胞壁酰二肽类似物的生物学活性筛选及其对乙肝表面抗原的佐剂性研究

发布时间：2018-02-16 19:09

本文关键词： 乙型肝炎疫苗佐剂胞壁酰二肽胞壁酰二肽类似物细胞免疫　出处：《中国药品生物制品检定所》2006年硕士论文　论文类型：学位论文

【摘要】：我国属于乙型肝炎高流行地区之一，乙型肝炎表面抗原(HBsAg)携带者高达1亿之多，其中慢性乙肝患者有3000万人。目前对于乙肝治疗尚无有效的药物，接种疫苗是控制乙肝流行的基本措施。铝盐是乙肝疫苗的现行佐剂，大量研究表明，铝佐剂主要提高体液免疫和Th2类细胞免疫应答。 1974年，Lederer E．等人发现胞壁酰二肽(MDP)是弗式完全佐剂的最小有效结构单位，是灭活分枝杆菌细胞壁的成分之一。MDP具有广泛的生物学活性，但同时也表现出一些副作用如致热、代谢迅速等。为了提高其活性，降低副作用，，人们对其结构加以改造，合成了许多MDP衍生物及类似物，找到了一些活性较高、副作用较低的化合物，并对其构效关系进行了深入的研究。本文采用酶联免疫斑点分析(ELISPOT)方法对中国协和医科大学药物研究所合成的60种MDP类似物进行生物学活性筛选，将筛选出的2种MDP类似物(5#、17#化合物)作为HBsAg的佐剂，对其免疫原性做了进一步的研究。首先通过ELISPOT方法，对3ug HBsAg免疫BALB／c小鼠后的细胞免疫应答动态进行研究。结果显示，免疫后4天，产生较弱的细胞免疫应答，7天时，细胞免疫应答达到一定强度，14天时，达到高峰。同时研究了HBsAg免疫小鼠7天后脾淋巴细胞及CD8~+T细胞细胞免疫应答的剂量—效应关系，结果说明，随着免疫剂量的增加，能够增强机体的特异性细胞免疫应答强度。通过上述试验，确定了适宜的检测时间点和HBsAg的免疫剂量。然后将60种化合物与HBsAg MHC Ⅰ类多肽S_(28-39)及HBsAg联合免疫BALB／c小鼠后，ELISPOT方法检测小鼠脾淋巴细胞经多肽S_(28-39)刺激，分泌IFN-γ的水平，来评价化合物的免疫佐剂活性。经过初筛和复筛，最终确定了2种免疫佐剂活性最高的MDP类似物：5#、17#化合物。最后将筛选出的5#、17#化合物作为HBsAg的佐剂，对其免疫原性做了进一步的研究。在细胞免疫方面，ELISPOT检测小鼠脾淋巴细胞IFN-γ分泌水平的结果显示5#、17#化合物分别与HBsAg混合组均高于HBsAg组、疫苗组。通过Luminex方法对免疫后小鼠脾细胞经HBsAg刺激分泌的多细胞因子进行测定，探讨了5#、17#化合物作为HBsAg佐剂对HBsAg细胞免疫的影响。通过~(51)Cr释
[Abstract]:China belongs to one of the high prevalence areas of hepatitis B, and there are as many as 100 million carriers of hepatitis B surface antigen HBsAg, among which there are 30 million patients with chronic hepatitis B. there are no effective drugs for the treatment of hepatitis B at present. Aluminum salt is the current adjuvant of hepatitis B vaccine. A large number of studies show that aluminum adjuvant mainly improves humoral immunity and cellular immune response of Th2. In 1974, Lederer E. et al. Found that MDP) is the smallest effective structural unit of Freund complete adjuvant. It is one of the components of inactivated mycobacterium cell wall. MDP has a wide range of biological activities, but it also shows some side effects such as fever. In order to improve its activity and reduce its side effects, many MDP derivatives and analogues were synthesized, and some compounds with higher activity and lower side effects were found. The bioactivity of 60 MDP analogues synthesized by the Institute of Pharmacology of China Union Medical University was screened by Elisa. The immunogenicity of HBsAg was further studied by using two kinds of MDP analogues, named as the adjuvant of HBsAg. The dynamics of cellular immune response of BALB/c mice immunized with 3ug HBsAg were studied by ELISPOT method. The results showed that after 4 days of immunization, the cellular immune response reached a certain intensity at 14 days after 4 days of immunization, when a weak cellular immune response was produced at 7 days. At the same time, the dose-response relationship of spleen lymphocytes and CD8T cells immune response of mice immunized with HBsAg was studied after 7 days. The results showed that, with the increase of immune dose, It can enhance the specific cellular immune response. Through the above experiments, the appropriate detection time point and the immune dose of HBsAg were determined. Then, 60 compounds were immunized with HBsAg MHC class I polypeptides (SZ28-39) and BALB/c mice were immunized with HBsAg. The levels of IFN- 纬 secreted from spleen lymphocytes of mice were detected by Elisa method after immunizing BALB/c mice by SSP 28-39), to evaluate the immuno-adjuvant activity of the compounds, and to evaluate the immunoadjuvant activity of the compounds by screening and screening again. Finally, two MDP analogues with the highest immunological adjuvant activity were identified. Finally, the immunogenicity of the selected 5 #n17# compound as the adjuvant of HBsAg was further studied. In cellular immunity, the level of IFN- 纬 secretion in spleen lymphocytes of mice was detected by ELISPOT. The results showed that the level of IFN- 纬 secretion in the mixed group of 5 #n17# and HBsAg was higher than that in the HBsAg group. In the vaccine group, the polycytokines stimulated by HBsAg in the spleen cells of mice after immunization were determined by Luminex method, and the effect of 5 #N 17# compound as an adjuvant of HBsAg on the cellular immunity of HBsAg was investigated.
【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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