炭疽、鼠疫基因工程联合疫苗的前期研究

发布时间：2018-05-09 20:02

本文选题：炭疽 + 鼠疫　；参考：《中国人民解放军军事医学科学院》2009年博士论文

【摘要】： 炭疽和鼠疫都属于自然疫源性烈性传染病,这两种疾病的病原体炭疽芽孢杆菌和鼠疫耶尔森菌是极其重要的生物恐怖剂/生物战剂。美国、前苏联、日本等多个国家都曾把炭疽杆菌和鼠疫菌制成生物武器, 2001年美国发生的邮寄炭疽芽孢恐怖袭击事件再次提醒我们时刻不可对生物恐怖掉以轻心,我国存在较大范围的炭疽和鼠疫的自然疫源地,平战时都有可能同时面对炭疽和鼠疫的威胁。近年来随着抗生素的大量使用,多重耐药菌株的出现使得炭疽和鼠疫的预防显得更加重要。目前针对这两种疾病国内外已上市的疫苗有炭疽减毒活疫苗、炭疽吸附上清疫苗、鼠疫灭活全菌死疫苗、鼠疫减毒活疫苗等,但都存在副作用大等缺点。目前以rPA为基础构建的新型重组亚单位炭疽疫苗,以rF1和rV为基础构建的新型重组亚单位鼠疫疫苗都已进入临床试验阶段。炭疽芽孢杆菌和鼠疫耶尔森菌保护性抗原明确,诱导的免疫以体液免疫为主,研制炭疽、鼠疫基因工程联合疫苗可以简化免疫程序、增加使用者的依从性、降低生产成本并节约卫生资源,将为应对与炭疽和鼠疫相关的生物战/生物恐怖、重大传染病和突发公共卫生事件提供保障。本研究的目的是利用基因工程技术制备鼠疫保护性抗原,评价炭疽和鼠疫亚单位疫苗联合免疫的效果,为炭疽、鼠疫基因工程联合疫苗的研制奠定基础。大肠杆菌原核表达系统具有遗传背景清楚、技术操作简便、生产周期短、培养条件简单等特点,是最常用的外源蛋白表达系统之一。此前本室已成功在大肠杆菌表达系统中表达并纯化了炭疽保护性抗原PA。考虑到F1和V来自于原核细胞,分子量比较适中,本研究首先进行了F1和V抗原在大肠杆菌系统中的高效表达研究。F1抗原由于疏水性较强,因而对表达和纯化都提出了挑战。此前研究报道V抗原多采用融合GST标签亲和纯化后,再去除标签的方法进行纯化。我们考虑无标签蛋白与标签蛋白相比虽然纯化步骤更为复杂,但不必考虑标签去除以及成品中是否残存标签或蛋白酶,更便于进行成品的质量控制。经过多次尝试,最后选择使用pET32a(+)载体在大肠杆菌BL21(DE3)中表达了无标签的F1和V抗原,结果表明实现了无标签F1和V的高效表达。随后将摇瓶工艺放大5倍至14L生物反应器中,然后又放大5倍至42L生物反应器中,成功得到了优化的中试发酵生产工艺。在此基础上分别进行了三次中试发酵,结果显示发酵工艺稳定,具有良好的重复性。继而充分利用蛋白质的各种性质进行目的蛋白分离纯化。在V抗原纯化过程中,首先利用V抗原的疏水性质将V抗原从裂菌后的上清中捕获出来。然后利用V抗原在pH值高于等电点时带负电荷的性质,将捕获的V抗原通过弱阴离子柱DEAE层析进行进一步纯化,最后采用凝胶过滤层析纯化得到纯度95%的V抗原。蛋白免疫印迹结果显示纯化的V抗原中存在单体和二聚体两种形式。研究表明,F1抗原在生理条件下易聚集形成多聚体,且多聚体形式的F1抗原与单体F1抗原相比具有更好的免疫原性和保护效果。本研究首先采用盐析的方法进行F1抗原初步分离提纯,随后利用凝胶过滤层析进行进一步的精纯并交换缓冲液,结果获得纯度95%的F1抗原。蛋白N端氨基酸序列测定显示结果与预期完全一致。在此基础上,将蛋白使用氢氧化铝佐剂进行吸附,在小鼠、豚鼠和兔体内进行了免疫原性研究,观察抗原免疫后的体液免疫反应,并比较联合免疫组和单独免疫组之间体液免疫反应的差异。免疫原性的结果显示,在三种动物模型上,联合免疫组和单独免疫组相比具有相似的体液免疫反应,初次免疫后一至二周后动物血清中可检测到相应的抗原特异性IgG抗体,加强免疫后达到较高水平。抗体亚型分析结果显示,小鼠共免疫rPA、rF1+rV没有改变抗原特异性的IgG1/IgG2a比值,抗体反应趋向于Th2型;小鼠长期免疫原性研究显示抗体滴度维持超过一年之久,预示了联合疫苗具有良好的长期效果。在掌握了抗原免疫原性资料后,选择炭疽敏感动物模型兔进行了炭疽芽孢攻击保护效果的研究,选择鼠疫敏感动物模型小鼠进行了鼠疫菌攻击保护效果的研究。结果显示联合免疫后,兔可以抵抗皮下注射80LD50的毒力炭疽芽孢攻击,而佐剂对照组的兔在6天内全部死亡。小鼠模型结果显示联合免疫后,小鼠可抵抗皮下攻击高达60,000MLD鼠疫菌,佐剂对照组则于4天内全部死亡。在两种动物模型中,联合免疫组生存率/生存时间不低于单独免疫组。本研究利用基因工程技术制备了鼠疫的保护性抗原F1和V,与炭疽保护性抗原PA联合后,使用氢氧化铝佐剂进行吸附,在小鼠、豚鼠、兔模型上初步评价了抗原联合免疫的免疫效果和保护效果。结果表明联合免疫后可分别对毒力鼠疫耶尔森菌和炭疽芽孢攻击产生有效保护。此外,免疫后产生的抗体滴度在小鼠体内维持超过一年时间,预示联合疫苗具有良好的长期效果。
