血杆菌C4几丁质酶基因的克隆与表达

TAO Yong , JIN Hong , LONG Zhang-Fu , ZHANG Li , DING Xiu-Qiong , TAO Ke , LIU Shi-Gui
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引用次数: 9

摘要

几丁质酶Chi58是一种胞外几丁质酶,由血杆菌属菌株C4产生。采用基因特异性PCR引物检测菌株C4中chiA基因的存在。从C4基因组DNA中扩增出一个chiA片段(chiA- f),并用于从GenBank数据库中blast-search相关序列。通过比对和选择同源序列的高度保守区,设计了两对引物,利用巢式PCR扩增菌株C4几丁质酶的开放阅读框(ORF)。结果表明,Chi58 ORF由1 692个核苷酸组成,编码563个氨基酸残基的蛋白。成熟蛋白的分子量为58.544 kDa。Chi58 ORF是一种模块化酶,由信号肽序列、多囊肾病I结构域和糖基水解酶家族18结构域组成。C4的几丁质酶在氨基酸序列水平上与沙雷氏菌的几丁质酶a具有高度的相似性(88.9% ~ 99.6%)。将Chi58基因克隆到表达载体pET32a中,构建重组质粒pChi58,经IPTG诱导在大肠杆菌bl21 (DE3)细胞中表达。经SDS-PAGE鉴定,Trx-Chi58融合蛋白分子量为81.1 kDa。
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Cloning and Expression of a Chitinase Gene from Sanguibacter sp. C4

The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank dadabase. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%–99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.

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