Hz-Au多肽佐剂功能的研究

发布时间：2018-06-26 03:03

本文选题：佐剂 + 免疫原　；参考：《华中农业大学》2011年硕士论文

【摘要】：免疫佐剂与免疫原合并使用可以增强抗原免疫原性,增强机体免疫应答,或改变机体免疫应答类型。本研究从芽胞杆菌产物中分离纯化得到一种热稳定性高、毒性低的低分子量物质Hz-Au多肽,将其分别与模式抗原卵清白蛋白(OVA)、谷胱甘肽S转移酶蛋白(GST)联合免疫昆明(KM)小鼠,通过间接ELISA检测血清抗体来研究其免疫佐剂作用。同时分别对Hz-Au多肽的免疫剂量、免疫方式,生物安全性及免疫机理进行了研究,结果如下： (1) Hz-Au多肽提高小鼠对模式抗原OVA和GST免疫反应的研究。以OVA为免疫原免疫KM小鼠,Hz-Au多肽组抗OVA IgG抗体显著高于对照组(P0.01),其中腹腔免疫时抗体效价达到2×105,高于相同免疫条件下弗氏佐剂组；背部皮下注射免疫时抗体效价达到5×104,接近于弗氏佐剂组；口服免疫时抗体效价达到3000,与霍乱毒素无毒B亚单位(CTB)组相同。GST作为免疫原,Hz-Au多肽组特异性IgG抗体与对照组差异显著(P0.01),效价分别能达到2×105(腹腔免疫)、1×105(皮下免疫)和12000(口服免疫)。特异性IgG抗体亚型分析显示,IgG的提高以IgG1亚型增加为主,表明Hz-Au多肽偏向于诱导Th2型免疫应答。抗体的持续性研究显示,Hz-Au多肽佐剂激起的特异性抗体在小鼠体内可高水平稳定存在两个月以上,加强免疫后激发强烈记忆反应,效价可达4×105。 (2) Hz-Au多肽及免疫原的免疫剂量研究。注射免疫方式Hz-Au多肽的佐剂作用与其剂量呈正相关关系,即在100μg到5μg间免疫效果逐渐降低,但并不呈线性关系。口服免疫时,Hz-Au多肽佐剂作用在200μg到500μg之间最强,浓度升高或降低都将减弱其佐剂效果。最佳免疫剂量为,注射免疫Hz-Au多肽5μg,口服免疫Hz-Au多肽200μg。GST口服免疫结果显示,免疫效果随着免疫原剂量的减少而降低。 (3) Hz-Au多肽生物安全性研究。Hz-Au多肽对小鼠急性毒性试验结果表明,口服免疫30 mg仍无致死性,小鼠未出现任何异常生理反应,主要脏器未出现损伤或病变。小鼠免疫安全试验显示,注射部位未出现局部反应,未发生腹膜炎及肠黏膜损伤等情况。因此,Hz-Au多肽作为佐剂副反应极低,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。将Hz-Au多肽分别与DNA序列及合成短肽混合进行免疫,结果显示Hz-Au多肽能显著提高核酸和多肽免疫效果(P0.01),小鼠血清中特异性IgG抗体效价与无佐剂组比提高32倍以上 (5) Hz-Au多肽抗酶解作用研究。Hz-Au多肽对OVA. BSA. GST三种蛋白具有不同程度的抗胃蛋白酶和胰蛋白酶酶解作用,保护效果依次为GSTBSAOVA。 (6)抗原特异性淋巴细胞增殖实验。Hz-Au多肽可以提高小鼠细胞免疫的水平,在CTL肽或Th肽的刺激下,Hz-Au多肽可显著提高T细胞免疫应答,表现为OVA特异性T细胞的大量增殖,其效果好于无佐剂组。 (7)T淋巴细胞胞内细胞因子的检测。Hz-Au多肽既能诱导Th1型为主的Th1/Th2混合型细胞免疫应答反应(口服免疫),也能诱导Th2型为主的Th1/Th2混合型应答(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).
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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