分泌型表达EDA-EmIMP1的重组枯草芽胞杆菌的构建及其免疫原性研究
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摘要
枯草芽胞杆菌有着异源蛋白表达系统,纤维连接蛋白外域A(EDA)是一种能够提高相关蛋白的抗原性的佐剂,为了增强巨型艾美耳球虫的免疫相关蛋白1(IMP1)的抗原性,笔者对枯草芽胞杆菌分泌表达载体作为EDA分子佐剂重组亚单位疫苗的递呈载体的功能进行了研究,构建质粒pHT43-EDA-EmIMP1和pHT43-EmIMP1,将获得的重组质粒转化到枯草芽胞杆菌感受态中。将该重组菌对鸡饲喂,进行免疫学试验并做免疫效果评价。结果显示,该重组枯草芽胞杆菌可以成功表达目的蛋白,蛋白大小为70 ku。ELISA检测EDA组的效价最高,其滴度在3 200左右;三免EDA组的IFN-γ浓度显著高于其他组(P<0.05);三免EDA组和EmIMP1组的IL-12/70浓度显著高于PBS组(P<0.05);CD4+T细胞群的检测显示,EDA组的CD4+T细胞群比例显著高于枯草芽胞杆菌组和PBS组(P<0.05);EDA组、枯草芽胞杆菌组和EmIMP1组的卵囊排出量都显著低于攻虫组(P<0.05);病变计分可以看出,EDA组对肠道保护效果显著优于除空白组外的其他组(P<0.05)。以上各项免疫学指标表明,枯草芽胞杆菌分泌型表达载体作为EmIMP1的抗原递呈载体可以有效提高免疫原性,其中EDA佐剂组的免疫效果最好。
Bacillus subtilis has heterologous protein expression system.Fibronectin extra domain A(EDA)is an adjuvant that can enhance the antigenicity of relevant protein.To strengthen the antigenicity of immune-related protein1(IMP1)of E.maxima,the present study is conducted on the function of secreting expression vector of Bacillus subtilis as the delivery vector of recombinant subunit vaccine of molecular adjuvant EDA.Plasmids p HT43-EDA-EmIMP1 and p HT43-EmIMP1 was constructed and the recombinant plasmids was transformed into the competence of Bacillus subtilis,Next,the recombinant strain was fed on chickens in an immunological experiment and immunization effect evaluation was carried out.In result,the recombinant Bacillus subtilis can successfully express target protein,the size of which is 70 ku.According to ELISA,the titer of EDA group was highest,and reached to 3200.The concentration of IFN-γ of third immunity EDA group was prominently higher than other groups(P<0.05).The determination of CD4+T cell population showed that CD4+T cell population EDA group was obviously higher than that of Bacillus subtilis group and PBS group(P<0.05).The concentration of IL-12/70 of third immunity EDA group and EmIMP1 group were apparently higher than PBS group(P<0.05).The output of oocyst of EDA group,Bacillus subtilis group and EmIMP1 group were obviously lower than attacking larvae group(P<0.05). From lesion score,it can be seen that the protection effect on intestinal of EDA group was clearly better than other groups except blank group(P<0.05).In conclusion,from all immunological indexes,as the delivery vector of antigen of EmIMP1,secreting expression vector of Bacillus subtilis can effectively enhance immunogenicity.Furthermore,and the immune effect of EDA group is the best.
Bacillus subtilis has heterologous protein expression system.Fibronectin extra domain A(EDA)is an adjuvant that can enhance the antigenicity of relevant protein.To strengthen the antigenicity of immune-related protein1(IMP1)of E.maxima,the present study is conducted on the function of secreting expression vector of Bacillus subtilis as the delivery vector of recombinant subunit vaccine of molecular adjuvant EDA.Plasmids p HT43-EDA-EmIMP1 and p HT43-EmIMP1 was constructed and the recombinant plasmids was transformed into the competence of Bacillus subtilis,Next,the recombinant strain was fed on chickens in an immunological experiment and immunization effect evaluation was carried out.In result,the recombinant Bacillus subtilis can successfully express target protein,the size of which is 70 ku.According to ELISA,the titer of EDA group was highest,and reached to 3200.The concentration of IFN-γ of third immunity EDA group was prominently higher than other groups(P<0.05).The determination of CD4+T cell population showed that CD4+T cell population EDA group was obviously higher than that of Bacillus subtilis group and PBS group(P<0.05).The concentration of IL-12/70 of third immunity EDA group and EmIMP1 group were apparently higher than PBS group(P<0.05).The output of oocyst of EDA group,Bacillus subtilis group and EmIMP1 group were obviously lower than attacking larvae group(P<0.05). From lesion score,it can be seen that the protection effect on intestinal of EDA group was clearly better than other groups except blank group(P<0.05).In conclusion,from all immunological indexes,as the delivery vector of antigen of EmIMP1,secreting expression vector of Bacillus subtilis can effectively enhance immunogenicity.Furthermore,and the immune effect of EDA group is the best.
