分子佐剂chFc对巨型艾美耳球虫DNA疫苗在鸡上的免疫效果的影响
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摘要
将鸡免疫球蛋白Ig G的Fc片段(chFc基因)和Em IMP1基因连接,构建真核表达载体pVAX1-Em IMP1-chFc和p VAX1-Em IMP1,对chFc作为Em IMP1的分子佐剂的功能进行研究。结果显示:在ELISA检测中,chFc佐剂组效价的D450值最高,其抗体效价为3 200;chFc佐剂组的第3次免疫血清中IL-4质量浓度显著提高(P<0.05),chFc佐剂组的IFN-γ质量浓度最高;流式细胞仪检测结果显示,chFc组的CD4+和CD8+T淋巴细胞的比例都是最高的;chFc佐剂组卵囊排出量显著低于其他实验组;chFc佐剂组对肠道的保护效果显著优于其他实验组(P<0.05);chFc佐剂组增重显著高于其他实验组(P<0.05)。结果表明,chFc佐剂组有明显的优势,Em IMP1组次之,说明chFc能增强Em IMP1的免疫原性。
To research the biological and immunological functions of chicken Fc fragment(ch Fc)gene of immunoglobulin G(Ig G) as an molecular adjuvant of Em IMP1 gene,Em IMP1-ch Fc and Em IMP1 genes were amplified from the templates saved in our laboratory,then the target fragments were linked to the pVAX1 eukaryotic expression vector to yield to the expression plasmids pVAX1-Em IMP1-ch Fc and pVAX1-Em IMP1.And the plasmids were extracted from the Escherichia coli competent cells.The plasmids were intramuscularly injected into the chickens,and the immunological experiment was conducted to evaluate the immune effect of the vaccine.The results showed that,in the ELISA,the ch Fc adjuvant group had the highest D450 value,and the antibody titer was 3 200;the mass concentration of IL-4 in the third immune serum of ch Fc adjuvant group was significantly increased(P<0.05),and the mass concentration of IFNgamma in ch Fc adjuvant group was the highest;according to the proportion of CD4+and CD8+T lymphocytes detected by flow cytometry,the ratio of CD4+and CD8+T lymphocytes in ch Fc group was the highest; the oocyst outputs were significantly decreased in the adjuvant ch Fc group; the intestine lession score was lower in the adjuvant ch Fc group than that in other groups;and the weight gain of ch Fc adjuvant group was significantly higher than that in the experimental group(P<0.05).Therefore,the ch Fc adjuvant group had obvious advantages,and the Em IMP1 group was inferior to the ch Fc adjuvant group from the test results.The results indicated that the ch Fc adjuvant can enhance the immunogenicity of Em IMP1 based vaccine.
To research the biological and immunological functions of chicken Fc fragment(ch Fc)gene of immunoglobulin G(Ig G) as an molecular adjuvant of Em IMP1 gene,Em IMP1-ch Fc and Em IMP1 genes were amplified from the templates saved in our laboratory,then the target fragments were linked to the pVAX1 eukaryotic expression vector to yield to the expression plasmids pVAX1-Em IMP1-ch Fc and pVAX1-Em IMP1.And the plasmids were extracted from the Escherichia coli competent cells.The plasmids were intramuscularly injected into the chickens,and the immunological experiment was conducted to evaluate the immune effect of the vaccine.The results showed that,in the ELISA,the ch Fc adjuvant group had the highest D450 value,and the antibody titer was 3 200;the mass concentration of IL-4 in the third immune serum of ch Fc adjuvant group was significantly increased(P<0.05),and the mass concentration of IFNgamma in ch Fc adjuvant group was the highest;according to the proportion of CD4+and CD8+T lymphocytes detected by flow cytometry,the ratio of CD4+and CD8+T lymphocytes in ch Fc group was the highest; the oocyst outputs were significantly decreased in the adjuvant ch Fc group; the intestine lession score was lower in the adjuvant ch Fc group than that in other groups;and the weight gain of ch Fc adjuvant group was significantly higher than that in the experimental group(P<0.05).Therefore,the ch Fc adjuvant group had obvious advantages,and the Em IMP1 group was inferior to the ch Fc adjuvant group from the test results.The results indicated that the ch Fc adjuvant can enhance the immunogenicity of Em IMP1 based vaccine.
