HB株E.tenella AMA-1基因的克隆及免疫调节型真核表达载体构建  

Cloning of E.tenella HB strain AMA-1 gene and construction of immunoregulatory eukaryotic expression vector

作　　者：闫艳娟 李蕴玉[1] 张东林[1] 庞洪泽 张香斋[1] 李佩国[1] YAN Yan-juan;LI Yun-yu;ZHANG Dong-lin;PANG Hong-ze;ZHANG Xiang-zhai;LIei-guo(Hebei Normal University of Science&Technology,Hebei Province Key Laboratory of VeterinaryPreventive Medicine,Qinhuangdao,Hebei 066600,China)

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

出　　处：《中国兽医学报》2019年第11期2173-2178,共6页Chinese Journal of Veterinary Science

基　　金：河北省现代农业产业技术体系蛋肉鸡产业创新团队岗位资助项目(HBCT2018150206);2017年河北省硕士研究生创新资助项目(CXZZSS2017158)

摘　　要：为研究HB株柔嫩艾美耳球虫(E.tenella)AMA-1基因的免疫原性,根据GenBank收录的AMA-1基因序列设计了1对引物,对HB株E.tenella的AMA-1基因进行克隆,应用生物信息学分析软件预测AMA-1基因的核苷酸及编码的蛋白质的结构和功能。连接pPIC9载体,并与细胞因子IL-2串联,构建真核表达载体pPIC9-IL-2-AMA-1,转化毕赤酵母细胞GS115,进行蛋白表达及纯化验证。结果显示,HB株E.tenella AMA-1基因全长为1617bp,编码535个氨基酸,具有跨膜区及信号肽。经酶切和测序鉴定表明,真核表达载体pPIC9-IL-2-AMA-1构建成功。毕赤酵母转化结果表明,成功将重组载体转化到毕赤酵母中,PCR鉴定得到2200,2100bp 2条带。Western blot检测结果表明AMA-1在毕赤酵母中得到了很好的表达。本试验结果为AMA-1基因的功能和核酸疫苗的研制提供依据。The aims of the study was to discuss the biological information of the AMA-1 gene of E.tenella HB strain.Based on the published ORF of E.tenella AMA-1 gene,a pair of primers was designed.The AMA-1 gene of E.tenella HB strain was cloned.The bioinformatics analysis software were used to predict the structure and function of nucleotides of the AMA-1 gene and encoded proteins.The pPIC9 vector was ligated with the cytokine IL-2 to construct the eukaryotic expression vector pPIC9-IL-2-AMA-1,which was transformed into Pichia pastoris cell GS115 for protein expression and purification.The results showed that the HB strain E.tenella AMA-1 gene was 1 617 bp in length and encoded 535 amino acids with a transmembrane region and a signal peptide.The restriction endonuclease digestion and sequencing showed that the eukaryotic expression vector pPIC9-IL-2-AMA-1 was successfully constructed.Pichia pastoris transformation showed that the recombinant vector was successfully transformed into Pichia pastoris,and two fragments of 2 200 and 2 100 bp were obtained by PCR.Western blot verification showed that AMA-1 was well expressed in Pichia pastoris.The results provide a scientific basis for the function of the AMA-1 gene and the development of nucleic acid vaccines,laying the foundation for subsequent cell and animal experiments.

关 键 词：E.TENELLA HB株 AMA-1基因 真核表达载体 

分 类 号：S852.72[农业科学—基础兽医学]