摘要
提取人肝癌SMMC-7721细胞和小鼠NS0细胞总RNA,对热休克蛋白10基因(Hsp10)进行RT-PCR扩增、TA克隆和测序。结果表明,从上述2种细胞中获得的cDNA分别由312,313 bp组成,与报道的人HSPE1 mRNA(NM-002157)、鼠肌肉Hspe1 mRNA(NM-008303)序列同源性分别为99%,97%,命名为HSPE1-1和Hspe1-1,序列分析表明,二者之间在核苷酸水平上的同源性为92%,氨基酸水平上的同源性为97%。将上述2种cDNA进行双酶切、亚克隆,成功构建了重组表达载体pET-28a-HSPE1-1与pET-28a-Hspe1-1,转化大肠杆菌并进行了诱导表达。检测结果表明,pET-28a-HSPE1-1与pET-28a-Hspe1-1均能在大肠杆菌中表达,表达蛋白分子量大小约为14 kDa,与预期大小一致。Western-Blot结果证实了其为目的蛋白,经镍树脂柱纯化,获得了相应的重组蛋白。为进一步研究这2种蛋白的结构、功能及临床应用奠定了基础。
RT-PCR was used to study the expression of heat-shock 10 kDa protein.The results showed that there were two different HSP transcriptons of varied sizes in Homo liver cancer SMMC-7721 and Mus NS0.One HSP cDNA was composed of 312 bp（named HSPE1-1） and held 99% homology with HSPE1 mRNA reported（NM-002157）.The other HSP gene cDNA was consisted of 313 bp（named Hspe1-1） and had 97% homology with Hspe1 mRNA reported（NM-008303）.The homology between HSPE1-1 and Hspe1-1 was 92% in nucleotide sequence and 97% in amino acid sequence.Prokaryotic expression plasmids pET-28a-HSPE1-1 and pET-28a-Hspe1-1 for producing HSPE1 and Hspe1 proteins were constructed successfully.Its molecular weight was 14 kDa.Anti-His6 monoclonal antibody was used to demonstrate the identity of the proteins in Western Blot.The corresponding proteins were obtained after purification by Ni-NTA chromatography.The purified corresponding proteins would be helpfulin further studies of their molecular structure,biological function and clinical application.
出处
《华北农学报》
CSCD
北大核心
2011年第3期56-59,共4页
Acta Agriculturae Boreali-Sinica
基金
河南省科技攻关项目(0823004532084)
关键词
热休克蛋白
人热休克蛋白1
鼠热休克蛋白1
基因克隆
原核表达
Heat-shock 10 kDa protein（Hsp10）
Homo sapiens heat shock 10 kDa protein 1（chaperonin 10）（HSPE1）
Mus musculus heat shock protein 1（chaperonin 10）（Hspe1）
Gene cloning
Protein expression
