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

【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 has highest homology to that of higher animals. This gene was highly expressed in both lungs of Chinese giant salamander and E. coli BL21 in vitro.