表达胸腺肽和干扰素融合蛋白的重组乳酸乳球菌作为鸡新城疫病毒活疫苗免疫佐剂的初步研究  

Preliminary study on recombinant Lactococcus lactis expressing thymosin and interferon fusion protein as an immune adjuvant for chicken Newcastle disease virus live vaccine

作　　者：胡洪娇 王贺 刘增琪 崔子扬 胡馨匀 崔红玉[4] 王云峰 何高明[1] HU Hong-jiao;WANG He;LIU Zeng-qi;CUI Zi-yang;HU Xin-yun;CUI Hong-yu;WANG Yun-feng;HE Gao-ming(College of Animal Science and Technology,Shihezi University,Shihezi 832002,China;Harbin Guosheng Biotechnology Co.,Ltd,Harbin 150028,China;Clinical College of Medicine,Northern University of Hebei,Zhangjiakou 075000,China;Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832002 [2]哈尔滨国生生物科技股份有限公司,黑龙江哈尔滨150028 [3]河北北方大学临床医学院,河北张家口075000 [4]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2024年第4期392-400,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2023YFD180260、2022YFD1800802)。

摘　　要：为评估以滴鼻方式免疫表达鸡胸腺肽和鸡干扰素融合蛋白的重组乳酸乳球菌能否作为新城疫病毒(NDV)活疫苗的免疫佐剂及对其免疫效力的影响,本研究以表达鸡胸腺肽和鸡γ干扰素融合蛋白(cTα1-cIFN-γ,后简写为Tα1-IFN)的重组乳酸乳球菌(Tα1-IFN/r-L.lactis)与NDV活疫苗混合后(Tα1-IFN/r-L.lactis+NDV活疫苗)滴鼻免疫14日龄SPF鸡,同时设NDV活疫苗组、L.lactis+NDV活疫苗组及PBS阴性对照组。分别于免疫后不同时间(3 d、7 d、14 d、21 d、28 d)采血,于免疫后14 d、21 d、28 d分别检测各组鸡血清中的HI抗体效价;采用间接ELISA测定免疫后3 d、7 d及14 d各组鸡血清中主要细胞因子的分泌水平;分离各组鸡免疫后21 d及28 d外周血单个核细胞(PBMC)和外周血白细胞,分别通过CCK-8法检测各组鸡PBMC和白细胞中抗原递呈细胞(APC)的增殖活性。HI抗体检结果显示,分别在免疫后14 d、21 d,Tα1-IFN/r-L.lactis+NDV活疫苗组鸡的NDV HI抗体效价与NDV活疫苗组以及L.lactis+NDV活疫苗组均无显著差异;但免疫后28 d,该组鸡血清中的NDV HI抗体效价极显著高于其余两组(P<0.0001)。细胞因子检测结果显示,在免疫后各时间点Tα1-IFN/r-L.lactis+NDV活疫苗组鸡血清中IFN-γ及IL-2(Th1型细胞因子)和IL-4(Th2型细胞因子)的分泌水平基本均显著或者均极显著高于NDV活疫苗组、L.lactis+NDV活疫苗组及阴性对照组(14 d的IL4除外)。Tα1-IFN/r-L.lactis+NDV活疫苗组鸡血清中IL-1β(促炎因子)和IL-10(抗炎因子)的分泌水平在免疫后3 d、7 d、14 d均极显著高于其余各组(P<0.0001)。细胞增殖活性的检测结果显示,免疫后21 d和28 d,Tα1-IFN/r-L.lactis+NDV活疫苗组鸡PBMC的增殖活性均极显著高于其余各组;免疫后28 d,该组鸡PBMC的增殖活性均极显著高于L.lactis+NDV活疫苗组和阴性对照组(P<0.0001),但与NDV活疫苗组差异不显著(P>0.05);免疫后21 d和28 d,Tα1-IFN/r-L.lactis+NDV活疫苗组鸡外周血白细胞�To evaluate the potential of recombinant Lactococcus lactis expressing chicken thymosin and chicken interferon fusion protein as an immune adjuvant for Newcastle disease virus(NDV)live vaccine and its effect on immune efficacy,14-day-old SPF chickens were intranasally immunized with recombinant Lactococcus lactis expressing chicken thymosin and chicken gamma interferon fusion protein(cTα1-cIFN-γ,abbreviated as Tα1-IFN)mixed with live NDV vaccine(Tα1-IFN/r-L.lactis+live NDV vaccine),and the live NDV vaccine group,L.lactis+live NDV vaccine group and PBS negative control group were set up.Blood samples were collected at 3,7,14,21 and 28 days post-immunization(dpi),and the HI antibody titers in the serum of chickens in each group were detected at 14,21 and 28 dpi,respectively;the secretion levels of major cytokines in the serum of chickens in each group at 3,7 and 14 dpi were determined by indirect ELISA kits;peripheral blood mononuclear(PBMC)and peripheral blood leukocytes were isolated from chickens in each group at 21 and 28 dpi,and the proliferative activities of T-lymphocytes in PBMC and antigen-presenting cells(APCs)in leukocytes were detected by CCK-8 assay.The results showed that there was no significant difference in NDV HI antibody titers between Tα1-IFN/r-L.lactis+NDV live vaccine group and NDV live vaccine group as well as L.lactis+NDV live vaccine group at 14 and 21 dpi,respectively.However,at 28 dpi,the NDV HI antibody titer in Tα1-IFN/r-L.lactis+NDV live vaccine group was extremely significantly higher than that of the other two groups(P<0.0001).The results of cytokine assay showed that the secretion levels of IFN-γ,IL-2(Th1 cytokine)and IL-4(Th2 cytokine)in chicken sera of the Tα1-IFN/r-L.lactis+NDV group were significantly higher than those of the NDV group,the L.lactis+NDV group and the negative control group at the corresponding timepoints after immunization.The secretion levels of IL-1β(pro-inflammatory factor)and IL-10(anti-inflammatory factor)in chicken sera of the Tα1-IFN/r-L.lactis+N

关 键 词：滴鼻佐剂 胸腺肽 干扰素 重组乳酸乳球菌 免疫增强剂 

分 类 号：S852.42[农业科学—基础兽医学]