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。
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.
出处
《生物技术通报》
CAS
CSCD
2008年第S1期271-275,281,共6页
Biotechnology Bulletin
基金
国家自然科学基金项目(No.30270981)
国家科技攻关计划课题(No.2004BA519A53)