引文
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[23]刘玲红,赵翠,郭浩,等.融合表达禽白血病env基因和IgG-Fc基因对鸡免疫效果研究[J].中国预防兽医学报,2017,39(8):654-657.LIU Ling-hong,ZHAO Cui,GUO Hao,et al.Research on the immune effect of the fusion expression of env and IgG-Fc genes on avian leukosis virus[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(8):654-657.(in Chinese)
[24]张玉杨,张改平,乔松林,等.猪IgGⅡ类Fc受体基因的真核表达及其在组织中的分布状况[J].中国兽医科学,2008,38(9):791-795.ZHANG Yu-yang,ZHANG Gai-ping,QIAO Song-lin,et al.Eukaryotic expression of swine immunoglobulin G Fc receptorⅡgene and its distribution of IgGFc receptorⅡin porcine tissues[J].Chinese Veterinary Science,2008,38(9):791-795.(in Chinese)
[25]OPERSCHALL E,SCHUH T,HEINZERLING L,et al.Enhanced protection against viral infection by coadministration of plasmid DNA coding for viral antigen and cytokines in mice[J].Journal of Clinical Virology,1999,13(1-2):17-27.
[2]SHIRLEY M W,LILLEHOJ H S.The long view:a selective review of 40 years of coccidiosis research[J].Avian Pathology,2012,41(2):111-121.
[3]BLAKE D P,TOMLEY F M.Securing poultry production from the ever-present Eimeria challenge[J].Trends in Parasitology,2014,30(1):12-19.
[4]孔繁瑶.家畜寄生虫学[M].北京:中国农业大学出版社,1997:331-332.KONG Fan-yao.Livestock Parasitology[M].Beijing:China Agricultural University Press,1997:331-332.(in Chinese)
[5]赵家龙,桂德芳.巨型艾美耳球虫研究之回顾[J].畜牧兽医科技信息,2013(5):14-16.ZHAO Jia-long,GUI De-fang.A review of the E.maxima study[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2013(5):14-16.(in Chinese)
[6]刘群,杨勇,李博,等.鸡球虫病早熟弱毒苗实验室应用研究[J].中国兽医杂志,2003,39(2):11-13.LIU Qun,YANG Yong,LI Bo,et al.Study on the application of chicken coccidiosis vaccine in the laboratory[J].Chinese Journal of Veterinary Medicine,2003,39(2):11-13.(in Chinese)
[7]索勋,李国清.鸡球虫病学[M].北京:中国农业大学出版社,1998.SUO Xun,LI Guo-qing.Chicken Coccidiosis[M].Beijing:China Agricultural University Press,1998.(in Chinese)
[8]郝文波,罗树红.核酸疫苗---寄生虫疫苗的新希望[J].国外医学(寄生虫病分册),1996(5):197-201.HAO Wen-bo,LUO Shu-hong.Nucleic acid vaccineNew hope for parasite vaccine[J].Parasitic Diseases Foreign Medical Sciences,1996(5):197-201.(in Chinese)
[9]孙树汉.核酸疫苗[M].上海:第二军医大学出版社,2000.SUN Shu-han.Nucleic Acid Vaccine[M].Shanghai:Second Military Medical University Press,2000.(in Chinese)
[10]WATINE G J,MCMANUS D P.Nucleic acids vaccines of the future[J].Parasitology Today,1995,11:113.
[11]MCCLUSKIE M J,BRAZOLOT MILLAN C L,GRAMZINSKI R A,et al.Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates[J].Molecular Medicine,1999,5(5):287-300.
