卤虫泌壳腺特异表达蛋白SGEG1的原核表达
首发时间:2019-04-19
摘要:卤虫(丰年虾)是一种能在极端环境下生存的小型海洋动物,其休眠卵可以在干燥、缺氧的环境中保持长久活力,并且在合适条件下发育成幼虫,卤虫依靠其休眠状态渡过极端环境。卤虫泌壳腺特异表达蛋白(Shell Gland-specifically Expresses Gene, SGEG)是休眠卵壳蛋白的重要组成部分,是卵生卤虫卵囊泌壳腺分泌型细胞上特异性表达的基因,参与卤虫在极端环境中的抗逆休眠机制。本文将卤虫SGEG1基因成熟肽序列克隆到带有6×His标签的原核表达载体pET-28a(+)中,构建含pET-28a(+)-SGEG1重组质粒,转化至大肠杆菌 E.coli BL21(DE3)中,诱导表达重组蛋白。通过对诱导的IPTG浓度、诱导时间和蛋白电泳检测方式的优化,确定最佳表达条件和蛋白电泳检测方法。本研究成功构建pET-28a(+)-SGEG1重组载体,实现分子量约为10 KDa小分子重组SGEG1蛋白的高效表达,其融合蛋白主要以包涵体形式存在,为开展SGEG1蛋白的体外制备奠定了基础。
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Prokaryotic Expression of SGEG1 Protein from Artemia Diapause Cysts
Abstract:Artemia is a small marine animal survived in extreme environments. Its diapause cysts can maintain long-term vitality in dry and anoxic environment and develop into larvae under suitable condition. Artemia passed through the extreme environment by its dormancy state. The shell gland-specifically expresses gene (SGEG) is specifically expressed in secretory cells of shell gland of oviparous Artemia\'s ovisac and SGEG peptide is an important componenProkaryotic Expression of SGEG1 Protein from Artemia Diapause Cystst of diapause cysts shell protein. In this study, the SGEG1 gene sequence of deduced mature peptide was cloned into the prokaryotic expression vector pET-28a (+) with 6×His tag. A recombinant plasmid containing pET-28a (+)-SGEG1 was successfully constructed and transformed into E. coli BL21(DE3). The fusion expression of recombinant SGEG1 (rSGEG1) protein was induced. Meanwhile, the induced IPTG concentration, induction time of rSGEG1 and the method of protein electrophoresis were optimized. The recombinant SGEG1 peptide with a molecular weight of about 10 KDa was highly expressed,and the fusion protein mainly existed in the form of inclusion body, which supported a foundation for the preparation of SGEG1 protein in vitro.
Keywords: Artemia SGEG1 prokaryotic expression
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