太平洋牡蛎i型溶菌酶基因的克隆与重组高效表达  

Molecular Cloning and Recombinant Expression of i-type Lysozyme from Oyster Crassostrea gigas

作　　者：谢三群[1] 丛丽娜[1] 张欢[1] 王丹[1] 

机构地区：[1]大连工业大学生物工程学院,大连116034

出　　处：《生物技术通报》2011年第4期127-132,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31072224);辽宁省教育厅创新团队项目(LT2010012);大连市科技计划项目(2009J31SC034)

摘　　要：在双壳类软体动物牡蛎体内,溶菌酶(Lysozyme)在实现宿主免疫防御,破坏和消除侵入体内的病原中发挥着重要的作用。根据GenBank已有的太平洋牡蛎溶菌酶的全长cDNA序列(GenBank:AB179775),通过RT-PCR技术,从太平洋牡蛎(Crassostrea gigas)中克隆得到溶菌酶(简称为CgLys)基因的开放阅读框(open reading frame,ORF)序列。生物信息软件分析表明,其ORF为414 bp,编码137个氨基酸(aa),前20个aa为信号肽,成熟肽由117个aa组成,其分子量为13.2 kD。通过构建分子系统发育树对其同源性进行分析比较,初步推断该CgLys属于i型溶菌酶。将该CgLys基因的成熟肽亚克隆进原核表达载体pET-32a(+)中,构建重组质粒pET32a(+)-CgLys,再转化至大肠杆菌BL21(DE3)pLysS。该基因工程菌经IPTG诱导发酵后,成功高效地表达了重组CgLys蛋白,其分子质量约为18 kD。该重组CgLys蛋白主要存在于细菌裂解液的上清液中,即以可溶性蛋白形式存在。上述结果将显著简化后续的蛋白纯化过程,为今后扩大规模生产牡蛎溶菌酶提供参考。In bivalve mollusks including oysters,lysozymes play an important role in the host defense mechanisms against invading microbes.Based on the full-length cDNA of C.gigas lysozyme in GenBank（GenBank：AB179775）,the lysozyme gene with open reading frame（ORF）from C.gigas was cloned by the RT-PCR method and named Cg-lysozyme（CgLys in short）.Bioinformatic software analysis showed that the ORF with the length of 414 bp encoded 137 amino acids（aa）,including a signal peptide of 20 aa and at N-terminus and a mature peptide of 117 aa,and the molecular weight of the mature protein was of 13.2 kD.Phylogenetic analysis showed that the lysozyme from C.gigas clustered with the i-type lysozyme of other bivalve species.The DNA fragment of mature CgLys was subcloned into pET-32a（＋）expression vector to construct the recombinant plasmid of pET32a（＋）-CgLys.Next,the recombinant plasmid was transformed into Escherichia coli BL21（DE3）pLysS and then induced by isopropylthio-β-D-galactoside（IPTG）.As a result,a new anticipated fusion protein of CgLys（ca.18 kD）was strongly expressed on the gel of SDS-PAGE.Further analysis showed that the expressed fusion protein existed as a soluble form.These results could simplify purification steps and facilitate the production of oyster lysozyme in a large scale.

关 键 词：无脊椎动物 太平洋牡蛎 i型溶菌酶 重组表达 纯化 

分 类 号：Q78[生物学—分子生物学]