摘要： 提取人肝癌SMMC-7721细胞和小鼠NSO细胞总RNA,对热休克蛋白10基因(Hspl0)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(NM002157)、鼠肌肉Hspel mRNA(NM-008303)序列同源性分别为99%,97%,命名为HSPEl-1和Hspel-Ⅰ,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1 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.
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
Abstract: 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.
Key words:
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
中图分类号:
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WANG Li, DU Xiao-ming, WANG Lin-lin, SHI Xi-bao, TENG Man, LI Gong-quan, QUAN Kai, GUO Jun-qing, ZHANG Gai-ping. Cloning, Sequencing and Prokaryotic Expression of Homo Sapiens Heat Shock 10 kDa Protein and Mus Heat Shock 10 kDa Protein[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(3): 56-59. doi: 10.7668/hbnxb.2011.03.012.