重组鸡白细胞介素-6/2融合蛋白对新城疫(LaSota株)活疫苗免疫增强作用研究
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摘要
为探究重组鸡白细胞介素-6/2融合蛋白(rChIL-6-Linker-ChIL-2,重组融合蛋白)对新城疫病毒(NDV)活疫苗(LaSota株)的免疫增强作用,本研究将90只SPF鸡随机分为6组,分别为PBS对照组、NDV弱毒苗对照组、rChIL-6-Linker-ChIL-2免疫增强组、rChIL-6蛋白免疫增强组、rChIL-2蛋白免疫增强组及rChIL-6+rChIL-2混合蛋白免疫增强组,将鸡白细胞介素重组融合蛋白水剂与LaSota株联合接种于SPF鸡,分别采用MTS法、流式细胞术、ELISA和HA/HI法检测接种前后不同时间各组鸡外周血及脾淋巴细胞增殖活性、鸡外周血中CD3+CD4+/CD3+CD8+T淋巴细胞的百分含量、血清中Th1/Th2型细胞因子表达水平及免疫抗体水平等指标。结果显示,接种后7～21d时,与NDV弱毒苗对照组相比,同时接种重组融合蛋白组鸡淋巴细胞增殖活性、外周血CD3+CD4+/CD3+CD8+T淋巴细胞的百分含量比值及血清中重组鸡IL-4蛋白(rChIL-4)、ChIL-6、ChIL-2、重组鸡IFN-α蛋白(ChIFN-α)、ChIFN-γTh1/Th2型细胞因子表达水平明显提升;HI免疫抗体较NDV弱毒苗对照组提高了1.0～1.9个滴度,较单一rChIL-6、rChIL-2对照组分别提高了0.2～0.7、0.2～1.1个抗体滴度。综上所述,rChIL-6-Lin-ker-ChIL-2融合蛋白对NDV(LaSota株)活疫苗在鸡体内具有明显的免疫增强效果。
In order to study the immunopotentiation effect of recombinant chicken interleukin-6/2 fusion protein(rChIL-6-Linker-ChIL-2)on Newcastle disease vinus(NDV)live vaccine(LaSota vaccine),90 SPF chickens were chosen and randomly distributed into six groups which were the PBS control group,NDV live vaccine control group,rChIL-6-Linker-ChIL-2 immunopotentiation group,rChIL-6 protein immunopotentiation group,rChIL-2 protein immunopotentiation group andthe rChIL-6+rChIL-2 fusion protein immunopotentiation group.The interleukin fusion protein fluid and NDV live vaccine(LaSota vaccine)were inoculated into SPF chickens.The indexes of proliferation activities of peripheral blood and splenic lymphocytes,percentages of the CD3+CD4+/CD3+CD8+T-lymphocytes in peripheral blood,the contents of Th1/Th2-type cytokines and the specific immune antibody level were detected by assays of MTS,FCM,ELISA and HA/HI.The results showed that compared with the indexes of NDV vaccine inoculation control group,the lymphocytes proliferation activity,the percentage of CD3+CD4+/CD3+CD8+T-lymphocytes,and the contents of Th1/Th2-type cytokines such as ChIL-4,ChIL-6,ChIL-2,ChIFN-αand ChIFN-γof chickens in rChIL-6-Linker-ChIL-2 inoculation group were promoted obviously at7 to 21 dpi,and the HI immune antibodies increased about 1.0 to 1.9 titer,which was 0.2 to 0.7 titer higher than that of rChIL-6 group and 0.2 to 1.1 titer higher than rChIL-2 that of group.In conclusion,the rChIL-6-Linker-ChIL-2 protein had an obvious immunopotentiation effect on NDV live vaccine(LaSota)in chickens.