[Abstract]:Anthrax and plague are all natural foci of infectious diseases. The pathogens of the two diseases, Bacillus anthracis and Jerson, are extremely important bioterrorism agents / biological agents. The United States, the former Soviet Union, Japan and other countries have made the Bacillus anthracis and Yersinia pestis into biological weapons, and the anthrax spores were mailed in the United States in 2001. The terrorist attacks remined us once again that we could not take care of biological terror at all times. There is a wide range of natural foci of anthrax and plague in our country. It is possible to face the threat of anthrax and plague at the same time in peacetime. In recent years, with the extensive use of antibiotics, the emergence of multiple resistant strains has made the prevention of anthrax and plague appear. More important. At present, the vaccine has been listed at home and abroad of these two diseases, including anthracnose attenuated live vaccine, anthrax adsorbed supernatant vaccine, plague inactivated whole bacteria vaccine, plague attenuated live vaccine and so on, but there are many disadvantages such as large side effects. The new recombinant subunit anthrax vaccine based on rPA is based on rF1 and rV. The new recombinant subunit plague vaccine has entered the clinical trial stage. The protective antigen of Bacillus anthracis and Yersinia pestis is clear, the induced immunity is mainly humoral immunity, and anthrax is developed. The combined vaccine of Yersinia pestis gene engineering can simplify the immune procedure, increase the compliance of the users, reduce the cost of production and save the health resources. The aim of this study is to prepare the plague protective antigen by genetic engineering technology, to evaluate the effect of combined immunization with anthrax and the plague subunit vaccine, and to develop a joint vaccine for anthrax and plague gene engineering. Lay the foundation.
