细粒棘球绦虫EgG1Y162-1/2重组蛋白诱导小鼠免疫应答特点的比较
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摘要
目的研究和对比经生物信息学分析后重组构建的EgG1Y162-1/2蛋白诱导小鼠产生免疫应答反应的特点。方法随机将60只小鼠分5组,根据所注射抗原的不同分为EgG1Y162-1组、EgG1Y162-2组、GST对照组、佐剂组和正常对照组。每2周免疫1次,共免疫4次。采集免疫前后的小鼠血清进行Elisa检测抗体效价;流式细胞术检测不同时间点淋巴细胞亚群(CD4~+T细胞和CD8~+T细胞)的动态变化;免疫10周后用细粒棘球蚴感染攻击小鼠,对比小鼠脾淋巴细胞体外刺激后的增殖反应、脾细胞凋亡率和小鼠囊重抑制率的检测结果。结果免疫后第10周,EgG1Y162-2组抗体滴度为1∶20 480高于EgG1Y162-1组抗体滴度1∶5 120。经原头蚴感染攻击的EgG1Y162-2组的淋巴细胞亚群(CD4~+T细胞、CD8~+T细胞)百分比均高于EgG1Y162-1组,且差异有统计学意义(P <0.05),但CD4~+T/CD8~+T细胞比值差异无统计学意义(P>0.05)。免疫后的EgG1Y162-2组小鼠脾淋巴细胞在体外被特异性刺激后增殖水平高于EgG1Y162-1组,差异有统计学意义(P <0.05)。检测显示EgG1Y162-2组的脾细胞凋亡率低于EgG1Y162-1,差异有统计学意义(P <0.05)。疫苗保护实验中发现EgG1Y162-1组的小鼠囊重抑制率比EgG1Y162-2组低,差异有统计学意义(P <0.05)。结论EgG1Y162-2抗原比EgG1Y162-1抗原具有更强的免疫优势作用,本研究不仅证明了生物信息学分析表位的准确性,也提示该表位信息丰富的抗原值得作为疫苗的保护性抗原进行深入研究。
Objective The purpose of this study was to contrast and investigate the immunological effects induced by the EgG1Y162-1/2recombinant protein that predicted by bioinformatics methods in mice.Methods 60 mice were randomly divided into 5groups:EgG1Y162-1group,EgG1Y162-2group,GST group,FCA group and normal saline group(NS),which were injected by different vaccine according to trial grouping method.Immunization was performed once every 2weeks for 4times.Blood samples were collected before and after the immunization.The dynamic changes of lymphocyte subsets(CD4~+T cells and CD8~+T cells)at different time were detected by flow cytometry.After 10 weeks of immunization,the proliferation response of splenic lymphocytes stimulated was detected by MTT,and the apoptosis rate of spleen cells was detected.In the end,the inhibition rate of cystic weight in mice in each group were calculated.Result After 10 weeks,the level of IgG in EgG1Y162-2group(1∶21 480)was higher than EgG1Y162-1(1∶5120)group.The level of CD4~+T and CD8~+T cells were significantly increased after the infection of Echinococcus granulosus protoscoleces(PSCs)(P <0.05).There was no statistically significant difference in the level of CD4~+T/CD8~+T cells(P >0.05).After immunization,the proliferation level of spleen lymphocytes in mice from EgG1Y162-2 group was higher than that of EgG1Y162-1group after specific stimulation in vitro,and the difference was statistically significant(P <0.05).The apoptosis rate of spleen cells in EgG1Y162-2group was lower than EgG1y162-1group,and the difference was statistically significant(P <0.05).In the vaccine protection experiment,the rate of cystic weight inhibition in EgG1y162-1group was lower than that of EggG1y162-1group,and the difference was statistically significant(P <0.05).Conclusion EgG1y162-2antigen has stronger immune dominance than EgG1y162-1antigen.It's not only proves the accuracy of the prediction by bioinformatics method but also suggests that the antigen with abundant epitope information is worthy of in-depth study as a protective antigen of vaccine.
Objective The purpose of this study was to contrast and investigate the immunological effects induced by the EgG1Y162-1/2recombinant protein that predicted by bioinformatics methods in mice.Methods 60 mice were randomly divided into 5groups:EgG1Y162-1group,EgG1Y162-2group,GST group,FCA group and normal saline group(NS),which were injected by different vaccine according to trial grouping method.Immunization was performed once every 2weeks for 4times.Blood samples were collected before and after the immunization.The dynamic changes of lymphocyte subsets(CD4~+T cells and CD8~+T cells)at different time were detected by flow cytometry.After 10 weeks of immunization,the proliferation response of splenic lymphocytes stimulated was detected by MTT,and the apoptosis rate of spleen cells was detected.In the end,the inhibition rate of cystic weight in mice in each group were calculated.Result After 10 weeks,the level of IgG in EgG1Y162-2group(1∶21 480)was higher than EgG1Y162-1(1∶5120)group.The level of CD4~+T and CD8~+T cells were significantly increased after the infection of Echinococcus granulosus protoscoleces(PSCs)(P <0.05).There was no statistically significant difference in the level of CD4~+T/CD8~+T cells(P >0.05).After immunization,the proliferation level of spleen lymphocytes in mice from EgG1Y162-2 group was higher than that of EgG1Y162-1group after specific stimulation in vitro,and the difference was statistically significant(P <0.05).The apoptosis rate of spleen cells in EgG1Y162-2group was lower than EgG1y162-1group,and the difference was statistically significant(P <0.05).In the vaccine protection experiment,the rate of cystic weight inhibition in EgG1y162-1group was lower than that of EggG1y162-1group,and the difference was statistically significant(P <0.05).Conclusion EgG1y162-2antigen has stronger immune dominance than EgG1y162-1antigen.It's not only proves the accuracy of the prediction by bioinformatics method but also suggests that the antigen with abundant epitope information is worthy of in-depth study as a protective antigen of vaccine.
