喷雾干燥法制备噬菌体展示的1型糖尿病微球疫苗

发布时间：2018-04-01 07:15

本文选题：1型糖尿病疫苗　切入点：喷雾干燥　出处：《中国协和医科大学》2009年硕士论文

【摘要】： 疫苗对乙型肝炎、艾滋病、类风湿性关节炎、癌症及其他感染性疾病的防治和治疗发挥着重要的作用,预防和治疗传染性疾病及人类非传染性重大疾病最有效的方法是接种疫苗。疫苗包括预防性疫苗和治疗性疫苗,随着生物技术的发展,未来的疫苗将以化学合成和重组DNA技术为主的肽类和蛋白质亚单位疫苗以及核酸疫苗为主,但由于其在体内的快速降解和消除而使它们存在的药剂学缺陷。目前普遍采用的接种方式为注射,这种方式存在制作费用高、耐受性差和需专业技术人员操作等问题。另外,由于单独接种疫苗引起的免疫应答微弱,为提高疫苗的免疫原性,需多次接种或应用佐剂以诱导出更理想的免疫应答。可降解的聚合物微球是一种具有佐剂效应的疫苗控释系统,它可以提供大量抗原并保护其免受生理条件下的快速降解,通过微球对所包埋抗原的持续或脉冲释放行为,有效模拟传统疫苗多次免疫程序,在相当长时间内持续提供抗原。因而可降解的聚合物微球作为一种有效的靶向递送系统,减少接种次数和免疫原的使用总量,因而可以改进病人的适应性、降低用药成本。许多传染性疾病是通过病原微生物与消化道、呼吸道和生殖道的表面黏膜接触而引起的,口服疫苗给药系统(orally administered vaccine deliverysystem,OAVDS)使用方便且直接刺激易感染部位(胃肠道黏膜)产生抗体,在抵御病原体入侵,尤其对最初在黏膜部位的病原体感染发挥重要作用,同时对儿童免疫、地区疾病的防治亦表现出特别的优势,而且OAVDS在减少接种次数、降低接种脱漏率、提高疫苗贮运管理效率及简化接种方式等方面具有重要意义。以胃肠道黏膜为输送部位的OAVDS,是目前疫苗递送研究的热点之一。但由于受胃肠道因素的影响,多数蛋白类药物易失活且吸收差,致使生物利用度低,其口服给药系统研究仍面临挑战。为了解决胃肠道因素对疫苗活性影响,提高疫苗的免疫效果,增强疫苗的稳定性、靶向性及延长疫苗作用的效期,目前新型的疫苗载体一微球载体可有效避免胃肠道环境对疫苗的降解作用,在实现整体式摄取后,于免疫效应部位引发高效的应答反应,最终实现疫苗的单剂量接种。目前,已有流感、乙肝和百日咳杆菌、支原体肺炎、破伤风等多种疫苗采用微粒形式作为口服接种的载体,源于肠毒性大肠杆菌移生因子抗原的口服微球疫苗已进入临床前试验。本研究首次采用水溶性壳聚糖、海藻糖作为保护剂配方,通过喷雾干燥技术制备了噬菌体展示的1型糖尿病壳聚糖微球疫苗。扫描电镜下观察其形态及粒径大小,滴度法检测其活性及热稳定性。结果通过喷雾干燥法制备了粒径大小为5-50μm左右的壳聚糖微球疫苗,收率在35%-53%左右,滴度检测显示喷雾干燥后实验组样品的滴度与对照组相比均降低,并且实验组的滴度随着入口温度的升高先升高而后降低,热稳定性分析显示喷雾干燥样品37℃保存一周和25℃保存6个月后,滴度下降的幅度明显小于对照组,6个月后实验中滴度最小降幅为21.86%、最大降幅为36.34%,而对照组下降了57.79%,与对照组相比制备的壳聚糖微球疫苗具有更好的热稳定性。此外,通过对结果的正交分析,我们确定了最佳的喷雾干燥工艺A4B1C3D2,即壳聚糖的浓度为1%、壳聚糖与海藻糖的重量比为5:1、进口温度为160℃、空气流速为3.0m/s。
[Abstract]:The vaccine of hepatitis B, AIDS, rheumatoid arthritis, prevention and treatment of cancer and other infectious disease plays an important role in the prevention and treatment of infectious diseases and non infectious diseases of major human method is the most effective vaccine. The vaccine including preventive vaccines and therapeutic vaccines, with the development of biotechnology, the future the vaccine will be based on the chemical synthesis and recombinant DNA technology based peptide and protein subunit vaccine and nucleic acid vaccine, but because of its rapid degradation in vivo pharmaceutics and elimination and make them the defects of inoculation. Widely used for injection, this method has high production costs, poor tolerance and problems the operation of specialized technical personnel. In addition, the immune responses induced by vaccination alone is weak, in order to improve the immunogenicity of the vaccine, vaccination should be used repeatedly or adjuvant The immune response induced by better. Biodegradable polymer microsphere is a kind of adjuvant vaccine delivery system, which can provide a lot of antigen and protect it from rapid degradation under physiological conditions, the microspheres for embedded antigen continuous or pulsed release behavior, effective simulation of traditional vaccine program. Continue to provide antigen in quite a long time. So the biodegradable polymer microspheres as an effective targeting delivery system, reduce the use of the original total inoculation times and immunity, thus can improve the adaptability of patients, reduce drug costs.
