艰难梭菌基因重组乳链菌口服活菌疫苗的应用基础研究

发布时间：2018-07-15 08:47

【摘要】：研究背景：艰难梭菌(Clostridium difficile)是西方工业化国家医院内感染的重要致病菌之一。临床上约10％～20％的抗生素相关性腹泻、75％抗生素相关性肠炎和近100％的伪膜性肠炎与此菌有关，统称为艰难梭菌相关性疾病(Clostridium difficile associated diseases，CDAD)，每年造成数十亿美元的经济损失。随着我国工业的进一步发展以及人们对抗生素的使用日益普遍，其发病率有增加的趋势。CDAD主要发生于长期服用抗生素者，也可发生于其他影响肠道正常菌群，降低抵抗艰难梭菌定殖的情形，如用抗肿瘤药物、长期住院、免疫缺陷等，特别是那些有免疫功能降低者和／或老年人。CDAD临床表现复杂多样，从无症状携带者到爆发性结肠炎。重症患者预后差，死亡率高达40％。甲硝唑和万古霉素是治疗该病的主要药物，，但近年来，国内外均出现同时耐甲硝唑、万古霉素的艰难梭菌，给艰难梭菌的治疗增加了困难，因此进行免疫防治研究十分重要。艰难梭菌疫苗的研究目前已取得了很大进展，并出现了各种类型的疫苗，但由于种种原因，限制了其应用。乳酸链球菌(Streptococcus lactis；Lactococcus lactis，以下简称乳链菌)是一种对人体无害，较为安全的食品工业菌，近年来乳链菌因不产生内毒素，及产生的蛋白酶远少于大肠杆菌等优点而被大量用于表达多种生物多肽或蛋白，及口服疫苗的研究工作，并且被证实是安全有效的，这一方法为口服疫苗的研制开辟了一个全新的领域。目前国内外均未见艰难梭菌重组乳链菌口服活菌疫苗的报道。目的及意义：由于乳链菌表达载体尚未商品化，无法通过购买或捐赠获得。因而我们参照一些成熟的乳链菌表达系统构建方法及Genebank公布的序列，设计并构建具有自主知识产权的乳链菌表达系统；分别用这套表达系统表达了艰难梭菌A毒素羧基末端重复序列(14CDTA)、破伤风毒素C片段(TETC)及TETC—14CDTA
[Abstract]:Background: Clostridium difficile is one of the most important nosocomial pathogens in western industrialized countries. Clinically, about 10% of antibiotics associated with diarrhea and 75% of antibiotic associated enteritis and nearly 100% of pseudomembranous enteritis are associated with this bacterium, collectively known as Clostridium difficile associated disease (CDAD), causing billions of dollars of economic losses each year. With the further development of industry in China and the increasing use of antibiotics, the incidence of CDAD has an increasing trend. CDAD mainly occurs in patients who take antibiotics for a long time, but also in other bacteria that affect normal intestinal flora. To reduce the colonization of resistance to Clostridium difficulty, such as antitumor drugs, long-term hospitalization, immune deficiency, especially those with reduced immune function and / or the elderly. CDAD has complex clinical manifestations, ranging from asymptomatic carriers to explosive colitis. The prognosis of severe patients was poor, and the mortality was as high as 40%. Metronidazole and vancomycin are the main drugs for the treatment of this disease, but in recent years, there are Clostridium difficult to resist metronidazole and vancomycin at home and abroad, which makes it more difficult to treat Clostridium difficult, so it is very important to carry out immunological control research. Great progress has been made in the research of Clostridium difficulty vaccine, and various kinds of vaccines have appeared, but due to various reasons, its application is limited. Streptococcus lactis Lactococcus lactis (hereinafter referred to as Lactococcus lactis) is a harmless and relatively safe food industry bacterium. In recent years, Streptococcus lactis does not produce endotoxin. The protease produced is much less than Escherichia coli and is widely used to express a variety of biological polypeptides or proteins, as well as oral vaccine research work, and has been proven to be safe and effective, This method opens up a new field for oral vaccine development. At present, there is no report of live oral vaccine of Clostridium difficulty recombinant lactobacillus at home and abroad. Objective and significance: because Streptococcus lactis expression vector has not been commercialized, it can not be obtained through purchase or donation. Therefore, we design and construct the Lactobacillus expression system with independent intellectual property rights according to some mature methods of construction of Lactobacillus expression system and the sequence published by Genebank. The expression system was used to express the carboxyl terminal repeat sequence (14CDTA), tetanus toxin C fragment (TETC) and TETC-14 CDTA of Clostridium diffracta respectively.
【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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