微囊化大肠杆菌口服灭活疫苗对大鼠免疫效果的研究

doi:10.11843/j.issn.0366-6964.2017.02.014

摘要/Abstract

摘要：

不同血清型的致病性大肠杆菌是引起幼畜腹泻的主要病因之一。笔者拟对采用微囊包被技术制备的羊大肠杆菌灭活口服疫苗对大鼠的免疫效果进行评价。将制备的羊大肠杆菌蜂胶佐剂灭活苗作为芯材，天然高分子材料海藻酸钠为壁材，采用喷雾干燥法制备微囊化口服疫苗。微囊化口服疫苗的含菌量为7.52×10¹¹菌•g^－1。100只成年健康大鼠随机分为对照组、注射组和微囊包被口服疫苗组（再分为基础剂量、10倍基础剂量和20倍基础剂量三组），免疫后采用微量凝集试验和本室建立的间接ELISA方法检测免疫血清抗体效价并对细胞和黏膜免疫进行检测。微量凝集试验检测结果显示，免疫后7 d各试验组即可产生抗体，免疫后28 d时抗体效价达到高峰，随后开始下降。间接ELISA方法检测结果显示，免疫后14 d各试验组特异抗体转阳，并持续到28 d。口服组的抗体效价均显著高于对照组（P<0.05），而与注射组无差异（P >0.05），且各口服剂量组之间差异不显著（P>0.05）。免疫后35 d口服试验组特异抗体转为阴性，而注射组可维持到35 d；T淋巴细胞转化结果显示，免疫7 d后，三个口服剂量组的SI值均显著高于对照组和注射组（P<0.05）。免疫后口服各剂量组的肠黏膜分泌型免疫球蛋白A（sIgA）含量均高于同期的对照组和注射组，21 d后sIgA含量达到高峰。微囊包被口服疫苗能够产生与注射组同样的体液免疫，同时还可以刺激大鼠机体产生较好的细胞免疫和黏膜免疫。

关键词: 大肠杆菌, 口服疫苗, 微囊包被, 免疫, 大鼠

Abstract:

The pathogenic E. coli of different serotypes is a major sort of the causative agents of the diarrhea of young animals. In the present study a microencapsulated E. coli inactivated oral vaccine was prepared and its immune effect in rats was evaluated. The prepared ovine pathogenic E. coli inactivated vaccine with propolis-adjuvant as the core material was microencapsulated by natural alginate polymer as wall material, in which 7.52×10¹¹ bacteria•g^－1 dry powder were encapsulated. A hundred Wistar rats were divided into randomly 3 groups, i.e., the control, injection immunization and oral immunization groups, in which the oral immunization group was futher-divided in to the basal dose, 10-fold dose and 20-fold dose groups. After the inoculation the specific antibody was tested by the micro-agglutination test and the indirect ELISA, and the cellular immunity and mucosal IgA were detected. The micro-agglutination test showed that the specific antibody was produced from day 7 after the inoculation and reached to the peak at day 28, then declined thereafter. The indirect ELISA test showed that the antibody was positive from day 14 to day 28 after inoculation. The antibody titers in oral vaccine groups were significantly higher than that in the control (P<0.05), but not in the injection group (P>0.05), and were not significantly between the oral vaccine groups (P>0.05). The antibody titers in the oral vaccine groups declined to negative level at day 35 after inoculation whereas the injection group remained to be antibody-positive；T lymphocyte transformation test showed that SI index in 3 oral vaccine groups was higher than that in the control or the injection group significantly (P<0.05). The sIgA levels in oral vaccine groups were also significantly higher that either in the control or injection group, reaching to the peak at day 21 after inoculation. These data suggest that microencapsulated E. coli inactivated oral vaccine could produce the humoral immunity equivalent to the injected vaccine but with better cell and mucosal immunity in rats.

Key words: Escherichia coli, oral vaccine, microcapsule coating, immunization, rat

中图分类号:

S855.1

夏瑞阳，徐新龙，董明娣，陈俐静，祁来芳，王玲，姚刚. 微囊化大肠杆菌口服灭活疫苗对大鼠免疫效果的研究[J]. 畜牧兽医学报, 2017, 48(2): 307-315.

XIA Rui-yang，XU Xin-long，DONG Ming-di，CHEN Li-jing，QI Lai-fang，WANG Ling，YAO Gang. Evaluation of the Immune Effect of Microencapsulated E. coli Inactivated Oral Vaccine in Rats[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(2): 307-315.