The prokaryotic expression system of Escherichia coli has the characteristics of clear genetic background, simple operation, short production cycle, simple culture conditions, and is one of the most commonly used foreign protein expression systems. This room has successfully expressed and purified the protective antigen PA. of anthrax in the Escherichia coli expression system, considering that F1 and V are derived from the prokaryotic cells. The high expression of F1 and V antigen in the Escherichia coli system was first carried out in this study. The.F1 antigen was highly hydrophobic, so the expression and purification were challenged. The previous study reported that the V antigen was purified by fusion GST label affinity purification and then the label was removed. We consider no label. Although the purification step is more complex than the protein label protein, it is not necessary to consider the label removal and the remaining labels or proteases in the finished product. The pET32a (+) carrier is selected to express the unlabeled F1 and V antigen in the Escherichia coli BL21 (DE3) after several attempts. The results show that it is realized. The efficient expression of unlabeled F1 and V.
Then the shake flask process was amplified by 5 times to the 14L bioreactor, and then amplified by 5 times to the 42L bioreactor, the optimized fermentation process was successfully obtained. On this basis, three pilot fermentation processes were carried out respectively. The results showed that the fermentation process was stable and had good refolding. Then the various properties of protein were fully utilized. V antigen was purified by using the hydrophobic properties of V antigen to capture the V antigen from the supernatant of the cracked bacteria in the process of purification of the antigen. Then the V antigen was purified by the DEAE layer of the weak anion column and purified by the V antigen at the pH value higher than the isoelectric point. Finally, the gel filtration was used. The purity of 95% V antigen was purified by chromatography. The results of protein immunoblotting showed that there were two forms of monomer and two polymer in the purified V antigen. The study showed that the F1 antigen was easily aggregated under physiological conditions, and the F1 antigen of the polymer form had better immunogenicity and protective effect compared with the monomer F1 antigen. The method of salting out was used to separate and purify the F1 antigen preliminarily, then further purified and exchanged by gel filtration chromatography. The results obtained the purity of 95% F1 antigen. The results of the amino acid sequence of protein N were in full agreement with the expected results.
On this basis, the protein was adsorbed with aluminum hydroxide adjuvant, and the immunogenicity was studied in mice, guinea pigs and rabbits. The humoral immune response after antigen immunization was observed and the difference of humoral immune response between the combined immune group and the individual immune group was compared. The results of immunogenicity showed that the immunogenicity was combined with the three animal models. The pestilence group had a similar humoral immune response compared with the individual immune group. The corresponding antigen specific IgG antibody could be detected in the animal serum for one to two weeks after the initial immunization, and the immunization reached a high level. The antibody subtype analysis showed that the mice were immunized with rPA and rF1+rV did not change the specific IgG1/IgG2a ratio of the antigen, and the antibody was not changed. The response tended to be Th2 type. Long term immunogenicity study showed that the antibody titer lasted for more than a year, indicating that the combined vaccine had good long-term effects.
After mastering the antigen immunogenicity data, the rabbit model of anthrax sensitive animal model was selected to study the protective effect of the anthrax spore attack. The mouse pestis sensitive animal model mice were selected to carry out the study on the protection effect of the Yersinia pestis attack. The results showed that the rabbit could resist the attack of the 80LD50 anthracnose spore under the subcutaneous injection. The rabbits in the control group died within 6 days. The mice model showed that the mice could resist the subcutaneous attack as high as 60000MLD pestis, and the adjuvant control group died in 4 days after the combined immunization. In the two animal models, the survival rate / survival time of the combined immune group was not lower than that of the single immune group.
In this study, the protective antigen F1 and V of plague were prepared by genetic engineering. After combined with the protective antigen PA of anthrax, the adsorption was carried out by aluminum hydroxide adjuvant. In mice, guinea pigs and rabbits, the immune effect and protective effect of antigen combined immunization were preliminarily evaluated. The results showed that the combined immunization could be used for the virulence of Yersinia pestis, Jerson. Bacteria and anthrax spore attacks produce effective protection. In addition, the antibody titer produced after immunization is maintained in mice for more than one year, indicating that the combined vaccine has a good long-term effect.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

【参考文献】