摘要
从藏绵羊全血中提取基因组总DNA，用所设计的引物以聚合酶链式反应扩增出细胞型朊蛋白（PrP^c）基因。测序分析表明，所克隆的羊PrP^c基因片段大小为771bp，包含了羊朊蛋白基因的完整编码区序列，其与国内外报道的序列基本相同。将目的基因与经EcoRⅠ和XhoⅠ酶切的载体pGEX-4T-1连接后转化宿主菌BL21（DE3），挑选重组阳性菌用IPTG诱导表达，收集不同培养时间的菌液进行SDS-PAGE和Western-blotting。结果表明，PrP基因在大肠杆菌中成功表达，并能被抗牛PrP^c单抗4C11识别。凝胶薄层扫描结果显示，表达蛋白约占菌体总蛋白的309／6～45％，目的蛋白以包涵体的形式存在，包涵体经变性裂解、纯化和复性后得到具有一定生物学活性的目的蛋白。
Total genomic DNAs were isolated from peripheral whole-blood of Tibet sheep. The PrP^c gene was amplified by polymerase chain reaction using the two primers. Sequencing of the PCR-derived fragment showed that the 771 bp fragment contained an entire PrPc coding region, which was the same as the published sequences. The interest gene was ligated into a vector pGEX-4T-1 pre-digested with EcoRⅠand Xho Ⅰ respectively, then transformed into E. coli BL21（DE3） cells. Recombinant plasmids were iden- tified by restriction analysis and PCR as well as sequencing. The bacteria containing recombinant pGEX-4T-1 were induced with IPTG and samples of the culture were collected at different time. The samples were examined by SDS-PAGE and Western-blot after being properly treated. The results showed that the interest gene was expressed successfully in E. coli and could be recognized by 4C11 against bovine Prp^c. The amount of the target protein accounted for 30%-45% of total bacterial proteins. It was proved that the expressed protein was in the form of inclusion bodies. Target protein with some bioactivities was gained by denaturing and purifying and refolding.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2006年第11期859-863,共5页
Chinese Veterinary Science
基金
农业部畜禽病毒学重点实验室基金资助项目(2002-01)
兰州兽医研究所所长基金资助项目(2004-2005)
关键词
重组朊蛋白
藏绵羊
原核表达
recombinant prion protein
Tibet sheep
prokaryotic expression
