凡纳滨对虾溶菌酶基因在大肠杆菌中的表达和活性检测

Expression of Litopenaeus vannamei lysozyme gene in Escherichia coli and evaluation of its lytic activity

将凡纳滨对虾(Litopenaeus vannamei)血淋巴细胞中提取的总RNA,经RT-PCR扩增溶菌酶基因(LvLys基因)的开放阅读框,将其克隆至pMD18-T并测序。用限制性内切酶BamHⅠ和HindⅢ双酶切取目的基因并与表达载体pET-28a(+)连接,构建重组质粒pET-28(a+)/LvLys,转移到BL21(DE3)中诱导表达。经亲和纯化得到凡纳滨对虾重组溶菌酶。抑菌活性检测表明重组溶菌酶对大肠杆菌TOP10有一定抑制作用。

The total RNA extracted from the hemocyte sample ofLitopenaeus vannamei （L. Vannamei） was used to amplify the sequence encoding an open reading frame for lysozyme gene （called LvLys gene） by RT-PCR. The sequence was then cloned into pMD18-T vector and was sequenced . The recombinant plasmid was sequenced and digested by BamH Ⅰ and Hind Ⅲ. The target gene was subsequently connected to the pET-28a （＋） vector, which was digested with the corresponding restriction endonuclease. The recombinant plasmid pET-28a（＋）/LvLys was transformed into Escherichia coli BL21（DE3） and then induced by isopropylthio-β-D-galactoside （IPTG） and purified through chelating affinity, finally recombinant LvLys protein was analyzed and lytic activity was assayed. The recombinant protein showed a certain antibacterial activity against Escherichia TOP10.