嗜水气单胞菌微囊口服疫苗的制备及其免疫效应研究

发布时间：2018-06-27 06:40

本文选题：嗜水气单胞菌 + 微囊口服疫苗　；参考：《南京农业大学》2008年硕士论文

【摘要】：嗜水气单胞菌(Aeromonas hydrophila,Ah)是淡水鱼类的重要病原菌,给淡水养殖业带来了巨大的经济损失,同时也给人类健康带来潜在威胁。嗜水气单胞菌可引起淡水鱼类的出血性败血症,具有高死亡率。因此,规模化养殖场嗜水气单胞菌免疫与防控尤为重要。口服免疫是规模化鱼类养殖中具有良好应用前景的免疫方式。但是由于胃肠复杂环境对疫苗抗原成分的降解,其免疫效果往往不佳。本研究的目的是利用生物相容性好的高分子聚合物制备包裹有全菌抗原的微囊传递系统,开发嗜水气单胞菌口服疫苗。以海藻酸钠(Sodium alginate,SA)和壳聚糖(Chitosan)为材料,利用改良喷雾-离子交联技术制备嗜水气单胞菌微囊(Microsphere,MS)口服疫苗。通过对影响微囊粒径的细菌浓度、海藻酸钠浓度、喷雾速度及搅拌速度等主要技术参数进行正交筛选,确定最优的制备技术路线,并对该口服疫苗微囊的形态、直径、包被效率、载药量、体外模拟释放性、抗原稳定性、安全性、储存稳定性等特性进行检测。所确定的最优微囊制备参数为:SA浓度2%(W/V),细菌浓度5×10~9 cfu/mL,搅拌速度800rpm,喷雾速度7mL/min;微囊圆整,平均粒径为11.010±0.145μm,粒径分布较均匀,83%的微囊直径小于15μm;包被率约(96.52±0.17)%;载菌量为(2.41±0.32)×10~8 cfu/mg;微囊在模拟胃肠条件下稳定,表现出较好的突释性与缓释性;微囊中细菌抗原性保持良好,疫苗安全性与储存稳定性较好。结果表明所开发的嗜水气单胞菌微囊传递系统各项特性较理想,有进一步开发的前景。为了评价所研制的嗜水气单胞菌海藻酸钠-壳聚糖微囊(Ah-ACMS)口服疫苗在动物体内的免疫效果,采用不同剂量、不同剂型(有无佐剂)的Ah-ACMS疫苗分别口服免疫BALB/C小鼠,对免疫小鼠的血清抗体水平、巨噬细胞吞噬活性、淋巴细胞主要细胞因子相对表达量及淋巴细胞转化率等多个免疫指标进行了检测分析,并通过攻毒试验对疫苗的保护效率进行了评价。初次免疫四周后,高、中、低口服疫苗免疫组的各项免疫学参数与细菌口服对照组及空白对照组均差异显著(P＜0.05),高、中剂量口服疫苗组间大部分免疫参数差异不显著(P＞0.05)。Ah+SA+Ch口服组与空白对照组的脾淋巴细胞主要细胞因子表达及巨噬细胞吞噬活性等免疫指标差异显著。各免疫组小鼠的相对保护效率分别为:ACMS高剂量组46.7%;ACMS佐剂组53.3%;Ah腹腔注射组86.7%;而空白对照组与细菌口服对照组的小鼠100%死亡。结果表明所制备微囊口服疫苗对Ah感染具有一定的免疫抵抗力,所用口服佐剂(MonTanide IMS1312)能显著增强微囊口服疫苗的免疫效果,使小鼠的相对保护率提高了13.3%。由于哺乳动物与鱼类的体温、免疫系统及胃肠环境等生理参数有较大差异,为了科学评价所开发微囊口服疫苗的对水生动物的免疫效果,采用不同剂量,不同剂型(佐剂有无)的Ah-ACMs疫苗分别口服免疫银鲫,以腹腔注射福尔马林灭活的Ah(FKC)为阳性对照。初次免疫四周后,对免疫银鲫的血清抗体水平、巨噬细胞吞噬活性等免疫指标进行了检测分析,并通过攻毒试验对疫苗的保护效率进行了评价。低剂量的口服Ah-ACMS组的各项免疫指标与空白对照组之间有显著差异(P＜0.05)。佐剂可显著增强所开发微囊口服疫苗的免疫原性。低剂量Ah-ACMS组,佐剂Ah-ACMS组及腹腔注射组的相对保护率分别为:32.1%、50.0%、82.1%,而FKC口服对照组与空白对照组鲫鱼死亡率分别为96.7%和93.3%。结果表明所开发的Ah-ACMS疫苗对鲫鱼具有一定的免疫效应,可保护鲫鱼抵抗嗜水气单胞菌的感染。综上所述,本研究所开发的聚合物微囊抗原传递系统可有效保护全菌抗原的免疫原性,对复杂的胃肠环境耐受,可诱导小鼠及鲫鱼产生免疫应答。该研究以期能为后继的聚合物微囊口服疫苗的开发提供参考。有望进一步推广应用于其他鱼类病原菌口服疫苗的开发。
[Abstract]:Aeromonas hydrophila (Ah), an important pathogen of freshwater fishes, has brought huge economic losses to the freshwater aquaculture industry and also poses a potential threat to human health. Aeromonas hydrophila can cause hemorrhagic septicemia in freshwater fishes and have high death and death rates. Therefore, the immunization of Aeromonas hydrophila in large-scale farms. And prevention and control is particularly important. Oral immunization is a promising application of immunization in large-scale fish culture. However, the immune effect is often poor due to the degradation of antigen components in the complex gastrointestinal environment. The purpose of this study is to prepare microcapsules encapsulated with full bacterial antigen by good biocompatible polymer polymer. An oral vaccine for Aeromonas hydrophila was developed.
Taking sodium alginate (Sodium alginate, SA) and chitosan (Chitosan) as materials, the oral vaccine of Aeromonas hydrophila microcapsule (Microsphere, MS) was prepared by improved spray ion crosslinking technology. The main technical parameters, such as the concentration of bacteria, the concentration of alginate, the velocity of spray and the stirring speed, were screened by the orthogonal screening of the bacteria concentration affecting the microcapsule diameter. The optimal preparation technique was used to detect the morphology, diameter, encapsulation efficiency, drug loading, in vitro analogue release, antigen stability, safety and storage stability of the oral vaccine. The optimum microcapsule preparation parameters were SA concentration 2% (W/V), bacterial concentration 5 x 10~9 cfu/mL, stirring speed 800rpm, spray velocity 7mL/m In, the microcapsules were round and round, the average particle size was 11.010 + 0.145 mu m, the particle size distribution was more uniform, the diameter of the microcapsule was less than 15 mu m, the inclusion rate was about (96.52 + 0.17)%, and the carrier amount was (2.41 + 0.32) x 10~8 cfu/mg, and the microcapsules were stable under simulated gastrointestinal conditions and showed good release and sustained release; the bacteria antigenicity in microcapsules was good and vaccine safety and vaccine safety The storage stability was good. The results showed that the Aeromonas hydrophila microcapsule delivery system had better characteristics and had further development prospects.
