E.tenella河北株重组IL-2-EtMIC-2 DNA疫苗的构建及保护力分析  

Construction and protective efficacy evaluation of the recombinant IL-2-EtMIC-2 DNA vaccine against Eimeria tenella of Hebei strain

作　　者：张召兴[1] 张香斋[1] 李蕴玉[1] 丁咚 张志强[1] 吴同垒[1] 贾青辉[1] 张艳英[1] 李佩国[1] 

机构地区：[1]河北科技师范学院河北省预防兽医学重点实验室,河北秦皇岛066604 [2]沧州职业技术学院,河北沧州061001

出　　处：《中国兽医学报》2018年第1期77-81,共5页Chinese Journal of Veterinary Science

基　　金：河北省自然科学基金资助项目(C2014407043);河北省教育厅重点资助项目(ZD2014071);河北省现代农业产业技术体系蛋鸡产业创新团队岗位资助项目(HBCT2013090203)

摘　　要：为了构建E．tenella河北株重组IL-2-EtMIC-2DNA疫苗，并确定其抗球虫效力。采用RT-PCR方法分别克隆出鸡IL-2和E．tenella河北株EtMIC-2基因，并连接到pMD18-T载体上进行测序；将目的基因克隆到pcDNA3．0载体，构建重组质粒peDNA3．0-IL-2-EtMIC-2，并转染293T细胞，用Westernblot方法验证表达。将构建的重组IL-2-EtMIC-2DNA疫苗以50，100，150ug／只的剂量分别在14，21日龄胸肌注射免疫试验鸡，除阴性对照组外，28日龄时经口灌服孢子化E．tenella卵囊4×10 4个，研究其对E．tenella感染后的免疫保护效果。结果表明：成功构建了重组质粒pcDNA3．0-IL-2-EtMIC-2，且能在293T细胞中表达出融合蛋白；3个免疫组的ACI比阳性对照组分别提高了97．63％，127．02％和129．81％，其中免疫剂量为100，150ug时，ACI均达160以上，具有良好的免疫保护效果。In this study,recombinant IL-2-EtMIC-2 DNA vaccine was constructed and protective efficacy against E. tenella of Hebei strain was verified. EtMIC-2 gene of E. tenella and chicken IL-2 gene were cloned by RT-PCR and ligated into the pMD18-T vector and sequenced. The target genes were cloned into pcDNA3.0 vector to construct the prokaryotic expression plasmid pcD- NA3.0-IL-2-EtMIC-2 and then was transformed into 293T cells to verify its expression by Western blot. Chickens were immunized with the DNA vaccine with 50,100,150 ug/each via pectoral muscle injection at 14,21 days old, respectively. Except the negative control group, the other chickens were inoculated with 4 × 10 4 sporulated E. tenella oocysts per bird at 28 days old and the immunoprotection of DNA vaccine against E. tenella was analyzed. The results indicated that the recombinant plasmid pcDNA3.0-IL-2-EtMIC-2 was successfully constructed and the fusion proteins were expressed in 293T cells. The comprehensive evaluation of ACI value in 3 immunity groups were increased by 97.63% ,127.02% ,129.81% respectively,compared with control group. Both of ACI were up to 160 when the immune dose was 100 and 150 ug each chicken. So,the result indicates the IL-2-EtMIC-2 DNA vaccine could provide a good immunoprotection to chicken challenged E. tenella oocysts.

关 键 词：柔嫩艾美耳球虫 EtMIC-2基因 IL-2基因 DNA疫苗 

分 类 号：S852.7[农业科学—基础兽医学]