引文
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[15]ZHANG Z G,YANG J,BARFORD D.Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system[J],Methods Suppl,2016,8:13-25.
[16]LI Y H,ZHANG F B,MOHAMMED H,et al.Construction and identification of the recombinant plasmid pET30aEgA31-Eg95of Echinococcus granulosu[J].Exp Ther Med,2014,7:204-208.
[17]WANG Y,SUN M,HE M,et al.Weak binder for MHCmolecule is a potent Mycobacterium tuberculosis-specific CTL epitope in the context of HLA-A24allele[J].Microb Pathog,2012,53:162-167.
[18]李玉娇,王晶,赵慧,等.细粒棘球绦虫Eg95抗原表位的生物信息学预测[J].中国人兽共患病学报,2011,27(5):892-896,900.
[19]ZHANG F B,LU X B,GUO N,et al.The prediction of T-and B-combined epitope of Ag85Bantigen of Mycobacterium tuberculosis[J].Int J Clin Exp Med,2016,9:1408-1421.
[20]李玉娇,杨晶,赵慧,等.细粒棘球绦虫EgA31重组蛋白的抗原表位分析预测[J].中国寄生虫学与寄生虫病杂志,2012,30(1):78-80.
[2]HIGUITA N I A,BRUNETTI E,MCClOSKEY C,et al.Cystic Echinococcosis[J].J Clin Microbiol,2016,54(3):518-523.
[3]SARKAR S,ROY H,SAHA P,et al.Cystic echinococcosis:A neglected disease at usual and unusual locations[J].Trop Parasitol,2017,7(1):51-55.
[4]CARMENA D,CARDONA G A.Echinococcosis in wild carnivorous species:epidemiology,genotypic diversity,and implications for veterinary public health[J].Vet Parasitol,2014,202:69-94.
[5]ESTEVES A,PORTILLO V,EHRLICH R.Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus[J].Biochim Biophys Acta,2003,1631:26-34.
[6]曹春宝,马秀敏,丁剑冰,等.细粒棘球蚴egGlYl62抗原基因的克隆及蛋白质序列分析[J].中国病原生物学杂志,2008,3(12):903-906.
[7]MA X M,ZHAO H,ZHANG F B,et al.Activity in mice of recombinant BCG-EgG1Y162vaccine for Echinococcus granulosus infection[J].Hum Vacc Immunother.2016,12(1):170-175.
[8]ZHANG F B,LI S Y,ZHU Y J,et al.Immunization of mice with egG1Y162-1/2provides protection against Echinococcus granulosus infection in BALB/c mice[J].Mol Immunol,2018,94:183-189.
[9]ESTEVES A,PORTILLO V,EHRLICH R.Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus[J].Biochim Biophys Acta,2003,1631:26-34.
[10]曹春宝,马秀敏,丁剑冰,等.细粒棘球蚴egGlYl62抗原基因的克隆及蛋白质序列分析[J].中国病原生物学杂志,2008,3(12):903-906.
[11]CHAUHAN V,FAROOQ U.Identification of T cell and Bcell epitopes derived from EG95 antigen of Echinococcus granulosus using in silico approach for therapeutic vaccine development[J].Indo Am J Pharm Res,2016,6:4639-4645.
[12]安梦婷,吾拉木·马木提,马海梅,等.细粒棘球蚴AgB1、AgB2、AgB4抗原表位的特性分析[J].新疆医科大学学报,2016,39(12):1541-1546.
[13]刘晓霞,马海梅,朱明,等.细粒棘球蚴egG1Y162疫苗对小鼠免疫应答的研究[J].中国人兽共患病学报,2013,29(3):226-231.
[14]MENG M,HE S Y,ZHAO G H,et al.Evaluation of protective immune responses induced by DNA vaccines encoding Toxoplasmagondii surface antigen 1(SAG1)and 14-3-3protein in BALB/c mice[J].Parasite Vector,2012,5:273-277.
[15]ZHANG Z G,YANG J,BARFORD D.Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system[J],Methods Suppl,2016,8:13-25.
[16]LI Y H,ZHANG F B,MOHAMMED H,et al.Construction and identification of the recombinant plasmid pET30aEgA31-Eg95of Echinococcus granulosu[J].Exp Ther Med,2014,7:204-208.
[17]WANG Y,SUN M,HE M,et al.Weak binder for MHCmolecule is a potent Mycobacterium tuberculosis-specific CTL epitope in the context of HLA-A24allele[J].Microb Pathog,2012,53:162-167.
[18]李玉娇,王晶,赵慧,等.细粒棘球绦虫Eg95抗原表位的生物信息学预测[J].中国人兽共患病学报,2011,27(5):892-896,900.
[19]ZHANG F B,LU X B,GUO N,et al.The prediction of T-and B-combined epitope of Ag85Bantigen of Mycobacterium tuberculosis[J].Int J Clin Exp Med,2016,9:1408-1421.
[20]李玉娇,杨晶,赵慧,等.细粒棘球绦虫EgA31重组蛋白的抗原表位分析预测[J].中国寄生虫学与寄生虫病杂志,2012,30(1):78-80.