鼠伤寒沙门菌cya基因的克隆表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.06.007

摘要/Abstract

摘要： 研究旨在克隆鼠伤寒沙门菌cya基因并对其进行生物信息学分析。以鼠伤寒沙门菌SL1344株为模板PCR扩增并克隆了鼠伤寒沙门菌cya基因，构建原核表达质粒pET-32a-cya，转化大肠杆菌BL21（DE3）感受态细胞，用1 mmol/L IPTG对其进行诱导表达、纯化，同时利用相关的生物信息学软件对其进行生物信息学分析。结果显示，鼠伤寒沙门菌cya基因大小为1 185 bp。经SDS-PAGE和Western blotting分析显示，在原核表达系统中主要以包涵体形式存在，蛋白分子质量大小约为58 ku。生物信息学分析表明，Cya蛋白由394个氨基酸组成，理论分子质量为44.97 ku，分子式为C₂₀₂₆H₃₁₄₆N₅₆₂O₅₇₈S₁₁，等电点（pI）为7.01，其中带负电荷残基总数（Asp+Glu）43个，带正电荷残基总数（Arg+Lys）42个。Cya蛋白无信号肽与跨膜区，为膜内蛋白，含有4个N-糖基化位点、1个O-糖基化位点、27个磷酸化位点、13个线性B细胞结合位点、11个T细胞结合位点。二级结构中α-螺旋、延伸链、β-转角、无规则卷曲所占比例分别为38.07%、19.80%、5.58%和36.55%。本试验结果为进一步研究鼠伤寒沙门菌Cya蛋白的功能及建立以Cya蛋白为抗原的免疫鉴别检测方法奠定了基础。

关键词: 鼠伤寒沙门菌; cya基因; 原核表达; 生物信息学分析

Abstract: The aim of this study was to clone and bioinformatics analyze cya gene of Salmonella Typhimurium.cya gene was amplified from the Salmonella Typhimurium SL1344 strain by PCR and the sequence was cloned into pET-32a (+) vector for expression in Escherichia coli BL21 (DE3) cells and induced by addition of 1 mmol/L IPTG.At the same time,online bioinformatics software was used to analyze its bioinformatics.The results showed that cya gene from Salmonella Typhimurium was 1 185 bp.SDS-PAGE and Western blotting results showed that Cya expressed in the prokaryotic expression system was a inclusion body protein with a molecular weight of about 58 ku.Bioinformatics analysis results showed that cya gene of Salmonella Typhimurium was encoding a protein of 394 amino acids.The theoretical molecular weight of Cya protein was 44.97 ku,the molecular formular was C₂₀₂₆H₃₁₄₆N₅₆₂O₅₇₈S₁₁,and the isoelectric point(pI) was 7.01.The total number of negatively charged residues (Asp+Glu) and positively charged residues (Arg+Lys) were 43 and 42,respectively.Cya protein was a membrane protein without signal peptide and transmembrane region,containing 4 N-glycosylation sites,1 O-glycosylation sites,27 phosphorylation sites,13 linear B-cell epitopes and 11 T-cell epitopes.The secondary structure of Cya protein contained alpha helix (38.07%),extended chain (19.80%),beta turn (5.58%) and random coil (36.55%).The results laid a foundation for further study on the function of Cya protein of Salmonella Typhimurium and the establishment of an immunoassay method using Cya protein as antigen.

Key words: Salmonella Typhimurium; cya gene; prokaryotic expression; bioinformatics analysis

中图分类号:

S852.6

茹鹏辉, 路霞, 陈耀华, 廖成水, 余祖华, 程相朝, 张春杰. 鼠伤寒沙门菌cya基因的克隆表达及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(6): 1948-1956.

RU Penghui, LU Xia, CHEN Yaohua, LIAO Chengshui, YU Zuhua, CHENG Xiangchao, ZHANG Chunjie. Cloning,Expression and Bioinformatics Analysis of cya Gene from Salmonella Typhimurium[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 1948-1956.

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