In order to study the immunopotentiation effect of recombinant chicken interleukin-6/2 fusion protein(rChIL-6-Linker-ChIL-2)on Newcastle disease vinus(NDV)live vaccine(LaSota vaccine),90 SPF chickens were chosen and randomly distributed into six groups which were the PBS control group,NDV live vaccine control group,rChIL-6-Linker-ChIL-2 immunopotentiation group,rChIL-6 protein immunopotentiation group,rChIL-2 protein immunopotentiation group andthe rChIL-6+rChIL-2 fusion protein immunopotentiation group.The interleukin fusion protein fluid and NDV live vaccine(LaSota vaccine)were inoculated into SPF chickens.The indexes of proliferation activities of peripheral blood and splenic lymphocytes,percentages of the CD3+CD4+/CD3+CD8+T-lymphocytes in peripheral blood,the contents of Th1/Th2-type cytokines and the specific immune antibody level were detected by assays of MTS,FCM,ELISA and HA/HI.The results showed that compared with the indexes of NDV vaccine inoculation control group,the lymphocytes proliferation activity,the percentage of CD3+CD4+/CD3+CD8+T-lymphocytes,and the contents of Th1/Th2-type cytokines such as ChIL-4,ChIL-6,ChIL-2,ChIFN-αand ChIFN-γof chickens in rChIL-6-Linker-ChIL-2 inoculation group were promoted obviously at7 to 21 dpi,and the HI immune antibodies increased about 1.0 to 1.9 titer,which was 0.2 to 0.7 titer higher than that of rChIL-6 group and 0.2 to 1.1 titer higher than rChIL-2 that of group.In conclusion,the rChIL-6-Linker-ChIL-2 protein had an obvious immunopotentiation effect on NDV live vaccine(LaSota)in chickens.
引文
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[13]程俊贞,闫若潜,吴志明,等.重组鸡干扰素/白细胞介素-2融合蛋白体内抗传染性法氏囊病病毒作用研究[J].中国预防兽医学报,2014,36(2):140-145.CHENG J Z,YAN R Q,WU Z M,et al.The anti-infection bursal disease virus effect of recombinant chicken infection alph/chicken interleukin-2 fusion proten in SPF chickens[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(2):140-145.(in Chinese)
[14]BIRKHOLZ A M,KRONENBERG M.Antigen specificity of invariant natural killer T-cells[J].Journal of Biomedical Science,2015,38(6):470-483.
[15]BORJIGIN J,LI X,SNYDER S H.The pineal gland and melatonin:Molecular and pharmacologic regulation[J].Annual Review of Pharmacology and Toxicology,1999,39(1):53-65.
[16]SAMSOM J N,VOERMANS J J M,DE BRIN M G M,et al.Direct cytolytic activity of PBMC from pigs infected with porcine reproductive and respiratory syndrome[J].Veterinary Research,2000,31:44.
[17]WU G,WU N,LI T,et al.Total glucosides of peony ameliorates Sj9gren’s syndrome by affecting Th1/Th2cytokine balance[J].Experimental and Therapeutic Medicine,2016,11(3):1135-1141.
[18]陈洁,曹筱筱,徐蓉,等.银屑病血热及血瘀证患者外周血Th1/Th2细胞表达差异研究[J].中国中西医结合杂志,2014,34(1):46-50.CHEN J,CAO X X,XU R,et al.Research on different expressions of peripheral blood Th1/Th2cells in psoriasis patients of blood heat syndrome and of blood stasis syndrome[J].Chinese Journal of Integrated Traditional and Western Medicine,2014,34(1):46-50.(in Chinese)
[19]刘健,万磊,盛长健,等.佐剂性关节炎大鼠肺功能变化与Th1/Th2细胞、调节性T细胞的相关性研究[J].细胞与分子免疫学杂志,2011,27(1):56-60.LIU J,WAN L,SHENG C J,et al.The corrslative study on pulmonary function changes and Th1/Th2cells and regulatory T cells in adjuvant artharitis rats[J].Chinese Journal of Cellular and Molecular Immunology,2011,27(1):56-60.(in Chinese)
[20]姜永厚,陈奖励,宋秀龙,等.鸡新城疫病毒F基因和鸡IL-2重组DNA疫苗的构建[J].中国预防兽医学报,2001,23(2):2-5.JIANG Y H,CHEN J L,SONG X L,et al.Construction of the recombinant plasmid coexpressing NDV Fgene and chicken IL-2[J].Chinese Journal of Preventive Veterinary Medicine,2001,23(2):2-5.(in Chinese)
[21]徐向明,朱善元,张泉,等.鸡白细胞介素2对LaSota弱毒苗免疫效果促进作用的初步研究[J].中国家禽,2008,30(13):21-23.XU X M,ZHU S Y,ZHANG Q,et al.Preliminary study of immunization effect of LaSota vaccine facilitated by chicken interleukin-2[J].China Poultry,2008,30(13):21-23.(in Chinese)
[22]牛泽,任晓峰,曾岩,等.重组鸡白细胞介素-2对禽流感灭活疫苗免疫增强作用的研究[J].中国家禽,2010,32(3):20-23.NIU Z,REN X F,ZENG Y,et al.Immunopotentiation of recombinant chicken interleukin-2on avian influenza inactivated vaccine[J].China Poultry,2010,32(3):20-23.(in Chinese)
[2]DIEHL S,ANGUITA J,HOFFMEYER A,et al.Inhibition of Th1differentiation by IL-6is mediated by SOCS1[J].Immunity,2000,13(6):805-815.
