大鲵胸腺素β-4全长基因的分离与表达研究  

Isolation and expression analysis of thymosin β-4 in Andrias davidianus

作　　者：艾闽[1,2] 杨辉[1,2] 胡伟[1,2] 李锋刚[1,2] 刘小林[1,2] 王立新[1,2] 

机构地区：[1]西北农林科技大学动物科技学院水产科学系,陕西杨凌712100 [2]陕西省农业分子生物学重点实验室,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2013年第1期1-8,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：陕西省农业科技攻关项目(2012K01-18);西北农林科技大学基本科研业务费专项(QN2011062);西北农林科技大学后稷学者专项(Z11021007)

摘　　要：【目的】对大鲵胸腺素β-4基因进行生物信息学分析和原核表达载体构建,为大鲵胸腺素β-4蛋白生理活性的深入研究奠定基础。【方法】从大鲵皮肤cDNA文库中分离获得了大鲵胸腺素β-4基因全长序列,采用多种生物软件对其进行生物信息学分析,用定量PCR研究胸腺素β-4基因在不同组织的表达情况,并以pET32a为载体构建该基因原核表达载体。【结果】胸腺素β-4基因全长共699bp,其中开放阅读框135bp,5′-UTR 87bp,3′-UTR为474bp,该基因编码45个氨基酸,表达蛋白的理论分子质量为5 141.59u,等电点为5.34;结构分析发现,大鲵胸腺素β-4蛋白氨基酸序列的第7～43位处有"THY"特征模体,没有跨膜螺旋区、细胞膜外在跨膜结构域。氨基酸序列进化关系表明,大鲵与人、大鼠、牛、猩猩、鸡、倭蛙等动物亲缘关系较近,氨基酸同源性高于85%;与猪和爪蟾的亲缘关系次之,氨基酸同源性均为83%;而与虹鳟、大菱鲆、斑马鱼等鱼类的亲缘关系较远,氨基酸同源性低于80%。荧光定量PCR检测结果表明,胸腺素β-4基因在大鲵肺中的相对表达量最高。SDS-PAGE电泳结果表明,构建的大鲵胸腺素β-4原核表达重组菌株pET32a-Tβ4,在37℃条件下,用终浓度1.0mmol/mL IPTG诱导4h后,大鲵胸腺素β-4基因获得了较高的表达。【结论】分离获得了大鲵胸腺素β-4基因全长cDNA序列,该基因氨基酸序列与人等高等动物的同源性最高,能在大鲵肺中及大肠杆菌中获得高效表达。[Objective] Bioinformatics analysis and prokaryotic expression vector construction were conducted for investigating the physiological activity of Chinese giant salamander＇ s thymosin β-4 gene. [Method] In this case,the thymosin β-4 cDNA sequence was isolated and identified from skin cDNA library of Chinese giant salamander,and a variety of biological softwares were used to analyze the bioinformatics of Chinese giant salamander＇ thymosin β-4 gene. Then the real-time PCR was applied to study the expression in various tissues, and the prokaryotic expression vector was reconstructed with pET32a. [Result] The full- length of thymosin β-4 gene of Chinese giant salamander was 699 bp,among which the ORF, 5＇-UTR, and 3 ＇-UTR were 135 bp（88--223 bp） ,87 bp,and 474 bp,respectively. The gene code 45 contained amino acids with a deduced molecular weight of 5 141.59 u and an isoelectric point of 5.34. The results of structure a- nalysis showed that the thymosin β-4 protein contained a ＂THY＂ specific motif, but neither transmembrane helix nor transmembrane domain existed. The analysis of genetic relationship with amino acids of thymosin β-4 showed that Chinese giant salamander was close to human, rat, cattle, Pongo satyrus, chicken and frog （the amino acids homology was higher than 85%）,followed by swine and xenopus laevis（the amino acids homology was higher than 83%）, while far away from rainbow, turbot and zebrafish（the amino acids ho- mology was under 80%）. The result of fluorescent quantitation PCR indicated that the highest-expression of thymosin β-4 gene was in the lungs of Chinese giant salamander. SDS-PAGE showed the recombinant thymosin β-4 protein of Chinese giant salamander was highly expressed by the reconstruct bacterial strain pET32a-Tβ4 induced by 1.0 mmol/mL IPTG at 37 ℃ for 4 h. [Conclusion] In this study,the full-length cDNA sequence of thymosinβ-4 gene was isolated and identified from the skin cDNA library of Chinese gi- ant salamander. The detected amino acids sequence

关 键 词：大鲵 胸腺素β-4基因 分离与表达 

分 类 号：Q786[生物学—分子生物学] S917[农业科学—水产科学]