CAJ | 학술논문

本研究旨在克隆小尾寒羊钙粘和蛋白1(Calcium and integrin binding protein 1,CIB1)基因cDNA全长,并在原核载体中表达.参照已发表的CIB1基因的核苷酸序列,设计了1对特异性引物,采用RT-PCR法,从小尾寒羊睾丸组织中扩增获得CIB1基因全长cDNA.将其克隆到pMD19-T载体,并进行测序分析.将该基因编码区重组于融合表达质粒pET32a中,构建了重组原核表达载体(pET32a-CIB1).将其转化到BL21(DE3)pLysS宿主菌中,用IPTG进行诱导表达.结果表明,克隆的CIB1基因cDNA与GenBank上登录的牛、猪、猕猴、人、小鼠、大鼠、黑猩猩等动物该基因序列的同源性达90%以上,编码氨基酸的同源性在93%以上,并且该序列包含有完整的开放阅读框,大小为576 bp.实现了高效特异性融合表达,表达产物的分子质量约为38 ku.本研究结果为进一步研究CIB1蛋白功能打下良好的基础.
본연구지재극륭소미한양개점화단백1(Calcium and integrin binding protein 1,CIB1)기인cDNA전장,병재원핵재체중표체.삼조이발표적CIB1기인적핵감산서렬,설계료1대특이성인물,채용RT-PCR법,종소미한양고환조직중확증획득CIB1기인전장cDNA.장기극륭도pMD19-T재체,병진행측서분석.장해기인편마구중조우융합표체질립pET32a중,구건료중조원핵표체재체(pET32a-CIB1).장기전화도BL21(DE3)pLysS숙주균중,용IPTG진행유도표체.결과표명,극륭적CIB1기인cDNA여GenBank상등록적우、저、미후、인、소서、대서、흑성성등동물해기인서렬적동원성체90%이상,편마안기산적동원성재93%이상,병차해서렬포함유완정적개방열독광,대소위576 bp.실현료고효특이성융합표체,표체산물적분자질량약위38 ku.본연구결과위진일보연구CIB1단백공능타하량호적기출.
In order to clone cDNA of Small Tail Han sheep CIB1 gene, and express the fusion protein in E. coli, a pair of specific primers were designed according to the nucleotide sequence of the published CIB1 gene. Full-length cDNA of CIB1 gene was cloned from Small Tail Han sheep by RT-PCR. CIB1 was cloned into pMD19-T vector and identified by sequencing. CIB1 gene was cloned into pET32a vector to construct a prokaryotic expression vector pET32a-CIB1 and transformed into BL21(DE3)pLysS cells. The recombinant plasmid pET32a-CIB1 was induced in Escherichia coli using IPTG. The results showed that the identity at nucleotide level were more than 90% with CIB1 gene from cattle, pig, rhesus monkey, human, mouse, rat, chimpanzee and so on, and the amino acid similarity were higher than 93% correspondingly. The CIB1 cDNA containing a 576 bp open reading frame (ORF) encoding 191 amino acids. The expressed recombinant fusion protein is 38 ku in size. These results provided basis for the future research on ovine CIB1 protein function.