摘要
从健康BALB/c小鼠脑组织提取总RNA,逆转录获得cDNA。根据GenBank公布的视锥蛋白样基因1(vsnl1)基因序列设计引物,以鼠脑cDNA为PCR模板扩增vsnl1。将目的片段克隆至pMD18-T载体,经酶切和测序鉴定重组质粒pMDT-v。将vsnl1基因亚克隆至6×His原核表达载体pROEX-HTb,转化大肠杆菌DH5α,用IPTG诱导表达和Ni-NTA柱纯化,并采样进行SDS-PAGE分析。结果表明:RT-PCR扩增产生了与vsnl1大小吻合的目的片段,测序及BLAST比对提示与已公布序列的同源性为99%,将序列提交GenBank。SDS-PAGE显示,经IPTG诱导,重组质粒pROEX-v转化菌表达了约22 ku的蛋白,与预期分子量大小一致。
Total brain RNA was extracted from a healthy BALB/c mouse and reversely transcribed to cDNA. Primers were designed according to published vsnll sequence by GenBank and used to amplify target gene by PCR. The acquired vsnll fragment was inserted into pMD18-T vector. Recombinant pMDT-v plasmid was confirmed through restriction enzyme analyses and DNA sequencing. Then vsnll was subcloned into pROEX-HTb, a prokaryotic expression plasmid with a 6 × His tag, and the acquired pROEX-v was transformed into E. coli DH5α. Sequence alignment by online BLAST software revealed that target gene with 576 bp in length was of 99% similarity with reported sequence. SDS-PAGE analysis showed that an about 22 ku recombinant protein was expressed in transformed E. coli with pROEX-v under induction of IPTG, and that molecular weight of target protein was the same as expected.
出处
《扬州大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2008年第2期11-15,共5页
Journal of Yangzhou University：Agricultural and Life Science Edition
基金
上海重点基金项目(03DZ19703)
