本文選題：佐劑 + 免疫原　； 參考：《華中農(nóng)業(yè)大學(xué)》2011年碩士論文

【摘要】：免疫佐劑與免疫原合并使用可以增強(qiáng)抗原免疫原性,增強(qiáng)機(jī)體免疫應(yīng)答,或改變機(jī)體免疫應(yīng)答類型。本研究從芽胞桿菌產(chǎn)物中分離純化得到一種熱穩(wěn)定性高、毒性低的低分子量物質(zhì)Hz-Au多肽,將其分別與模式抗原卵清白蛋白(OVA)、谷胱甘肽S轉(zhuǎn)移酶蛋白(GST)聯(lián)合免疫昆明(KM)小鼠,通過間接ELISA檢測血清抗體來研究其免疫佐劑作用。同時分別對Hz-Au多肽的免疫劑量、免疫方式,生物安全性及免疫機(jī)理進(jìn)行了研究,結(jié)果如下： (1) Hz-Au多肽提高小鼠對模式抗原OVA和GST免疫反應(yīng)的研究。以O(shè)VA為免疫原免疫KM小鼠,Hz-Au多肽組抗OVA IgG抗體顯著高于對照組(P0.01),其中腹腔免疫時抗體效價達(dá)到2×105,高于相同免疫條件下弗氏佐劑組；背部皮下注射免疫時抗體效價達(dá)到5×104,接近于弗氏佐劑組；口服免疫時抗體效價達(dá)到3000,與霍亂毒素?zé)o毒B亞單位(CTB)組相同。GST作為免疫原,Hz-Au多肽組特異性IgG抗體與對照組差異顯著(P0.01),效價分別能達(dá)到2×105(腹腔免疫)、1×105(皮下免疫)和12000(口服免疫)。特異性IgG抗體亞型分析顯示,IgG的提高以IgG1亞型增加為主,表明Hz-Au多肽偏向于誘導(dǎo)Th2型免疫應(yīng)答�？贵w的持續(xù)性研究顯示,Hz-Au多肽佐劑激起的特異性抗體在小鼠體內(nèi)可高水平穩(wěn)定存在兩個月以上,加強(qiáng)免疫后激發(fā)強(qiáng)烈記憶反應(yīng),效價可達(dá)4×105。 (2) Hz-Au多肽及免疫原的免疫劑量研究。注射免疫方式Hz-Au多肽的佐劑作用與其劑量呈正相關(guān)關(guān)系,即在100μg到5μg間免疫效果逐漸降低,但并不呈線性關(guān)系�？诜庖邥r,Hz-Au多肽佐劑作用在200μg到500μg之間最強(qiáng),濃度升高或降低都將減弱其佐劑效果。最佳免疫劑量為,注射免疫Hz-Au多肽5μg,口服免疫Hz-Au多肽200μg。GST口服免疫結(jié)果顯示,免疫效果隨著免疫原劑量的減少而降低。 (3) Hz-Au多肽生物安全性研究。Hz-Au多肽對小鼠急性毒性試驗(yàn)結(jié)果表明,口服免疫30 mg仍無致死性,小鼠未出現(xiàn)任何異常生理反應(yīng),主要臟器未出現(xiàn)損傷或病變。小鼠免疫安全試驗(yàn)顯示,注射部位未出現(xiàn)局部反應(yīng),未發(fā)生腹膜炎及腸黏膜損傷等情況。因此,Hz-Au多肽作為佐劑副反應(yīng)極低,具有很高的生物安全性。 (4) Hz-Au多肽提高核酸及多肽免疫原性的研究。將Hz-Au多肽分別與DNA序列及合成短肽混合進(jìn)行免疫,結(jié)果顯示Hz-Au多肽能顯著提高核酸和多肽免疫效果(P0.01),小鼠血清中特異性IgG抗體效價與無佐劑組比提高32倍以上 (5) Hz-Au多肽抗酶解作用研究。Hz-Au多肽對OVA. BSA. GST三種蛋白具有不同程度的抗胃蛋白酶和胰蛋白酶酶解作用,保護(hù)效果依次為GSTBSAOVA。 (6)抗原特異性淋巴細(xì)胞增殖實(shí)驗(yàn)。Hz-Au多肽可以提高小鼠細(xì)胞免疫的水平,在CTL肽或Th肽的刺激下,Hz-Au多肽可顯著提高T細(xì)胞免疫應(yīng)答,表現(xiàn)為OVA特異性T細(xì)胞的大量增殖,其效果好于無佐劑組。 (7)T淋巴細(xì)胞胞內(nèi)細(xì)胞因子的檢測。Hz-Au多肽既能誘導(dǎo)Th1型為主的Th1/Th2混合型細(xì)胞免疫應(yīng)答反應(yīng)(口服免疫),也能誘導(dǎo)Th2型為主的Th1/Th2混合型應(yīng)答(腹腔免疫)。
[Abstract]:The combined use of immune adjuvant and immunogen can enhance the immunogenicity of the antigen, enhance the immune response of the body, or change the type of immune response. A low molecular weight substance Hz-Au polypeptide with high thermal stability and low toxicity was isolated and purified from the product of Bacillus spore, and was separated from the model antigen ovalbumin (OVA), Gu Guanggan, respectively. Peptide S transferase protein (GST) combined with immunized Kunming (KM) mice, the immune adjuvant was studied by indirect ELISA detection of serum antibodies. The immunological dose, immune mode, biological safety and immune mechanism of Hz-Au polypeptide were studied. The results were as follows:
(1) Hz-Au polypeptide increased the immunological response of mice to model antigen OVA and GST. The anti OVA IgG antibody of Hz-Au polypeptide group was significantly higher than that of the control group (P0.01) with OVA as immunogen, and the antibody titer reached 2 * 105 in the abdominal immune system, which was higher than that of the Freund's adjuvant group under the same immune condition; the antibody titer of the back subcutaneous immunization was reached. To 5 * 104, close to the Freund adjuvant group, the titer of the antibody reached 3000 in oral immunization, and the same.GST as the non toxic B subunit (CTB) of cholera toxin as the immunogen. The specific IgG antibody in the