【摘要】：目的: 制備納米氫氧化鋁佐劑,吸附抗淋病PorB、LTB-PorB重組蛋白疫苗,通過鼻飼途徑免疫雌性BALB/c小鼠,分析其免疫活性,探討納米氫氧化鋁佐劑能否輔佐蛋白疫苗誘導(dǎo)產(chǎn)生更有效的特異性體液免疫和細(xì)胞免疫,尤其是高效的黏膜免疫,為尋找經(jīng)黏膜途徑感染病原體的疫苗遞送載體進(jìn)行有益探索和提供實(shí)驗(yàn)依據(jù)。 方法: 1.采用微乳液法制備納米氫氧化鋁顆粒,檢測(cè)其粒徑大小并進(jìn)行透射電鏡測(cè)定,同時(shí)進(jìn)行細(xì)胞毒性試驗(yàn)和動(dòng)物毒性試驗(yàn)以評(píng)價(jià)其作為疫苗載體的安全性; 2.將課題組已構(gòu)建的原核表達(dá)重組質(zhì)粒pET-30a/LTB、pET-30a/porB和pET-30a/LTB-PorB在大腸桿菌BL21中誘導(dǎo)表達(dá)重組蛋白,鑒定后大量制備并純化; 3.吸附試驗(yàn)比較納米氫氧化鋁和常規(guī)鋁佐劑吸附重組蛋白后的穩(wěn)定性; 4.納米氫氧化鋁顆粒吸附重組疫苗蛋白,鼻飼途徑免疫雌性BALB/c小鼠,檢測(cè)重組蛋白誘導(dǎo)的體液免疫和細(xì)胞免疫水平。 結(jié)果: 1.采用微乳液法成功制備了納米氫氧化鋁顆粒,經(jīng)檢測(cè)其粒徑分布范圍為100-200nm,峰值為148.12nm。經(jīng)細(xì)胞及動(dòng)物毒性試驗(yàn)分析,制備的納米氫氧化鋁顆粒符合本次試驗(yàn)的要求; 2.納米氫氧化鋁(Nano)分別吸附的PorB(Nano-P)和LTB-PorB(Nano-L-P)重組蛋白通過鼻飼途徑免疫雌性BALB/c小鼠,其血清特異性IgG和生殖道黏膜特異性sIgA水平隨免疫時(shí)間的增加呈上升趨勢(shì);第3次免疫后(第6w) IgG A450值分別為0.724±0.076,0.854±0.146,明顯高于PBS組、Nano組、LTB組、PorB組、LTB-PorB組五組,有統(tǒng)計(jì)學(xué)意義(P0.01);第6周生殖道灌洗液sIgA450值分別為0.487±0.052,0.673±0.122,明顯高于其他五組,有統(tǒng)計(jì)學(xué)意義(P0.01);Nano-P組和Nano-L-P組脾淋巴細(xì)胞誘生的IL-4水平均明顯高于其他五組,有統(tǒng)計(jì)學(xué)意義(P0.01); IFN-γ水平與其他五組比較無統(tǒng)計(jì)學(xué)意義(P0.05);脾淋巴細(xì)胞刺激指數(shù)與其他五組比較有統(tǒng)計(jì)學(xué)意義(P0.05);Nano-P和Nano-L-P之間比較無統(tǒng)計(jì)學(xué)意義(P0.05);IgG2a比IgG1比值均小于1,提示納米氫氧化鋁吸附重組蛋白以誘導(dǎo)Th2型體液免疫應(yīng)答為主。 結(jié)論: 1.納米氫氧化鋁作為疫苗遞送載體,通過鼻飼途徑免疫小鼠,能促進(jìn)PorB和LTB-PorB重組蛋白產(chǎn)生較高水平的特異性體液免疫,尤其是生殖道的黏膜免疫。
[Abstract]:Objective: to prepare nano-aluminum hydroxide adjuvant, to adsorb the recombinant protein vaccine against gonorrhea, and to immunize female BALB/c mice by nasal feeding. To investigate whether nano-aluminum hydroxide adjuvant can induce more effective specific humoral and cellular immunity, especially mucosal immunity. In order to search for the vaccine delivery vector which infects pathogens through mucosal pathway and provide experimental basis. Methods: 1. Nanometer aluminum hydroxide particles were prepared by microemulsion method. The particle size was measured and the transmission electron microscopy (TEM) was carried out. Meanwhile, the cytotoxicity test and animal toxicity test were carried out to evaluate its safety as vaccine carrier. 2. The recombinant plasmids pET-30a / L LTBN pET-30a / porB and pET-30a/LTB-PorB were induced to express recombinant proteins in E. coli BL21, and the recombinant proteins were prepared and purified in large quantities after identification. The stability of recombinant protein adsorbed by nano-aluminum hydroxide and conventional aluminum adjuvant was compared. 4. The recombinant vaccine protein was adsorbed by nano-aluminum hydroxide particles and the female BALB/c mice were immunized by nasal feeding. The humoral immunity and cellular immunity induced by recombinant protein were detected. Results: 1. Nanometer aluminum hydroxide particles were successfully prepared by microemulsion method. The particle size distribution ranges from 100 to 200 nm and the peak value is 148.12 nm. According to the analysis of cytotoxicity test and animal toxicity test, the prepared nano aluminum hydroxide particles meet the requirements of this test. 2. The recombinant proteins of PorB (Nano-P) and LTB-PorB (Nano-L-P) adsorbed by nano-aluminum hydroxide (Nano) were immunized by nasal feeding to female BALB/c mice. The levels of serum specific IgG and reproductive tract mucosal specific sIgA increased with the increase of immune time. The value of IgG A450 was 0.724 鹵0.0766 鹵0.146 after the third immunization, which was significantly higher than that in the PBS group and PBS group (P 0.01), and the sIgA450 value of the reproductive tract lavage fluid was 0.487 鹵0.0520.673 鹵0.122 in the 6th week, which was significantly higher than that in the other five groups. The levels of IL-4 induced by splenic lymphocytes in Nano-P group and Nano-L-P group were significantly higher than those in other five groups (P0.01). There was statistical significance (P0.01), IFN- 緯 level was not significant compared with other five groups (P0.05), spleen lymphocyte stimulating index was statistically significant compared with other five groups (P0.05) there was no significant difference between P05 and Nano-L-P (P0.05) the ratio of IgG2a to IgG1 was less than 1, indicating that the ratio of IgG2a to IgG1 was lower than that of other five groups (P0.05). The recombinant protein adsorbed by nanometer aluminum hydroxide mainly induced Th2 humoral immune response. Conclusion: 1. Nano aluminum hydroxide as a vaccine delivery carrier immunized mice by nasal feeding could promote the production of specific humoral immunity of PorB and LTB-PorB recombinant proteins especially the mucosal immunity of reproductive tract.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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