单 位：
中国动物卫生与流行病学中心国家外来动物疫病诊断中心
摘 要：
根据已知的绵羊PRNP基因序列设计引物,扩增出完整的PrP前体蛋白基因,将获得的基因克隆到pGEM-T载体中,得到插入PrP前体蛋白基因的pGEM-T-OPrP质粒,经PCR、酶切、测序鉴定后,用EcoRI和NotI从pGEM-T-OPrP上切下目的片段后,连接到汉逊酵母表达载体pFMDHZ-α-A中,通过PCR、酶切、插入鉴定保证正确插入后,经电转化将重组质粒pFMDHZ-α-A-OPrP转入多形汉逊酵母中,随后通过甲醇诱导进行表达。本研究表达的绵羊PrP前体蛋白,为绵羊痒的检测及诊断、PrP的结构与功能研究奠定基础。
译 名：
Expression of the Ovine PrP Precursor Gene in Hansenula polymorpha
作 者：
Lan Zouran~(1,2)Wang Zhiliang~1 Zhao Yonggang~1 Li Jinming~1 Wu Xiaodong~1 (1 National Diagnostic Center for Exotic Animal Disease,National Animal Quarantine institute,MOA,Qingdao 266032; 2 Shandong Provincial Center for Animal Disease Control and Prevention,Jinan 250022)
关键词：
PrP;;Precursor;;Hansenula polymorpha;;Expression
摘 要：
Ovine PrP precursor gene was amplified by polymerase chain reaction(PCR)from ovine genomic DNA and inserted into pGEM-T vector,after identification of the positive pGEM-T-OPrP with inserted ovine PrP precursor gene by PCR,double digestion and sequencing,the cloned gene was digested with EcoRI/NotI and inserted into pFMDHZ-α-A vector at the multiple cloning sites between EcoRⅠand NotⅠ.subsequently,the recombinant plasmid pFMDHZ-α-A-OprP was introduced into Hansenula polymorpha by electroporation,Hansenula polymorpha with recombinant plasmid pFMDHZ-α-A-OprP was induced by methanol and analysised by SDS-PAGE and western blot,The results of SDS-PAGE and western blot showed that the ovine PrP precursor protein was successfully expressed in Hansenula polymorpha.The expression of PrP precursor gene provides the basis for researching on scrapie.
