CAJ | 학술논문

Chi58是Sanguibacter sp.strain C4产生的一种胞外几丁质酶.通过chiA的特异性PCR引物探测到菌株C4中存在几丁质酶,并将扩增到的几丁质酶基因片段(chiA-F)克隆、测序后,提交GenBank数据库进行同源性搜索.对从GenBank中获得的高同源性序列进行比对,并根据保守区域设计2对PCR引物进行嵌套PCR,扩增出Chi58基因的开放阅读框(ORF).测序结果表明该酶的ORF由1692个核苷酸组成,编码563个氨基酸,在N端有23个氨基酸的信号肽,其成熟蛋白的分子量应为58.544 kDa.对其推导氨基酸的序列分析表明Chi58与沙雷氏菌的几丁质酶(chiA)有高度同源性(88.9%～99.6%),其结构主要包括信号肽序列、PKD结构域和18家族糖苷水解酶结构域.将该基因克隆到pET32a(+)载体构建重组质粒pChi58,转入大肠杆菌BL-21(DE3)进行融合表达.经IPTG诱导后,可见分子量约81.1 kDa的融合蛋白的表达.
Chi58시Sanguibacter sp.strain C4산생적일충포외궤정질매.통과chiA적특이성PCR인물탐측도균주C4중존재궤정질매,병장확증도적궤정질매기인편단(chiA-F)극륭、측서후,제교GenBank수거고진행동원성수색.대종GenBank중획득적고동원성서렬진행비대,병근거보수구역설계2대PCR인물진행감투PCR,확증출Chi58기인적개방열독광(ORF).측서결과표명해매적ORF유1692개핵감산조성,편마563개안기산,재N단유23개안기산적신호태,기성숙단백적분자량응위58.544 kDa.대기추도안기산적서렬분석표명Chi58여사뢰씨균적궤정질매(chiA)유고도동원성(88.9%～99.6%),기결구주요포괄신호태서렬、PKD결구역화18가족당감수해매결구역.장해기인극륭도pET32a(+)재체구건중조질립pChi58,전입대장간균BL-21(DE3)진행융합표체.경IPTG유도후,가견분자량약81.1 kDa적융합단백적표체.
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 563amino 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.