Many infectious diseases by pathogenic microorganisms and gastrointestinal mucosal contact surface, respiratory and reproductive tract caused by the drug delivery system for oral vaccine (orally administered vaccine deliverysystem, OAVDS) is easy to use and easy to directly stimulate the infection site (gastrointestinal mucous membrane) to produce antibodies against the pathogens in the invasion, especially play an important role in the initial infection in the mucous membranes of pathogens, at the same time on children's immunity, prevention of disease in this area also showed a special advantage, and OAVDS in reducing inoculation times, reduce the rate of missed vaccination, has important significance to improving the management efficiency and reduce the inoculation of vaccine storage and transportation methods. In the gastrointestinal mucosa for delivery site OAVDS, is one of the hot spot vaccine delivery at present. But due to the influence of gastrointestinal factors, most protein drugs are easy to inactivation and poor absorption, resulting in low bioavailability, the Oral administration system research is still facing challenges. In order to solve the gastrointestinal influencing factors of vaccine activity, improve the immune effect of vaccine, vaccine enhanced stability, targeting and extend the validity of the vaccine, the vaccine vector model of the microspheres can effectively avoid the degradation of the gastrointestinal environment of the vaccine, to achieve the overall type ingestion on the immune effector trigger response efficiency, single dose vaccination and ultimately vaccines. At present, the existing influenza, hepatitis B and pertussis, mycoplasma pneumonia, tetanus vaccine using multiple carrier particles form as oral inoculation, source colonization factor antigen oral microsphere vaccine has entered clinical trials in the intestine the toxicity of Escherichia coli.
This is the first study to the water soluble chitosan, trehalose as the protective agent, were prepared by spray drying technique of type 1 diabetes chitosan microsphere vaccine phage display. Scanning electron microscopy to observe the morphology and particle size, determine its activity and thermal stability was prepared by spray drying process. The results of the particle size for about 5-50 m chitosan microsphere vaccine, the yield was about 35%-53%, was detected after spray drying experiment group sample titer was lower compared with control group, the experimental group and the titer increased with the temperature of the entrance first increased and then decreased with high thermal stability, analysis showed that spray drying samples stored at 37 week and 25 stored at 6 months after the titer decreased significantly than the control group, after 6 months the titer minimum decrease is 21.86% and the largest decline of 36.34%, while the control group decreased by 57.79%, and the control group Compared with the thermal stability of the prepared chitosan microsphere vaccine has better. In addition, through orthogonal analysis of the results, we determine the optimal A4B1C3D2 spray drying process, namely the concentration of chitosan was 1%, chitosan and trehalose in the weight ratio of 5:1, inlet temperature is 160 DEG C, the air flow rate is 3.0m/s.

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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