参考文献

［1］中国农业科学院哈尔滨兽医研究所. 动物传染病［M］. 哈尔滨：哈尔滨出版社, 1999.
Harbin Veterinary Research Institute. Animal infectious diseases［M］. Harbin: Harbin Publishing House, 1999.(in Chinese)
［2］郎洪武，杨汉春. 口蹄疫研究进展［J］. 中国兽药杂志, 2005, 39(10): 16-21.
LANG H W, YANG H C. A review on foot-and-mouth disease［J］. Chinese Journal of Veterinary Drug, 2005, 39(10): 16-21. (in Chinese)
［3］WANG J, MEERS J, SPRADBROW P B, et al. Evaluation of immune effects of fowlpox vaccine strains and field isolates［J］. Vet Microbiol, 2006, 116(1-3):106-119.
［4］LIN D G, SUN S S, DU L J, et al. Natural and experimental infection of dogs with pandemic H1N1/2009 influenza virus［J］. J Gen Virol, 2012, 93(Pt 1): 119-123.
［5］WALKER R I. Considerations for development of whole cell bacterial vaccines to prevent diarrheal diseases in children in developing countries［J］. Vaccine, 2005, 23(26): 3369-3385.
［6］唐丽杰，欧笛，葛俊伟，等. 表达猪传染性胃肠炎病毒 S 基因的重组乳酸乳球菌的构建及免疫原性分析［J］. 微生物学报, 2007, 47(2): 340-344.
TANG L J, OU D, GE J W, et al. Construction of recombinant Lactococcus lactis expressing porcine transmissible gastroenteritis spike glycoprotein and analysis of immunogenicity［J］. Acta Microbiologica Sinica, 2007, 47(2): 340-344. (in Chinese)
［7］BARAS B, BENOIT M, POULAIN-GODEFROY O, et al. Vaccine properties of antigens entrapped in microparticles produced by spray-drying technique and using various polyester polymers［J］. Vaccine, 2000, 18(15): 1495-1505.
［8］LEBENS M, SUN J B, SADEGHI H, et al. A mucosally administered recombinant fusion protein vaccine against schistosomiasis protecting against immunopathology and infection［J］. Vaccine, 2003, 21(5-6): 514-520.
［9］马蕾，姚刚，杨光，等. 微囊化与非微囊化胆囊收缩素卵黄抗体对小鼠促生长免疫效果的比较研究［J］. 中国畜牧兽医, 2013, 40(12): 216-219.
MA L, YAO G, YANG G, et al. Comparative study of microencapsulated and unmicroencapsulated CCK-IgY on the growth immuning effect in mice［J］. China Animal Husbandry and Veterinary Medicine, 2013, 40(12): 216-219. (in Chinese)
［10］WANG G, CHEN J, SHI Y. Preparation of microencapsulated xanthophyll for improving solubility and stability by nanoencapsulation［J］. J Food Eng, 2013, 117(1): 82-88.
［11］AGHBASHLO M, MOBLI H, RAFIEE S, et al. Energy and exergy analyses of the spray drying process of fish oil microencapsulation［J］. Biosystems Eng, 2012, 111(2): 229-241.
［12］SCHER H B, RODSON M, LEE K S. Microencapsulation of pesticides by interfacial polymerization utilizing isocyanate or aminoplast chemistry［J］. Pest Manage Sci, 1998, 54(4): 394-400.
［13］FRIEDMAN-RUDOVSKY J. Chagas disease. With novel paint, chemist aims to vanquish the vinchuca［J］. Science, 2012, 336(6082): 666-667.
［14］STOJAKOVIC D, BUGARSKI B, RAJIC N. A kinetic study of the release of vanillin encapsulated in Carnauba wax microcapsules［J］. J Food Eng, 2012, 109(3): 640-642.
［15］WANG Q X, YAN D D, MAO L G, et al. Efficacy of 1, 3-dichloropropene plus chloropicrin gelatin capsule formulation for the control of soilborne pests［J］. Crop Protection, 2013, 48: 24-28.
［16］SHAKWEH M, BESNARD M, NICOLAS V, et al. Poly (lactide-co-glycolide) particles of different physicochemical properties and their uptake by peyer's patches in mice［J］. Eur J Pharm Biopharm, 2005, 61(1-2): 1-13.
