分子佐剂eda对巨型艾美耳球虫亚单位疫苗的影响
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摘要
纤维连接蛋白外域A(fibronectin extra domain A,eda)可以作为一种佐剂,而免疫相关蛋白1(IMPⅠ)是巨型艾美耳球虫中的一种免疫保护抗原。本试验将eda基因和EmIMPⅠ基因连接,构建真核表达载体pVAXⅠ-eda-EmIMPⅠ和pVAXⅠ-EmIMPⅠ,对eda作为EmIMPⅠ的分子佐剂的功能进行研究。首先,根据本实验室保存的含eda-EmIMPⅠ与EmIMPⅠ的载体为模板进行PCR扩增目的片段,将目的片段分别连接到真核表达载体pVAXⅠ中,构建质粒pVAXⅠ-eda-EmIMPⅠ和pVAXⅠ-EmIMPⅠ,然后将质粒转入大肠杆菌感受态Trans1-T1中,大量提取质粒。将提取的质粒通过肌肉注射到鸡体内,进行免疫学试验,对疫苗的免疫效果进行评价。结果显示:在ELISA检测中,eda佐剂组,IgG效价的D值最高,其抗体滴度为6 400,血清中IL-10含量明显多于其他组,IFN-γ细胞因子的提升最高。eda佐剂组在卵囊计数方面,减少卵囊排出量仅次于空白组。肠道病变计分中,eda佐剂组计分仅次于空白组,对肠道保护效果最好。从增重来看,eda佐剂组也是攻虫组当中增重最多的一组。所以,从各项检测结果来看,eda佐剂组有明显的优势,EmIMPⅠ组从检测结果上看差于eda佐剂组,说明含eda佐剂能使EmIMPⅠ增强其免疫原性。
Fibronectin extra domain A(eda)can be an adjuvant.Immune mapped protein-1(IMP1)is an immune protective antigen in E.maxima.We linked the eda gene to the EmIMPⅠ gene and constructed the expression vector pVAXⅠ-eda-EmIMPⅠto research the biological and immunological functions of eda as a molecular adjuvant of EmIMPⅠ gene.Firstly,the template containing eda-EmIMPⅠ and EmIMPⅠ saved in laboratory was used for PCR amplification fragment.Link the target fragment to the pVAXⅠ eukaryotic expression vector,structuring the plasmid pVAXⅠ-eda-EmIMPⅠand pVAXⅠ-EmIMPⅠ.And then transferred into Ec.oli competent Trans1-T1 and extracting plasmids.The extracted plasmid was intramuscularly injected into the chicken,and the immunological experiment was conducted to evaluate the immune effect of the vaccine.The result displayed,in ELISA test,the Dvalue of IgG of eda group was the highest,and the antibody titer was 6 400.The levels of IL-10 in the serum of the eda adjuvant group were significantly higher than that in the other groups.The increase of IFN-γcytokines was the highest.The eda adjuvant group was only second to the blank group in the count of coccidiumsoocust.In the score of intestinal diseases,eda adjuvant group was second only to the blank control group,which had the best effect on intestinal protection.In terms of weight gain,eda adjuvant group was also the most heavily weighted group in the attack group.Therefore,from the results of various tests,the eda adjuvant group had obvious advantages,and the EmIMPⅠ group was inferior to the eda adjuvant group from the test results.It is indicated that an eda adjuvant can make EmIMPⅠenhance its immunogenicity.
Fibronectin extra domain A(eda)can be an adjuvant.Immune mapped protein-1(IMP1)is an immune protective antigen in E.maxima.We linked the eda gene to the EmIMPⅠ gene and constructed the expression vector pVAXⅠ-eda-EmIMPⅠto research the biological and immunological functions of eda as a molecular adjuvant of EmIMPⅠ gene.Firstly,the template containing eda-EmIMPⅠ and EmIMPⅠ saved in laboratory was used for PCR amplification fragment.Link the target fragment to the pVAXⅠ eukaryotic expression vector,structuring the plasmid pVAXⅠ-eda-EmIMPⅠand pVAXⅠ-EmIMPⅠ.And then transferred into Ec.oli competent Trans1-T1 and extracting plasmids.The extracted plasmid was intramuscularly injected into the chicken,and the immunological experiment was conducted to evaluate the immune effect of the vaccine.The result displayed,in ELISA test,the Dvalue of IgG of eda group was the highest,and the antibody titer was 6 400.The levels of IL-10 in the serum of the eda adjuvant group were significantly higher than that in the other groups.The increase of IFN-γcytokines was the highest.The eda adjuvant group was only second to the blank group in the count of coccidiumsoocust.In the score of intestinal diseases,eda adjuvant group was second only to the blank control group,which had the best effect on intestinal protection.In terms of weight gain,eda adjuvant group was also the most heavily weighted group in the attack group.Therefore,from the results of various tests,the eda adjuvant group had obvious advantages,and the EmIMPⅠ group was inferior to the eda adjuvant group from the test results.It is indicated that an eda adjuvant can make EmIMPⅠenhance its immunogenicity.
