毕赤酵母提高产肠毒素大肠杆菌灭活疫苗免疫保护效果的研究  

A study on improving the immune protection effects of ETEC inactivated vaccine by Pichia pastoris

作　　者：李莹慧 张迪[1] 张兆天 刘迎迎 朱悦 贾垌 卢海强[2] 李妍[1] LI Ying-hui;ZHANG Di;ZHANG Zhao-tian;LIU Ying-ying;ZHU Yue;JIA Tong;LU Hai-qiang;LI Yan(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Collcge of Food Science and Technology,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北农业大学食品科技学院,河北保定071001

出　　处：《中国预防兽医学报》2022年第12期1324-1331,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：河北省自然科学基金(C2020204072);中国博士后科学基金(2019M661954)。

摘　　要：为探究毕赤酵母提高产肠毒素大肠杆菌(ETEC)灭活疫苗免疫保护效果的作用,本研究将48只BALB/c小鼠随机均分为4组,分别为毕赤酵母活菌+疫苗组(仔猪大肠埃希氏菌K88、K99、987P三价灭活疫苗)、毕赤酵母灭活菌+疫苗组、疫苗组及生理盐水(对照组)。免疫后15 d各组随机取6只小鼠采血后剖杀采集其十二指肠样品及肠液,分别采用ELISA检测各组小鼠血清K88ac(菌毛)IgA和K88ac IgG特异性抗体水平与十二指肠液中K88ac分泌型IgA(SIgA)抗体水平;采用q PCR检测十二指肠组织紧密连接蛋白Occludin、ZO-1及黏蛋白MUC2基因的转录水平;制备小鼠十二指肠组织切片,观察其肠道结构完整性,采用Image J 1.41软件定量分析小鼠肠绒毛高度与隐窝深度;对各组小鼠脾脏称重,计算脾脏指数。采用腹腔注射ETEC K88ac菌株(3×10^(7)cfu/只)方式对每组剩余小鼠攻毒,连续5 d观察各组小鼠的临床症状,统计小鼠的腹泻率和死亡率并称量其体质量。通过以上检测指标综合分析毕赤酵母对提高灭活疫苗保护效果的作用。抗体检测结果显示,与疫苗组及对照组相比,添加毕赤酵母活菌与灭活菌均可极显著(P<0.01)提高小鼠血清K88ac IgA、K88ac IgG及十二指肠液中K88ac SIgA特异性抗体水平,且活菌效果极显著(P<0.01)或者显著(P<0.05)优于灭活菌及其他组。qPCR结果显示,与疫苗组相比,添加毕赤酵母活菌或灭活菌均可显著提高小鼠十二指肠紧密连接蛋白基因的转录水平(P<0.05),极显著提高小鼠十二指肠黏蛋白的转录水平(P<0.01)。组织切片观察可见,活菌+疫苗组小鼠肠道肠壁绒毛排列相对整齐,绒毛结构基本完整,隐窝清晰,优于灭活菌+疫苗组及其他组。软件分析结果显示,与对照组相比,毕赤酵母菌+疫苗组小鼠十二指肠绒毛高度极显著(活菌,P<0.01)或者显著(灭活菌,P<0.05)升高,且活菌效果极显著优于灭活菌(P<0.01);活菌+疫苗组小鼠十二指�To explore the effects of Pichia pastoris as an immune adjuvant on improving the immune protection of enterotoxigenic Escherichia coli(ETEC) inactivated vaccines. In this study, 48 BALB/c mice were randomly divided into 4 groups and intramuscularly injected with live yeast + vaccine(Piglet E. coli K88, K99, 987P trivalent inactivated vaccine), inactivated yeast +vaccine, vaccine and normal saline. Fifteen days post immunization, 6 mice of each group were randomly selected for collecting serum and then necropsied to collect their duodenal samples and intestinal fluid. ELISA was used to detect the serum K88ac(fimbriae) IgA and K88ac IgG specific antibody levels and K88ac IgA(SIgA) antibody levels in duodenal fluid of mice in each group. qPCR was used to detect the transcription levels of tight junction proteins Occludin, ZO-1 and MUC2 genes in duodenal tissue;duodenal tissue sections of mice were prepared to observe the structural integrity of the intestine, and Image J 1.41 software was used to quantitatively analyze the villi height and crypt depth of mice;the spleen weights of mice in each group were used to calculate the spleen index. The remaining mice in each group were challenged with 3×10^(7)cfu of ETEC K88ac strain by intraperitoneal injection, and the clinical symptoms of mice in each group were observed for 5 days, and the diarrhea rate and mortality of mice were counted and their body weights were weighed. The effects of Pichia pastoris on improving the protective effects of inactivated vaccine was comprehensively analyzed by the above detection indicators. The results of specific antibody levels showed that compared with the vaccine group and the control group, the addition of live yeast and inactivated yeast significantly(P<0.01) increased the levels of K88ac IgA, K88ac IgG in serum and K88ac SIgA specific antibody in duodenal fluid of mice, and the live yeast effects were extremely significant(P<0.01) or significantly(P<0.05) superior to inactivated yeast and other groups. q PCR results showed that

关 键 词：产肠毒素大肠杆菌 毕赤酵母 特异性抗体 紧密连接蛋白 

分 类 号：S855.12[农业科学—临床兽医学]