小尾寒羊CIB1基因全长cDNA克隆及原核表达

畜牧兽医学报 ›› 2009, Vol. 40 ›› Issue (12): 1712-1717.doi:

小尾寒羊CIB1基因全长cDNA克隆及原核表达

于艳^1，2，王楚端¹，李宏滨³，魏彩虹³，孙丹⁴，陆建⁵，刘开东⁶，吕雁飞⁶，杜立新³*

1.中国农业大学动物科技学院，北京 100193；2.山东中医药大学第二附属医院生殖医学中心，济南 250001；3.中国农业科学院北京畜牧兽医研究所，北京 100193；4.辽宁师范大学生命科学院，大连 116029；5.扬州大学动物科学与技术学院，扬州 225009；6.青岛农业大学动物科技学院，青岛 266109

收稿日期:2008-12-22 修回日期:1900-01-01 出版日期:2009-12-24 发布日期:2009-12-24
通讯作者: 杜立新

CIB1 cDNA Cloning from Small Tail Han Sheep and Its Expression in E.coli

YU Yan ^{1, 2}, WANG Chuduan ¹, LI Hongbin ³, WEI Caihong ³, SUN Dan⁴, LU Jian ⁵, LIU Kaidong ⁶, LV Yanfei ⁶, DU Lixin ³*

1. College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; 2. Reproductive Medical Center, The Second Hospital Affiliated to Shandong University of Traditional Chinese Medicine, Jinan 250001, China; 3. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 4. College of Life Sciences, Liaoning Normal University, Dalian 116029, China; 5. College of Animal Science and Technology, Yangzhou University,Yangzhou 225009, China; 6. College of Animal Science＆Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China

Received:2008-12-22 Revised:1900-01-01 Online:2009-12-24 Published:2009-12-24
Contact: DU Lixin

摘要/Abstract

摘要： 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。

Abstract: 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.

于艳;王楚端;李宏滨;魏彩虹;孙丹;陆建;刘开东;吕雁飞;杜立新. 小尾寒羊CIB1基因全长cDNA克隆及原核表达[J]. 畜牧兽医学报, 2009, 40(12): 1712-1717.

YU Yan;;WANG Chuduan;LI Hongbin;WEI Caihong;SUN Dan;LU Jian;LIU Kaidong;LV Yanfei;DU Lixin . CIB1 cDNA Cloning from Small Tail Han Sheep and Its Expression in E.coli[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2009, 40(12): 1712-1717.

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