黄色瘤胃球菌纤维小体脚手架蛋白ScaC基因的克隆及其在大肠杆菌中的表达

doi:10.16431/j.cnki.1671-7236.2019.07.005

Abstract

Abstract:

To obtain the modules used to assembly the cellulosomes,the total microbial genomic DNA of Tan sheep were used to amplify the scaffoldin coding gene ScaC using a pair of primers designed according to the published sequences of Ruminococcus flavefaciens ScaC gene (GenBank accession No.:JN109634.1).The scaffoldin coding gene was amplified,cloned and sequenced,and the nucleotide and amino acid sequences were analyzed.Meanwhile,the expression of the target genes in E.coli was analyzed.The results showed that two scaffolding protein coding genes were successfully cloned using a pair of primers.One of them was 867 bp in length,encoding 288 amino acids,named as ScaC2,and the other was 870 bp in length,encoding 289 amino acids,named as ScaC7.Nucleotide sequence analysis results showed that the similarity of ScaC2 and ScaC7 genes was 74.1%.The similarity of ScaC2 and ScaC7 genes with R.flavefaciens isolate AGY80P318 and DA640P037 ScaC genes were 99% respectively.Conservative amino acid sequence analysis showed that both ScaC2 and ScaC7 amino acid sequences had typical scaffold protein domain,including a type Ⅰ adhesion domain and a type Ⅰ anchorage domain.Both genes were successfully expressed in Escherichia coli BL21 (DE3) and the expression products showed a molecular weight of 33 ku by SDS-PAGE.The two genes could be used as basic materials for the reconstruction of mini-cellulosomes in the future.

Key words: cellulosome; scaffoldin; cloning; expression

CLC Number:

S816.3

CAO Pinghua, LI Xiaoxia, ZHAO Longmei, WU Xiaohong, XU Huiying, MA Yulong. Cloning of Scaffoldin Gene ScaC from Cellulosome in Ruminococcus flavefaciens and Its Expression in Escherichia coli[J]. , 2019, 46(7): 1917-1925.

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Cloning of Scaffoldin Gene ScaC from Cellulosome in Ruminococcus flavefaciens and Its Expression in Escherichia coli

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