八字地老虎β-N-乙酰葡萄糖胺糖苷酶基因cDNA序列的克隆及原核表达研究

cDNA cloning and prokaryotic expression of β-N acetylglucosaminidase from Xestia c-nigrum

[目的]克隆八字地老虎β-N-乙酰葡萄糖胺糖苷酶基因的cDNA序列并进行原核表达。[方法]以八字地老虎预蛹期幼虫为材料提取总RNA,利用RT-PCR和RACE技术克隆基因cDNA序列,将cDNA序列编码区连接到原核表达载体pET21b并转化BL21进行原核表达,并使用His-tag纯化试剂盒将目的蛋白进行纯化。[结果]得到cDNA全长序列,含有2 488个碱基,包括一个1 785个碱基的开放阅读框,编码一个含594个氨基酸的蛋白,分子量为67.8 ku,等电点5.38,该序列登录GenBank并获得登录号FJ848784。诱导表达蛋白经SDS-PAGE电泳得到目的蛋白带,纯化结果也获得了目的蛋白带。[结论]获得了一条新的八字地老虎β-N-乙酰葡萄糖胺糖苷酶基因的cDNA序列并对其在原核表达系统中进行了成功表达和纯化。

[Objective] To clone β-N-acetylglucosaminidase cDNA sequence from Xestia c-nigrum Linnaeus and express this sequence in prokaryotic expression system. [Methods] Total RNA was isolated from the prepupal X. c-nigrum. The cDNA sequence was cloned by RT-PCR and rapid amplification of cDNA ends（RACE）. The encoding region was subcloned into the expression vector pET21b and expressed in BL21 cells. The recombinant protein with 6）〈 His tag was purified. [Results] The β-N-acetylglucosaminidase full-length cDNA sequence was obtained. The cDNA sequence of 2 488 base pairs in length contained an open reading frame of 1 785 base pairs, encoding for a polypeptide of 594 amino acid residues, with a predicted molecular weight of 67.8 ku and pI 5.38. The cDNA sequence has been deposited in GenBank with accession No. FJ848784. [Conclusion] A novel β-N- acetylglucosaminidase cDNA sequence was successfully cloned, expressed and purified from Xestia c-nigrum.