[3]LIAO W,LIN J X,LEONARD W J.Interleukin-2at the crossroads of effector responses,tolerance,and immunotherapy[J].Immunity,2013,38(1):13-25.
[4]HILTON L S,BEAN A G,LOWENTHAL J W.The emerging role of avian cytokines as immunotherapeutics and vaccine adjuvants[J].Veterinary Immunology and Immunopathology,2002,85(3-4):119-128.
[5]张书阳,谢彩华,闫若潜,等.鸡白细胞介素-6-2嵌合基因的融合表达及其表达产物活性的研究[J].中国兽医科学,2014,41(9):974-980.ZHANG S Y,XIE C H,YAN R Q,et al.Expression of chicken interleukin-6-2fusion gene in vitro and study on bioactivities of the expressed fusion protein[J].China Animal Husbandry&Veterinary Medicine,2014,41(9):974-980.(in Chinese)
[6]李勤楠,闫若潜,吴志明,等.重组鸡白细胞介素-6-2融合蛋白对鸡T淋巴细胞和B淋巴细胞免疫活性的影响[J].中国兽医科学,2015,45(12):1283-1288.LI Q N,YAN R Q,WU Z M,et al.Effect of recombinant chicken interleukin-6-2fusion protein on the immunological activity of T and B lymphocytes in chicken[J].Chinese Veterinary Science,2015,45(12):1283-1288.(in Chinese)
[7]闫若潜,谢彩华,吴志明,等.重组鸡白细胞介素6对不同种类疫苗的免疫增强作用[J].中国兽医科学,2010,40(5):494-500.YAN R Q,XIE C H,WU Z M,et al.Immunopotentiation effect of the recombinant chicken interleukin-6on different kinds of vaccines[J].Chinese Veterinary Science,2010,40(5):494-500.(in Chinese)
[8]杨晓璐,闫若潜,李勤楠,等.重组鸡α干扰素/白细胞介素-2融合蛋白体外活性评价方法研究[J].中国畜牧兽医,2015,42(12):3153-3159.YANG X L,YAN R Q,LI Q N,et al.Study on the activity estimation methods of recombinant chicken interferon-α/interleukin-2fusion protein in vitro[J].China Animal Husbandry&Veterinary Medicine,2015,42(12):3153-3159.(in Chinese)
[9]闫志玲,闫若潜,吴志明,等.重组鸡白细胞介素-6(rChIL-6)对传染性法氏囊灭活疫苗的免疫增强机理研究[J].中国兽医学报,2014,34(5):704-711.YAN Z L,YAN R Q,WU Z M,et al.The immuneenhancing mechanism of recombinant chicken interleukin-6(rChIL-6)protein for the infectious bursal virus inactivated vaccine[J].Chinese Journal of Veterinary Science,2014,34(5):704-711.(in Chinese)
[10]杨晓璐,闫若潜,吴志明,等.重组鸡α干扰素/白细胞介素-2融合蛋白对鸡外周血T淋巴细胞亚群的影响研究[J].中国畜牧兽医,2014,41(11):175-178.YANG X L,YAN R Q,WU Z M,et al.The influence for chickens’s lymphocyte subsets in peripheral blood after inoculated with the recombinant chicken interferon-α/chicken interleukin-2 complex protein[J].China Animal Husbandry&Veterinary Medicine,2014,41(11):175-178.(in Chinese)
[11]KIDD P.Th1/Th2balance:The hypothesis,its limitations,and implications for health and disease[J].Alternative Medicine Review,2003,8(3):223-246.