［17］黄嘉驷，苏艳，周军，等. 致病性大肠杆菌微胶囊口服疫苗的制备及对其免疫功能的评价［J］. 新疆农业大学学报, 2009，32(1): 31-34.
HUANG J S, SU Y, ZHOU J, et al. Preparation of the oral microencapsulated vaccine of Escherichia coli and its immunological function［J］. Journal of Xinjiang Agricultural University, 2009, 32(1): 31-34. (in Chinese)
［18］刘纪成. 鸡痢疾志贺氏菌灭活苗免疫抗体微量凝集试验方法的建立及应用［D］. 郑州: 河南农业大学, 2007.
LIU J C. Establishment and application of micro-agglutination test for detection of vaccine immunological antibodies against shigella infection in chicken［D］. Zhengzhou: Henan Agricultural University, 2007. (in Chinese)
［19］新疆农业大学. 用于微囊化大肠杆菌口服疫苗免疫抗体检测的检测方法及检测试剂盒：中国, 201610220735. 4［P］. 2016-4-11.
Xinjiang Agriculture University. Detection methods and kit of the oral microencapsulated vaccine of Escherichia coli: China, 201610220735. 4［P］. 2016-4-11. (in Chinese)
［20］郭曦，王继宏，肖雪珍，等. 4种中药多糖对小鼠脾淋巴细胞增殖的影响［J］. 中国兽医杂志, 2011, 47(8): 56-59.
GUO X, WANG J H, XIAO X Z, et al. Effect of four Chinese herbal polysaccharides on the proliferation of mouse splenic lymphocytes［J］. Chinese Journal of Veterinary Medicine, 2011, 47(8): 56-59. (in Chinese)
［21］张冰冰，赵魁，贺文琦，等.羔羊外周血单个核细胞转化MTT比色法最佳检测条件的筛选［J］.动物医学进展, 2011, 32(3): 65-68.
ZHANG B B, ZHAO K, HE W Q, et al. Study on optimual condition of MTT in PBMC transformation［J］. Progress in Veterinary Medicine, 2011, 32(3): 65-68. (in Chinese)
［22］杜丹，方立超，陈丙波，等. 乳酸杆菌和低聚异麦芽糖对抗生素相关腹泻大鼠肠粘膜SIgA的影响［J］. 放射免疫学杂志, 2005, 18(1): 37-40.
DU D, FANG L C, CHEN B B, et al. Effect of lactobacillus acidophilus combined with ISO-malto-oligsaccharide on the intestinal mucosal secretion of SIgA in rat models with antibiotic-associated diarrhea(AAD)［J］. Journal of Radioimmunology, 2005, 18(1): 37-40. (in Chinese)
［23］朱洪艳，赵红宇，周盛华，等.牛初乳中sIgA双抗夹心ELISA检测方法的建立［J］. 中国乳品工业, 2011, 39(3): 50-52.
ZHU H Y, ZHAO H Y, ZHOU S H, et al. Establishment of a sandwich ELISA for quantitive measurement of sIgA［J］. China Dairy Industry, 2011, 39(3): 50-52. (in Chinese)
［24］TAKAHASHI I. Mucosal immune system: the second way of the host defense［J］. Nihon Rinsho, 2007, 65: 102-108.
［25］姜燕，吕昌龙. 口服疫苗与肠粘膜免疫效应机制［J］. 国际免疫学杂志, 2006, 29(5): 273-276.
JIANG Y, LV C L. Effects and mechanism of oral vaccine on intestinal mucosal immunity［J］. International Journal of Immunology, 2006, 29(5): 273-276. (in Chinese)
［26］任燕，张小江，陶家发，等. 壳聚糖-海藻酸盐复合微囊疫苗的制备及其对斜带石斑鱼的口服免疫效果［J］. 中国生物制品学杂志， 2015, 28(2): 110-114.
REN Y, ZHANG X J, TAO J F, et al. Preparation and immune effect of chitosan-alginate microcapsules containing Vibrio harveyi antigen Ompk in orange-spotted groupers［J］. Chinese Journal of Biologicals, 2015, 28(2): 110-114. (in Chinese)
［27］FLICK-SMITH H C, EYLES J E, HEBDON R, et al. Mucosal or parenteral administration of microsphere-associated Bacillus anthracis protective antigen protects against anthrax infection in mice［J］. Infect Immun, 2002, 70(4): 2022-2028.
［28］REDFIELD R R, INNIS B L, SCOTT R M, et al. Clinical evaluation of low-dose intradermally administered hepatitis B virus vaccine. A cost reduction strategy［J］. JAMA, 1985, 254(22): 3203-3206.

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