Hz-Au polypeptide group was significantly different from the control group (P0.01), and the titer could reach 2 * 105 (abdominal cavity immunity), 1 x 105 (subcutaneous immunization) and 12000 (oral immunity). Specific I The analysis of gG antibody subtypes showed that the increase of IgG was mainly increased by IgG1 subtype, indicating that the Hz-Au polypeptide was biased towards the induction of Th2 type immune response. The persistence of the antibody showed that the specific antibodies induced by the Hz-Au polypeptide adjuvant could be stable for more than two months in the mice, and the strong memory reaction was stimulated after the strong immunization, and the titer could reach 4 * 1. Five
(2) study on the immunological dose of Hz-Au polypeptide and immunogen. The adjuvant effect of the immunized Hz-Au polypeptide has a positive correlation with its dose, that is, the immune effect between 100 and 5 mu g decreases gradually, but it does not have a linear relationship. When oral immunization, the effect of Hz-Au polypeptide adjuvant is between 200 and 500 micron g, and the increase or decrease of concentration will be weakened. The optimum immune dose was the immunization of Hz-Au polypeptide of 5 mu g and oral immunization of oral immunized Hz-Au polypeptide 200 u g.GST, which showed that the immune effect decreased with the decrease of the immunogenic dose.
(3) study on the biosafety of Hz-Au polypeptide, the results of acute toxicity test of.Hz-Au polypeptide in mice showed that the oral immune 30 mg still had no death, no abnormal physiological reaction and no injury or lesion in the main organs. Therefore, Hz-Au peptide as adjuvant has very low side effects and high biosafety.
(4) the Hz-Au polypeptide increased the immunogenicity of nucleic acid and polypeptide. The Hz-Au polypeptide was immunized with DNA sequence and synthetic peptide respectively. The results showed that the Hz-Au polypeptide could significantly improve the immune effect of nucleic acid and polypeptide (P0.01). The specific IgG antibody titer in the serum of mice was increased by more than 32 times more than that of the adjuvant without the adjuvant.
(5) anti enzymatic hydrolysis of Hz-Au polypeptide,.Hz-Au polypeptide has different degrees of anti pepsin and Trypsin Hydrolysis to OVA. BSA. GST protein, and the protective effect is GSTBSAOVA. in turn.
(6) the antigen specific lymphocyte proliferation experiment.Hz-Au polypeptide can improve the level of cell immunity in mice. Under the stimulation of CTL peptide or Th peptide, Hz-Au polypeptide can significantly increase the immune response of T cell, which shows a large proliferation of OVA specific T cells, and the effect is better than that in the non adjuvant group.
(7) the detection of.Hz-Au peptide in T lymphocyte cytokine can not only induce the Th1 type Th1/Th2 mixed cell immune response (oral immunity), but also induce the Th2 type Th1/Th2 mixed response (peritoneal immunity).
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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1 李菁菁;Hz-Au多肽佐劑功能的研究[D];華中農(nóng)業(yè)大學(xué);2011年

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