[12]李秋明,刘贤勇.浅析鸡球虫病疫苗[J].养禽与禽病防治,2012(12):2-4.LI Qiu-ming,LIU Xian-yong.Analysis of chicken coccidiosis vaccine[J].Poultry Husbandry and Disease Control,2012(12):2-4.(in Chinese)
[13]李学钊,宋鸿雁,刘根新,等.鸡球虫DNA疫苗研究进展[J].畜牧与兽医.2011,43(8):95-99.LI Xue-zhao,SONG Hong-yan,LIU Gen-xin,et al.Progress in research on DNA vaccine of chicken coccidia[J].Animal Husbandry&Veterinary Medicine,2011,43(8):95-99.(in Chinese)
[14]BLAKE D P,BILLINGTON K J,COPESTAKE S L,et al.Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite[J].PLoS Pathogens,2011,7(2):e1001279.
[15]DICKINSON B L,BADIZADEGAN K,WU Z,et al.Bidirectional Fc Rn-dependent Ig G transport in a polarized human intestinal epithelial cell line[J].The Journal of Clinical Investigation,1999,104(7):903-911.
[16]ROOPENIAN D C,AKILESH S.FcRn:the neonatal Fc receptor comes of age[J].Nature Reviews Immunology,2007,7(9):715-725.
[17]YOSHIDA M,KOBAYASHI K,KUO T T,et al.Neonatal Fc receptor for IgG regulates mucosal immune responses to luminal bacteria[J].The Journal of Clinical Investigation,2006,116(8):2142-2151.
[18]YE Li-lin,ZENG Rong-yu,BAI Yu,et al.Efficient mucosal vaccination mediated by the neonatal Fc receptor[P].Nature Biotechnology,2011,29(2):158-163.
[19]LINDEN C D,ROTH T F.IgG receptors on foetal chick yolk sac[J].Journal of Cell Science,1978,33:317-318.
[20]TRESSLER R L,ROTH T F.IgG receptors on the embryonic chick yolk sac[J].The Journal of Biological Chemistry,1987,262(32):15406-15412.
[21]SCH DEL F,AGUADO M T,LAMBERT P H.Nucleic Acid Vaccines,WHO,Geneva,17-18 May,1994[J].Vaccine,1994,12(16):1491-1492.
[22]柏家林,白喜龙,张从党,等.金黄色葡萄球菌Fc段受体结合蛋白A基因的克隆与原核表达[J].中国兽医科学,2012,42(5):505-510.BAI Jia-lin,BAI Xi-long,ZHANG Cong-dang,et al.Cloning and prokaryotic expression of Fc-binding protein A gene of Staphylococcus aureus[J].Chinese Veterinary Science,2012,42(5):505-510.(in Chinese)
[23]刘玲红,赵翠,郭浩,等.融合表达禽白血病env基因和IgG-Fc基因对鸡免疫效果研究[J].中国预防兽医学报,2017,39(8):654-657.LIU Ling-hong,ZHAO Cui,GUO Hao,et al.Research on the immune effect of the fusion expression of env and IgG-Fc genes on avian leukosis virus[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(8):654-657.(in Chinese)
[24]张玉杨,张改平,乔松林,等.猪IgGⅡ类Fc受体基因的真核表达及其在组织中的分布状况[J].中国兽医科学,2008,38(9):791-795.ZHANG Yu-yang,ZHANG Gai-ping,QIAO Song-lin,et al.Eukaryotic expression of swine immunoglobulin G Fc receptorⅡgene and its distribution of IgGFc receptorⅡin porcine tissues[J].Chinese Veterinary Science,2008,38(9):791-795.(in Chinese)
[25]OPERSCHALL E,SCHUH T,HEINZERLING L,et al.Enhanced protection against viral infection by coadministration of plasmid DNA coding for viral antigen and cytokines in mice[J].Journal of Clinical Virology,1999,13(1-2):17-27.