In order to evaluate the immune effect of the oral vaccine of Aeromonas hydrophila sodium alginate chitosan microcapsule (Ah-ACMS) in animals, the Ah-ACMS vaccine of different doses and different dosage forms (without adjuvant) was used to immunize BALB/C mice orally. The anti body level of the serum, the phagocytic activity of macrophage and the main lymphocyte of the mice were mainly fine. The relative expression of cytokine and lymphocyte transformation rate were detected and analyzed, and the protective efficiency of the vaccine was evaluated by the attack test. After the first four weeks, the immunological parameters of the high, middle and low oral immunization groups were significantly different from those in the oral control group and the blank control group (P < 0.05). The difference of most of the immune parameters between high and middle doses was not significant (P > 0.05) in the.Ah+SA+Ch oral group and the blank control group, the expression of main cytokines and the phagocytic activity of macrophages in the control group were significantly different. The relative protective efficiency of the mice in each immunization group was 46.7% in ACMS high dose group and 53.3% in the ACMS adjuvant group. The Ah intraperitoneal injection group was 86.7%, while the mice in the blank control group and the bacterial oral control group died in 100%. The results showed that the oral vaccine prepared by the prepared microcapsules had certain immune resistance to Ah infection, and the oral adjuvant (MonTanide IMS1312) could significantly enhance the immunization effect of the oral vaccine of microcapsules and increase the relative protection rate of the mice by 13.3%..
In order to evaluate the immune effect of the microcapsule oral vaccine for aquatic animals, the Ah-ACMs vaccine of different doses and different dosage forms (adjuvant or not) was taken orally to the inactivated Ah of the formalin in order to evaluate the immune effect of the microcapsule oral vaccine. FKC) was a positive control. After the first four weeks of immunization, the serum antibody level of the silver crucian carp and the phagocytic activity of macrophage were detected and analyzed. The protective efficiency of the vaccine was evaluated by the attack test. There was a significant difference between the immunization index and the blank control group in the low dose group of oral Ah-ACMS group (P < 0.05). The relative protection rate of the low dose Ah-ACMS group, the adjuvant Ah-ACMS group and the intraperitoneal injection group were 32.1%, 50%, 82.1%, respectively, while the mortality rate of the FKC oral control group and the blank control group was 96.7% and 93.3%. respectively, indicating that the Ah-ACMS vaccine developed by the Ah-ACMS was one of the crucian carp. The immunological effect can protect crucian carp against infection by Aeromonas hydrophila.
To sum up, the polymer microcapsule antigen transfer system developed in this study can effectively protect the immunogenicity of all bacterial antigens and can induce immune responses in mice and Carassius auratus in complex gastrointestinal environments. This study is expected to provide a reference for the development of subsequent oral vaccine for microcapsules of polymer microcapsules. Development of oral vaccine for the pathogenic bacteria of fish.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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