鼠疫亚单位疫苗和口服活载体疫苗的研究

发布时间：2018-11-03 18:40

【摘要】： 鼠疫是由鼠疫耶尔森菌(Y.pestis)引起的人畜共患烈性传染病,人间鼠疫包括肺鼠疫和腺鼠疫,腺鼠疫是通过跳蚤叮咬感染,Y.pestis迁移到肺部就会引起肺鼠疫感染,肺鼠疫能够通过气溶胶在人与人之间传播,在不予治疗的情况下死亡率高达100%。目前鼠疫仍是一个重要的公共卫生问题,世界上每年还有上千感染病例,主要分布在非洲、亚洲和南美洲。由于传统的鼠疫疫苗存在对肺鼠疫无效、副反应强烈等缺陷,至今世界还没有一种值得推广的鼠疫疫苗。一种理想的鼠疫疫苗应具备成本低、热稳定、能通过黏膜简便方法接种、能同时刺激机体产生体液和细胞免疫应答等特点,基于肺鼠疫的感染特点,激发黏膜免疫反应是预防肺鼠疫的研究重点。Y.pestis能产生很多由染色体或质粒编码的毒力因子,包括pgm、pst、Yops、F1抗原及V抗原等,由于这些毒力因子的致病作用机制尚不清楚,很难确定候选疫苗的组分。然而,诸多研究发现,F1和V抗原是鼠疫两大主要保护性抗原,也是鼠疫新型疫苗研究的重点。本研究就是以F1和V抗原为材料,进行鼠疫新型疫苗的探索,主要内容包括: 1.从Y.pestis EV76株的质粒中钓取编码F1和V抗原的基因片段,并将二者融合克隆到原核表达载体pET11c上,转入宿主菌E. coli BL21,用IPTG诱导表达重组融合蛋白rF1-V,纯化后加入氢氧化铝佐剂制备鼠疫候选亚单位疫苗。 2.将编码F1-V融合蛋白的基因,克隆到原核表达载体asd-pTrc99A,转入asd-大肠杆菌X6097,用电击转化经减毒沙门氏菌中间宿主asd- X3730转入减毒沙门氏菌终末宿主asd- X4072得到重组菌X4072(F1-V/99A),将菌体裂解液做Western-blot鉴定表达的F1-V融合蛋白。从而构建出稳定表达F1-V融合抗原的减毒沙门氏菌X4072(F1-V/99A),作为鼠疫口服疫苗候选株。 3.以小鼠为动物模型进行鼠疫候选DNA疫苗(裸质粒F1-V/pVAX1)的安全性检测,通过接种动物的大体观察和称重等方法,进行急性毒性和长期毒性试验;提取组织DNA,通过PCR方法,检测质粒F1-V/pVAX1在小鼠组织的分布和滞留时间;用ELISA和ELISPOT方法进行抗核抗体检测。初步验证裸质粒F1-V/pVAX1的安全性,为下一步口服活载体DNA疫苗的研究提供依据。 4.将编码F1-V融合抗原的基因克隆到真核表达载体asd-pVAX1,转入asd-大肠杆菌X6212,再依次将重组质粒转化asd-减毒沙门氏菌X3730、X4550,得到重组减毒沙门氏菌X4550(F1-V/AP),提取重组质粒转染COS-7细胞,用免疫组化和Western-blot
[Abstract]:Yersinia pestis is a zoonotic infectious disease caused by Yersinia pestis (Y.pestis). Human plague includes pneumonic plague and glandular plague, which is infected by flea bite, and the migration of Y.pestis to the lungs can cause pneumonic plague infection. Pneumonic plague can spread from person to person through aerosol, with a mortality rate of up to 100 kgs without treatment. Plague is still an important public health problem, with thousands of infections worldwide each year, mainly in Africa, Asia and South America. Because the traditional Yersinia pestis vaccine is ineffective to the pneumonic plague and has strong side effects, so far there is not a Yersinia pestis vaccine worth popularizing in the world. An ideal Yersinia pestis vaccine should have the characteristics of low cost, stable heat, simple method of inoculation through mucous membrane, stimulation of humoral and cellular immune response, etc., based on the characteristics of pneumonic plague infection. Y.pestis can produce many virulence factors encoded by chromosomes or plasmids, including pgm,pst,Yops,F1 antigen and V antigen. Because the pathogenicity of these virulence factors is unclear, it is difficult to determine the components of the candidate vaccine. However, many studies have found that F1 and V antigens are the two major protective antigens of plague, and they are also the focus of the study on the new vaccine of Yersinia pestis. In this study, F1 and V antigens were used as materials to explore a new vaccine for plague. The main contents were as follows: 1. The gene fragment encoding F1 and V antigen was isolated from the plasmid of Y.pestis EV76 strain and cloned into prokaryotic expression vector pET11c. The recombinant fusion protein rF1-V, was expressed by IPTG induced by E. coli BL21,. After purification, aluminum hydroxide adjuvant was added to prepare candidate subunit vaccine of Yersinia pestis. 2. The gene encoding F1-V fusion protein was cloned into prokaryotic expression vector asd-pTrc99A, into asd- Escherichia coli X6097. Asd- X3730, an intermediate host of attenuated Salmonella, was transformed into asd- X4072, the final host of attenuated Salmonella. The recombinant strain X4072 (F1-V/99A) was obtained by electroporation. The recombinant strain X4072 (F1-V/99A) was used as the F1-V fusion protein identified by Western-blot. Thus, attenuated Salmonella X4072 (F1-V/99A), which stably expressed F1-V fusion antigen, was constructed as an oral vaccine candidate for Yersinia pestis. 3. The safety of candidate DNA vaccine (naked plasmid F1-V/pVAX1) of Yersinia pestis was detected in mice as animal model. Acute and long-term toxicity tests were carried out by means of gross observation and weighing of inoculated animals. The distribution and retention time of plasmid F1-V/pVAX1 in mouse tissues were detected by PCR, and the antinuclear antibodies were detected by ELISA and ELISPOT methods. The safety of naked plasmid F1-V/pVAX1 was preliminarily verified, which provided the basis for the further study of oral live vector DNA vaccine. 4. The gene encoding F1-V fusion antigen was cloned into eukaryotic expression vector asd-pVAX1, into asd- Escherichia coli X6212, then transformed into asd- attenuated Salmonella X3730 X4550 in turn to obtain recombinant attenuated Salmonella X4550 (F1-V/AP). Recombinant plasmid was extracted and transfected into COS-7 cells. Immunohistochemistry and Western-blot were used.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392.1

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