摘要在病毒侵染和抗病毒免疫过程当中，病毒的抗原表位、受体以及受体结合 域起着至关重要的作用。S 蛋白（Spike）是作为 PEDV（PED Porcine Epidemic Diarrhea Virus）表面的一个重要结构蛋白，它既包含介导病毒侵入宿主细胞的 受体结合域，又拥有抗原表位能够介导机体使其产生病毒中和抗体，而 SD 区 域又是 S 蛋白上的一个主要抗原表位。鉴于此，该实验对 PEDV S 蛋白的 SD 区域进行克隆与鉴定，为诊断 PEDV 的方法以及研发新型疫苗提供更多的信息。83572
利用实验室已保存的 SD 基因，根据其设计正反引物，通过 PCR 扩增得到 足量的目的基因。本实验所选用的原核表达载体为 pET28a(+),将其通过 SDS 碱 裂解法进行抽提并单酶切鉴定。随后利用同源重组连接 SD 目的基因以及 pET28a(+)质粒，利用 BamHⅠ单酶切鉴定。将重组子转化 Top10 感受态细胞， 通过菌液 PCR 鉴定该重组子符合预期目标。将 pET28a(+)-SD 重组质粒进行测 序，得到结果与理论结果相符，为进一步研究 PEDV 抗原表位以及相关疫苗的 制备提供了一定的理论基础。
毕业论文关键词：PEDV；抗原表位；SD 区域；pET28a(+)质粒；同源重组
Abstract In the process of virus infection and antiviral immunity, viral antigen epitope and receptor and the receptor-binding domain plays a crucial role。 S protein (Spike) is an important structural protein on the surface of PEDV (PED Porcine Epidemic Diarrhea Virus), it contains the receptor-binding domain where mediated virus invaded host cells, and t has antigenic epitopes can be mediated by the body to produce virus-neutralizing antibody。 SD fragment is a major antigenic determinant on the S protein。 In view of this, Cloning and identification of the SD fragment of PEDV S protein can provision of information to the PEDV method for diagnosis as well as the development of new vaccines。
Used the saved SD laboratory gene, and accorded to the gene to design the forward primer and reverse primer。 Then we got plenty of target genes by PCR amplification。 This experiment selected pET28a (+) for prokaryotic expression vector, and extracted it through SDS alkaline Lysis method and identified it by single digestion。 Then connected SD gene with pET28a (+) plasmid using homologous recombination, used the BamH I to do the single enzyme digestion。 We put the reorganize into Top10 cells, identified it through PCR amplification of the recombinant bacteria liquid and the result matched expectations。 Sequenced the PET28a (+) –SD of recombinant plasmid, and the results conformed to the
theoretical results。 The result for further study of the PEDV epitope and related vaccine preparation provides a theoretical basis。
Keywords: PEDV；Epitope；SD fragment；PET28a(+ )plasmid；Homologous recombination
中英文对照表
缩写词 英文全称 中文全称
HCoV-229E Human coronavirus 229E 人冠状病毒 229E
FIPV Feline infectious peritonitis virus 猫传染性腹膜炎病毒
TGEV Porcine transmissible gastroenteri tis virus 猪传染性胃肠炎病毒
MHV Murine virus hepatitis 小鼠肝炎病毒
RCV Rat coronavirus 大鼠冠状病毒
HEV Hemagglutinating encepha lomyelitis virus 猪血凝性脑脊髓炎病毒
TBV PEDVS蛋白SD区域的克隆与鉴定:http://www.youerw.com/shengwu/lunwen_98624.html