松材线虫Hsp70基因的克隆与原核表达

 热激蛋白70(Hsp70)是已知热休克蛋白家族中最重要的-种, 它在细胞内的大量表达可以明显改善细胞的生存能力, 提高对环境胁迫或伤害的耐受性。采用RACE-PCR技术, 从松材线虫(Bursaphelenchus xylophilus)中克隆了Hsp70基因的全长cDNA序列(共2 061 bp)(GenBank登录号为:DQ785812)。其编码-个分子量为70 kD的642个氨基酸的蛋白序列, 含有3段Hsp70家族的签名序列。同源性分析表明, 氨基酸序列与其它真核生物的Hsp70序列具有很高的相似性, 并与秀丽隐杆线虫(Caenorhabditis elegans)热激蛋白70家族中的hsp-1基因编码的氨基酸序列更为相似。因此, 将克隆的松材线虫Hsp70基因命名为Bx-hsp-1。构建了-个原核表达载体Bx70pEASY-E1, 当IPTG终浓度为0.4~0.8 mmol/L时, 能诱导表达融合蛋白。Bx-hsp-1基因的克隆和表达, 将为松材线虫的生态适应性机理研究奠定基础。

Cloning and prokaryotic expression of Hsp70 from pine wood nematode (Bursaphelenchus xylophilus)

Heat shock protein 70(Hsp70) is one of the important heat shock proteins.Overexpression of Hsp70 may obviously improve the survivability of cell,enhance tolerance to stress or injury.The rapid amplification of cDNA ends(RACE) method was used for the isolation of full length cDNA(2 061 bp) of Hsp70 from Bursaphelenchus xylophilus(GenBank accession number: DQ785812).It encoded a 642-amino acid protein with a calculated molecular weight of 70 kD,which carried three important and intact Hsp70 signature sequences.The result of sequence similarity analysis revealed that the translated molecules showed high homo-logy to other eukarya,and it was the likely orthologue of the Caenorhabditis elegans protein,Hsp70A,also known as hsp-1.So,we had designated Bx-hsp-1 for B.xylophilus Hsp70 gene.The complete ORF encoding Bx-hsp-1 was inserted into pEASY-E1 and an expression vector Bx70pEASY-E1 was constructed.The Bx-hsp-1 protein was induced to express by 0.4-0.8 mmol/L IPTG.This study provided a fundamental condition supporting research on mechanisms of the ecological adaptability of B.xylophilus.