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

摘要/Abstract

摘要：

为获得纤维小体（cellulosome）的基本元件，本试验以滩羊瘤胃液微生物混合DNA为模板，根据黄色瘤胃球菌纤维小体的脚手架蛋白ScaC基因序列（GenBank登录号：JN109634.1）设计特异性引物，扩增纤维小体脚手架蛋白基因，并对其进行克隆、测序及核苷酸和氨基酸序列分析；同时构建脚手架蛋白基因原核表达载体，分析其在大肠杆菌中的表达情况。结果显示，试验利用1对引物同时成功克隆获得2个脚手架蛋白编码基因，其中一个基因全长867 bp，编码288个氨基酸，命名为ScaC2基因；另一个基因全长870 bp，编码289个氨基酸，命名为ScaC7基因。核苷酸序列分析表明，脚手架蛋白基因ScaC2与ScaC7的核苷酸序列相似性为74.1%，ScaC2基因与黄色瘤胃球菌AGY80P318及ScaC7基因与黄色瘤胃球菌DA640P037 ScaC基因核苷酸序列相似性均为99%；氨基酸保守序列分析显示，ScaC2和ScaC7氨基酸序列均具有典型的脚手架蛋白结构域，含有1个Ⅰ型黏附域和1个Ⅰ型锚定域。蛋白表达分析显示，ScaC2和ScaC7基因均能在大肠杆菌中实现可溶性表达，表达产物大小约为33 ku。本试验克隆获得的纤维小体的脚手架蛋白基因ScaC2和ScaC7可为后续人工纤维小体的构建提供基本材料。

关键词: 纤维小体; 脚手架蛋白; 克隆; 表达

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

中图分类号:

S816.3

曹平华, 李晓霞, 赵龙妹, 武晓红, 许会英, 马禹龙. 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[J]. 《中国畜牧兽医》, 2019, 46(7): 1917-1925.

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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