasmid and purified by affinity chromatography. The 6-week-old BALB/c mice were randomly divided into 7 groups with 6 mice in each group. Except for the control group, each group was given rSjLAP or rSjFBPA 100 渭 g / time for 3 times. Each time interval 2 weeks. According to the following groups: control group: subcutaneous injection of normal saline 2rSjLAP Freund's adjuvant group 3rSjFBPA Freund's adjuvant group 4rSjLAP rSjFBPA Freund's adjuvant group 5rSjLAP nano-C60 adjuvant group 6rSjFBPA nano-C60 adjuvant group 7rSjLAP rSjFBPA nano-C60 adjuvant group. After 5 weeks of immunization, The contents of IFN- 緯 and IL-4 in serum of mice were measured by ELISA method. Cercariae infected BALB/c mice with 30 鹵2 mice. After 6 weeks of cercariae infection, the mice were killed, the adult worms were collected, the rate of worm reduction was calculated, and the liver tissue eggs were collected. Results: different experimental groups of BALB/c mice, The results showed that the worm reduction rates induced by recombination proteins of 2t3 and 4 groups with Fowler's adjuvant group were: 1: 35.5P 0.05; 33.0 + P0.05 + 38.1and 7groups of recombinant proteins plus nano-C60 adjuvant were induced by recombination proteins and 7 groups of recombinant proteins and nano-C60 adjuvants, respectively. The worm reduction rates of groups 2 and 4 were 43.1P0.05P0.05P0.05 and 42.20.2N 42.6P 0.05N 48.9P 0.05p 0.05A, respectively, and the rate of egg reduction induced by 2C3 and 4 groups were 40.20.2ng / 42.6P 0.05 and 48.9p 0.05g / 5 respectively. The induced egg reduction rates of groups 2 and 4 were 40.20.2n and 42.6P 0.05g / 48.9p 0.05a respectively, and the rate of reduction of eggs induced by groups 2 and 4 were 42.20.2P 0.05 and 42.6P 0.05P 0.05a, respectively. The egg reduction rates of 41.6% P0.05 and 45.45% P0.05 and 46.9% P0.05U. Elisa were used to determine the level of IFN- 緯 in the control group, rSjFBPA adjuvant group and the control group, respectively. The level of PG / 緯 in the control group was 20.420.119.6IL-4 and the level of IL-4 was 192.0382.0518.0.The results showed that both the rSjFBPA adjuvant group and the control group could significantly increase the sensitivity of protein to schistosomiasis in BALB/c mice. The results showed that both the rSjLAP and rSjFBPA adjuvant could significantly increase the sensitivity of protein to Schistosoma japonicum in BALB/c mice. Conclusion BALB/c mice were immunized with 1% rSjLAP and rSjFBPA, plus Freund's adjuvant or nano-C60 adjuvant. Both Freund's adjuvant and nano-C60 adjuvant enhanced the immune effect of recombinant protein, and the results showed that the protective effect of Freund's adjuvant was not different from that of nano-C60 adjuvant. However, the low dose of nano-C60 adjuvant did not damage the cells and did little harm. These results suggest that rSjLAP and rSjFBPA may be the candidate vaccine molecules for schistosomiasis japonicum. The results showed that the level of IFN- 緯 did not change significantly in mice of all experimental groups, indicating that the immune responses of Th2 cells to the associated protein antigen in BALB/c mice infected with Schistosoma japonicum were mainly manifested by the response of Th2 cells.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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