引文
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[16]ARRIBILLAGA L,DURANTEZ M,LOZANO T,et al.A Fusion protein between streptavidin and the endogenous TLR4ligand eda targets biotinylated antigens to dendritic cells and induces T cell responses in vivo[J].Biol Med Res Int,2013,2013(1):1-9.
[17]MANSILLA C,CASALES E,CASARES N,et al.The extra domain a from fibronectin(eda)improves immunogenicity of NS3protein in a semliki forest virus(SFV)-based vaccine against hepatitis C[J].J Hepatol,2008,48(2):S228.
[18]RUDILLA F,FAYOLLE C,CASARES N,et al.Combination of a TLR4ligand and anaphylatoxin C5afor the induction of antigen-specific cytotoxic T cell responses[J].Vaccine,2012,30(18):2848-2858.
[19]CENEVA W H O.Nucleic acid vaccines[J].Vaccine,1994,12(16):1491-1550.
[2]SHIRLEY M W,LILLEHOJ H S.The long view:a selective review of 40years of coccidiosis research[J].Avian Pathol,2012,41(2):111-121.
[3]BLAKE D P,TOMLEY F M.Securing poultry production from the ever-present Eimeria challenge[J].Trends Parasitol,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.
[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.
[6]刘群,杨勇.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.
[7]索勋,李国清.鸡球虫病学[M].北京:中国农业出版社,1998:23-24.
[8]郝文波,罗树红,余新炳.核酸疫苗-寄生虫疫苗的新希望[J].国外医学寄生虫病学分册,1996,24(5):197-199.
[9]WAINE G J,MCMANUS D P.Nucleic acids vaccines of the future[J].Parasitol Today,1995,11(3):116.
[10]MCCLUSKI M J L,BRAZOLOT,MILLAN R A,et al.Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates[J].Mol Med,1999,5(5):287-300.
[11]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.
[12]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.
[13]BLAKE D P,BILLINGTON K J,COPESTAKE S L,et al.Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite[J],PLoSPathog,2011,7(2):e1001279.
[14]ARRIBILLAGA L,MARTINEZ M,VILLANUEVAL,et al.1163 eda-ST reptavidin fusion protein conjugated to biotinylated HCV-NS3protein induces strong T cell immune responses against NS3[J].J Hepatol,2013,58(S1):S473.
[15]MANSILLA C,GORRAIZ M,MARTINEZ M,et al.Immunization against hepatitis C virus with a fusion protein containing the extra domain A from fibronectin and the hepatitis C virus NS3protein[J].J Hepatol,2009,51(3):520-527.
[16]ARRIBILLAGA L,DURANTEZ M,LOZANO T,et al.A Fusion protein between streptavidin and the endogenous TLR4ligand eda targets biotinylated antigens to dendritic cells and induces T cell responses in vivo[J].Biol Med Res Int,2013,2013(1):1-9.
[17]MANSILLA C,CASALES E,CASARES N,et al.The extra domain a from fibronectin(eda)improves immunogenicity of NS3protein in a semliki forest virus(SFV)-based vaccine against hepatitis C[J].J Hepatol,2008,48(2):S228.
[18]RUDILLA F,FAYOLLE C,CASARES N,et al.Combination of a TLR4ligand and anaphylatoxin C5afor the induction of antigen-specific cytotoxic T cell responses[J].Vaccine,2012,30(18):2848-2858.
[19]CENEVA W H O.Nucleic acid vaccines[J].Vaccine,1994,12(16):1491-1550.