引文
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[18]岳敏杰.鸡干扰素α在枯草芽胞杆菌中的表达及活性检测[D].郑州:河南农业大学,2013.YUE Min-jie.Expression of chicken interferonαin Bacillus subtilis and its activity assay[D].Zhengzhou:Henan Agricultural University,2013.(in Chinese)
[19] WONG S L.Advances in the use of Bacillus subtills for the expression and secretion of heterologous proteins[J].Current Biology,1995,6(5):517-522.
[20]王振华.重组枯草芽胞杆菌SE1制剂对肉鸡生长、抗氧化能力及免疫功能的影响[J].中国预防兽医学报,2018,40(3):248-252.WANG Zhen-hua.Effect of recombinant Bacillus subtilis SE1 on the growth,antioxidant capacity and immune function of broilers[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(3):248-252.(in Chinese)
[21]罗正,张得玉,孙业贵,等.益生菌替代肉鸡饲料中促生长抗生素的田间试验[J].饲料工业,2014,35(13):22-24.LUO Zheng,ZHANG De-yu,SUN Ye-gui,et al.Field trials of probiotic replacing growth-promoting antibiotics in broiler production[J].Feed Industry,2014,35(13):22-24.(in Chinese)
[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]索勋,李国清.鸡球虫病学[M].北京中国农业出版社,1998.SUO Xun,LI Guo-qing.Chicken Coccidiosis[M].Beijing:China Agricultural University Press,1998.(in Chinese)
[7]孙树汉.核酸疫苗[M].上海:第三军医大学出版社,2000.SUN Shu-han.Nucleic Acid Vaccine[M].Shanghai:Second Military Medical University Press,2000.(in Chinese)
[8] WATINE G J,MCMANUS D P.Nucleic acids vaccines of the future[J].Parasitology Today,1995,11(3):113-116.
[9] MCCLUSKI M J L,BRAZOLOT M,ROBERT 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.
[10]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)
[11]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)
[12] 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.
[13] ARRIBILLAGA L,MARTINEZ M,VILLANUEVA L,et al.1163eda-streptavidin fusion protein conjugated to biotinylated hcv-ns3 protein induces strong T cell immune responses against ns3[J].Journal of Hepatology,2013(58):S473.
[14] 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 NS3 protein[J].Journal of Hepatology,2009,51(3):520-527.
[15] ARRIBILLAGA L,DURANTEZ M,LOZANO T,et al.A fusion protein between streptavidin and the endogenous TLR4 ligand EDA targets biotinylated antigens to dendritic cells and induces T cell responses in vivo[J].BioMed Research International,2013(1):1-9.
[16] MANSILLA C,CASALES E,CASARES N,et al.612 the extra domain a from fibronectin(eda)improves immunogenicity of ns3 protein in a semliki forest virus(sfv)-based vaccine against hepatitis c[J].Journal of Hepatology,2008,48(8):S228.
[17] RUDILLA F,FAYOLLE C,CASARES N,et al.Combination of a TLR4 ligand and anaphylatoxin C5a for the induction of antigen-specific cytotoxic T cell responses[J].Vaccine,2012,30(18):2848-2858.
[18]岳敏杰.鸡干扰素α在枯草芽胞杆菌中的表达及活性检测[D].郑州:河南农业大学,2013.YUE Min-jie.Expression of chicken interferonαin Bacillus subtilis and its activity assay[D].Zhengzhou:Henan Agricultural University,2013.(in Chinese)
[19] WONG S L.Advances in the use of Bacillus subtills for the expression and secretion of heterologous proteins[J].Current Biology,1995,6(5):517-522.
[20]王振华.重组枯草芽胞杆菌SE1制剂对肉鸡生长、抗氧化能力及免疫功能的影响[J].中国预防兽医学报,2018,40(3):248-252.WANG Zhen-hua.Effect of recombinant Bacillus subtilis SE1 on the growth,antioxidant capacity and immune function of broilers[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(3):248-252.(in Chinese)
[21]罗正,张得玉,孙业贵,等.益生菌替代肉鸡饲料中促生长抗生素的田间试验[J].饲料工业,2014,35(13):22-24.LUO Zheng,ZHANG De-yu,SUN Ye-gui,et al.Field trials of probiotic replacing growth-promoting antibiotics in broiler production[J].Feed Industry,2014,35(13):22-24.(in Chinese)