摘要
为研究堆型艾美耳球虫阳离子转运ATP酶(Eimeria acervulina Na+-K+-ATPase,Ea NKA)蛋白的生物学功能,根据Ea NKA基因(GenBank登录号:EU590120.1)设计1对特异性引物,以堆型艾美耳球虫孢子化卵囊cDNA文库为模板,对Ea NKA基因进行PCR扩增,将PCR扩增产物克隆入pUCM-T载体并进行测序鉴定,将测序获得的Ea NKA基因编码蛋白进行生物信息学分析。Ea NKA基因全长1157 bp,位于第81-368 bp之间,编码236个氨基酸,分子量约为25 kDa。编码蛋白主要位于内质网,不具有信号肽,编码蛋白主要位于内质网,不具有信号肽,有12个结构功能域,2个跨膜区,位于多肽N端的7个抗原表位。Blast分析显示其编码蛋白与堆型艾美耳球虫阳离子转运ATP酶相关蛋白序列(XP013248919、ACB97673)的同源性为100%。构建重组质粒p ET32a(+)-Ea NKA并进行测序鉴定,然后将重组质粒转化到大肠杆菌BL21感受态细胞,进行SDS-PAGE和Western blot表达鉴定。结果显示,构建的重组质粒可在原核细胞中成功表达45 kDa的融合蛋白。本研究成功获得Ea NKA基因的全长序列,并在原核细胞中成功表达,为今后研究该基因的功能及筛选基因工程疫苗候选分子奠定基础。
In order to study the biological function of Na+-K+-ATPase protein of Eimeria acervulina,(Eimeria acervulina Na+-K+-ATPase, Ea NKA), one pair of specific primers were designed according to the Ea NKA gene sequence deposited in GenBank(accession No. EU590120.1). The ORF of Ea NKA gene was amplified by PCR using the cDNA library of E. acervulina sporulated oocysts as a template. After sequence analysis, the structures and biochemical properties of Ea NKA protein were analyzed using bioinformatics software. The PCR fragment was ligated to the prokaryotic expression vector pET32 a(+) to construct the recombinant plasmid pET32 a(+)-Ea NKA. Then, the recombinant plasmid was transfected into BL21 cells for induction with IPTG. The results showed that the fulllength of Ea NKA was 1157 bp in length and ORF located between 81-788 bp, encoding 236 amino acids with a molecular weight of about25 kDa. The encoded protein mainly located in the endoplasmic reticulum and consisted of 12 structural domains, two transmembrane regions without a signal peptide. In addition, seven epitopes mainly located in the N-terminal polypeptide. Blast analysis showed that the encoded protein shared 100% sequence identity with ATPase-related protein of Eimeria acervulina sequence(XP013248919, ACB97673).Western blotting showed that a protein with molecular weight of 45 kDa in the transfected cells was strongly recognized by rabbit antiserum against E.acervulina sporulated oocysts. These results indicated that the full-length sequence of Ea NKA gene was obtained and expressed in prokaryotic cells, which laid the foundation for the screening of genetically engineered vaccine candidate molecules.
作者
阎晓菲
孔苗
韩红玉
董辉
黄兵
YAN Xiao-fei;KONG Miao;HAN Hong-yu;DONG Hui;HUANG Bing(College of Science and Technology, Xinjiang Agricultural University, Urumqi 830052, China;Key Laboratory for Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China)
出处
《中国动物传染病学报》
CAS
北大核心
2019年第3期65-73,共9页
Chinese Journal of Animal Infectious Diseases
基金
国家寄生虫种质资源共享服务平台(平台-TDRC-22)
新疆农业大学科学技术学院大学生科技创新基金项目(2017KCX01)
作者简介
阎晓菲,女,硕士,副教授,主要从事寄生虫分子生物学的研究;通信作者:黄兵,E-mail:huangbing232@163.com.
