金黄地鼠(Mesocricetus auratus)朊蛋白基因的克隆及其原核表达

Molecular cloning and prokaryotic expression of prion gene from Mesocricetus auratus

采用RT-PCR技术从金黄地鼠(Mesocricetus auratus)脑组织获得朊蛋白基因(Prion protein nucleic acid,PRNP)开放阅读框(Open reading frame,ORF),截去其N端信号肽(66 bp)和C端GPI锚定位点(69 bp)形成朊蛋白编码区PRNPx;将编码区重组于融合表达质粒Pet-DsbA中,并在大肠杆菌BL21(DE3)plysS中经IPTG诱导表达,并经Western-blot验证。结果表明,金黄地鼠PRNP基因ORF区全长为765 bp,编码254个氨基酸的前体蛋白,核苷酸序列与已发表金黄地鼠序列(M14054)同源性为99.87%,其第116个氨基酸发生了同义突变由GCT变为GCC;朊蛋白在大肠杆菌得到高效表达,产物是相对分子质量为47 000的融合蛋白。

Prions are infectious proteins that lead to transmissible spongiform encephalopathy （TSEs） or prion disease with fatal neurodegenerative disorders in human and animals,which are attributed by conversion of the normal cellular prion protein （PrPc） to an insoluble, protease resistant,and predominantly β-sheeted infectious form of the scrapie prion protein （prp^Sc）. The purpose of this study is to clone,identify and express the prion protein from golden hamster,PRNP was amplified by using PCR technique and PCR product was approximately 765 bp DNA segment. Clone vector pGEM-T-PRNP was successfully constructed with the PCR product that was cloned in pGEM-T- Vector by using T-A clone technique. A PRNP truncated fragment designed as PRNPx, was formed by removal of both N＇-terminal signal and C＇-terminal GPI sequences, PRNPx was cloned into expression vector Pet-DsbA, and transformed into fresh competent BL21 （DE3） plysS E. coli cell. The positive transformants determined by digesting and sequencing was induced with IPTG. The results of SDS-PAGE and western blotting showed that PRNPx was highly expressed as a 47 000 fusion protein,and the fusion protein can be recognized by a specific antibody AH6.