[12]谢彩华,闫若潜,崔保安,等.鸡白细胞介素-6的原核表达及其对疫苗免疫增强效果的研究[J].中国预防兽医学报,2008,30(7):557-561.XIE C H,YAN R Q,CUI B A,et al.Enhancment of immune respones to vaccines by recombinant chicken interleukin-6(ChIL-6)[J].Chinese Journal of Preventive Veterinary Medicine,2008,30(7):557-561.(in Chinese)
[13]程俊贞,闫若潜,吴志明,等.重组鸡干扰素/白细胞介素-2融合蛋白体内抗传染性法氏囊病病毒作用研究[J].中国预防兽医学报,2014,36(2):140-145.CHENG J Z,YAN R Q,WU Z M,et al.The anti-infection bursal disease virus effect of recombinant chicken infection alph/chicken interleukin-2 fusion proten in SPF chickens[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(2):140-145.(in Chinese)
[14]BIRKHOLZ A M,KRONENBERG M.Antigen specificity of invariant natural killer T-cells[J].Journal of Biomedical Science,2015,38(6):470-483.
[15]BORJIGIN J,LI X,SNYDER S H.The pineal gland and melatonin:Molecular and pharmacologic regulation[J].Annual Review of Pharmacology and Toxicology,1999,39(1):53-65.
[16]SAMSOM J N,VOERMANS J J M,DE BRIN M G M,et al.Direct cytolytic activity of PBMC from pigs infected with porcine reproductive and respiratory syndrome[J].Veterinary Research,2000,31:44.
[17]WU G,WU N,LI T,et al.Total glucosides of peony ameliorates Sj9gren’s syndrome by affecting Th1/Th2cytokine balance[J].Experimental and Therapeutic Medicine,2016,11(3):1135-1141.
[18]陈洁,曹筱筱,徐蓉,等.银屑病血热及血瘀证患者外周血Th1/Th2细胞表达差异研究[J].中国中西医结合杂志,2014,34(1):46-50.CHEN J,CAO X X,XU R,et al.Research on different expressions of peripheral blood Th1/Th2cells in psoriasis patients of blood heat syndrome and of blood stasis syndrome[J].Chinese Journal of Integrated Traditional and Western Medicine,2014,34(1):46-50.(in Chinese)
[19]刘健,万磊,盛长健,等.佐剂性关节炎大鼠肺功能变化与Th1/Th2细胞、调节性T细胞的相关性研究[J].细胞与分子免疫学杂志,2011,27(1):56-60.LIU J,WAN L,SHENG C J,et al.The corrslative study on pulmonary function changes and Th1/Th2cells and regulatory T cells in adjuvant artharitis rats[J].Chinese Journal of Cellular and Molecular Immunology,2011,27(1):56-60.(in Chinese)
[20]姜永厚,陈奖励,宋秀龙,等.鸡新城疫病毒F基因和鸡IL-2重组DNA疫苗的构建[J].中国预防兽医学报,2001,23(2):2-5.JIANG Y H,CHEN J L,SONG X L,et al.Construction of the recombinant plasmid coexpressing NDV Fgene and chicken IL-2[J].Chinese Journal of Preventive Veterinary Medicine,2001,23(2):2-5.(in Chinese)
[21]徐向明,朱善元,张泉,等.鸡白细胞介素2对LaSota弱毒苗免疫效果促进作用的初步研究[J].中国家禽,2008,30(13):21-23.XU X M,ZHU S Y,ZHANG Q,et al.Preliminary study of immunization effect of LaSota vaccine facilitated by chicken interleukin-2[J].China Poultry,2008,30(13):21-23.(in Chinese)
[22]牛泽,任晓峰,曾岩,等.重组鸡白细胞介素-2对禽流感灭活疫苗免疫增强作用的研究[J].中国家禽,2010,32(3):20-23.NIU Z,REN X F,ZENG Y,et al.Immunopotentiation of recombinant chicken interleukin-2on avian influenza inactivated vaccine[J].China Poultry,2010,32(3):20-23.(in Chinese)