猪传染性胃肠炎病毒微胶囊疫苗的制备及其免疫效果评价

doi:10.16431/j.cnki.1671-7236.2023.09.035

摘要/Abstract

摘要： 【目的】制备猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)灭活病毒包被的微胶囊,并评价其对新生仔猪口服黏膜免疫效果,以预防TGEV引起严重腹泻和造成经济损失。【方法】采用一步法以海藻酸钠和壳聚糖为微胶囊壁材,以灭活的TGEV为抗原制备TGEV微胶囊疫苗。以包封率、蛋白载量、体外释放速率为微胶囊质量评价指标,分别从成囊材料的配比及不同盐水溶液对微胶囊性能的影响方面对TGEV微胶囊的制备进行筛选,并评价TGEV微胶囊在不同环境的释放速率和室温储存耐受性。最后进行免疫学分析,对仔猪进行口服免疫,测定各项免疫指标,开展抗体效价检测和病毒中和试验。【结果】筛选出的制备微胶囊的最佳工艺条件为:海藻酸钠浓度1.5%,壳聚糖浓度1.2%,氯化钙浓度3.5%,灭活病毒抗原加入量为1~2 mL,在此工艺条件下制备的TGEV微胶囊在PBS和盐水溶液中第3天释放率均超过50%;第18天,释放率分别达到97%和88%。常温放置5个月最低释放率仍保持在80%以上。口服免疫仔猪,微胶囊高、低剂量组粪便样品中IgA抗体的D_{490 nm}值分别为0.97和1.49,IgG的抗体的D_{490 nm}值分别为1.03和1.75。病毒中和试验中,微胶囊高、低剂量组抗体IgG的病毒50%中和效价分别为1∶512和1∶256;微胶囊高、低剂量组抗体IgA的病毒50%中和效价分别为1∶128和1∶64。【结论】海藻酸钠-壳聚糖微胶囊不仅具有在肠道中释放灭活的TGEV抗原的缓释效果,且对于疫苗免疫具有佐剂辅助效果,可增加适应性黏膜免疫应答中的抗体效价,在肠道中释放灭活的TGEV可有效刺激黏膜和全身免疫反应。

关键词: 传染性胃肠炎病毒(TGEV); 微胶囊; 海藻酸钠; 壳聚糖; 黏膜免疫

Abstract: 【Objective】 The purpose of this experiment was to prepare microcapsules coated with inactivated Transmissible gastroenteritis virus (TGEV),and evaluate its oral mucosal immune effect on newborn piglets,so as to prevent TGEV from causing severe diarrhea and economic losses.【Method】 Sodium alginate and chitosan were used as the microcapsule wall materials,and inactivated TGEV was used as the antigen to prepare TGEV microcapsules by a one-step method.The quality evaluation indicators of microcapsules included encapsulation rate,protein loading,and in vitro release rate.The effects of the composition of the encapsulation materials and different saline solutions on the properties of the microcapsules were investigated to screen the optimal process conditions for TGEV microcapsule preparation.The release rate of TGEV microcapsules under different environmental conditions and their tolerance to room temperature storage were also evaluated.Finally,immunological analysis was conducted,oral immunity was administered to piglets,various immune indicators were measured,antibody titer testing and virus neutralization tests were conducted.【Result】 The optimal process conditions for preparing microcapsules were determined to be 1.5% sodium alginate,1.2% chitosan,3.5% calcium chloride,and 1-2 mL of inactivated virus antigen addition.Microcapsule prepared under these conditions had a release rate of over 50% on the third day in PBS and saline solutions.On the 18th day,the release rate reached 97% and 88%,respectively.The lowest release rate after 5 months of storage at room temperature was still above 80%.After oral immunization of piglets,the D_{490 nm} value of IgA antibody in the fecal sample of microcapsule high and low dose groups were 0.97 and 1.49,respectively,and the D_{490 nm} value of IgG antibody were 1.03 and 1.75,respectively.In virus neutralization experiments,the virus 50% neutralization titers of IgG antibody in microcapsule high and low dose groups were 1∶512 and 1∶256,respectively.The virus 50% neutralization titers of IgA antibody in microcapsule high and low dose groups with were 1∶128 and 1∶64,respectively.【Conclusion】 The sodium alginate-chitosan microcapsules not only had a sustained-release effect on the inactivated TGEV antigen in the intestine but also had an adjuvant effect on vaccine immunization,which could effectively stimulate mucosal and systemic immune responses in the adaptive mucosal immune response.

Key words: Transmissible gastroenteritis virus (TGEV); microcapsules; sodium alginate; chitosan; mucosal immunization

中图分类号:

S852.65^＋1

秦梓椋, 陈少俊, 何鑫淼, 王文涛, 夏继桥, 冯艳忠, 奈子达, 王超, 刘娣, 姜新鹏. 猪传染性胃肠炎病毒微胶囊疫苗的制备及其免疫效果评价[J]. 中国畜牧兽医, 2023, 50(9): 3790-3798.

QIN Ziliang, CHEN Shaojun, HE Xinmiao, WANG Wentao, XIA Jiqiao, FENG Yanzhong, NAI Zida, WANG Chao, LIU Di, JIANG Xinpeng. Preparation of Microencapsulated Vaccine Against Transmissible Gastroenteritis Virus and Its Immunological Efficacy in Piglets[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3790-3798.

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