| 羊源类鼻疽伯克霍尔德菌BPSL1467基因克隆、表达及其蛋白生物信息学分析 |
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| 引用本文： | 张萌萌,李宝宝,曹瑞勇,黄海峰,张振兴,杨小健,聂鑫,朱姝,王凤阳,杜丽.羊源类鼻疽伯克霍尔德菌BPSL1467基因克隆、表达及其蛋白生物信息学分析[J].中国畜牧兽医,2018,45(9):2401-2408. |
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| 作者姓名： | 张萌萌 李宝宝 曹瑞勇 黄海峰 张振兴 杨小健 聂鑫 朱姝 王凤阳 杜丽 |
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| 作者单位： | 海南大学热带农林学院动物科技学院, 海南省热带动物繁育与疫病研究重点实验室, 海口市动物基因工程重点实验室, 海口 570228 |
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| 基金项目： | 海南省重大科技计划项目（ZDKJ2016017-01）；国家肉羊产业技术体系（CARS-38）；中央领导地方科技发展专项资金项目（ZY2017HN07） |
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| 摘 要： | 试验旨在克隆和表达羊源性类鼻疽伯克霍尔德菌（Burkholderia pseudomallei，B.pseudomallea）的BPSL1467基因，并对其表达的蛋白进行生物信息学分析。以羊源类鼻疽伯克霍尔德菌（BPHN1株）基因组为模板，参照GenBank中类鼻疽伯克霍尔德菌K96243标准株BPSL1467基因序列设计引物，PCR扩增获得目的基因片段，将所得片段与pET-28a（+）载体连接，构建pET-28a（+）-BPSL1467重组质粒。将鉴定正确的pET-28a（+）-BPSL1467重组质粒转化大肠杆菌BL21（DE3）感受态细胞，通过IPTG诱导表达，SDS-PAGE和Western blotting鉴定表达产物。使用DNAMAN、ProtParam、SOPMA和Protscale相关生物信息学软件对BPSL1467基因编码的氨基酸序列进行分析。结果显示，本试验成功克隆了462 bp的BPSL1467基因，诱导表达重组蛋白大小约为22 ku，主要以包涵体的形式存在。BPSL1467蛋白分子式为C763H1209N203O217S6，分子质量为16 890.58 u；其不稳定系数为33.95，属于稳定蛋白；理论等电点（pI）为8.85，为碱性蛋白；总平均疏水性（GRAVY）为-0.190，为亲水性蛋白。该蛋白的二级结构中以无规则卷曲和α-螺旋为主。本试验结果为深入研究羊源类鼻疽伯克霍尔德菌的BPSL1467基因的分子作用机理提供了参考依据。
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| 关 键 词： | 类鼻疽伯克霍尔德菌 BPSL1467基因 克隆 原核表达 生物信息学分析 |
| 收稿时间： | 2018-01-22 |
Cloning,Expression and Protein Bioinformatics Analysis of BPSL1467 Gene of Burkholderia pseudomallei |
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| ZHANG Mengmeng,LI Baobao,CAO Ruiyong,HUANG Haifeng,ZHANG Zhenxing,YANG Xiaojian,NIE Xin,ZHU Shu,WANG Fengyang,DU Li.Cloning,Expression and Protein Bioinformatics Analysis of BPSL1467 Gene of Burkholderia pseudomallei[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2401-2408. |
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| Authors: | ZHANG Mengmeng LI Baobao CAO Ruiyong HUANG Haifeng ZHANG Zhenxing YANG Xiaojian NIE Xin ZHU Shu WANG Fengyang DU Li |
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| Institution: | Key Laboratory of Animal Genetic Engineering of Haikou City, Key Laboratory of Tropical Animal Reproduction & Breeding and Epidemic Disease Reseach of Hainan Province, College of Animal Science and Technology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China |
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| Abstract: | This study was aimed to clone and express BPSL1467 gene of Burkholderia pseudomallei (B.pseudomallea),and performe bioinformatics analysis of its protein.A pair of primers was designed according to the BPSL1467 gene sequence of B.pseudomallea K96243 strain in GenBank.BPSL1467 gene fragment of B.pseudomallea BPHN1 strain was amplified by PCR amplification.The BPSL1467 gene fragment was ligated into the pET-28a(+) vector to construct the pET-28a(+)-BPSL1467 recombinant plasmid.Then,the pET-28a(+)-BPSL1467 was transformed into E.coli BL21 (DE3) competent cells.The expressed product induced by IPTG was analyzed by SDS-PAGE and Western blotting.Bioinformatics analysis of BPSL1467 gene sequence was carried out using DNAMAN,ProtParam,SOPMA and Protscale softwares.The results showed that BPSL1467 gene was cloned with the length of 462 bp.The expressed recombinant protein was about 22 ku and was mainly in the form of inclusion body;The molecular weight of the BPSL1467 recombinant protein was 16 890.58 u (C763H1209N203O217S6);Its instability coefficient,theoretical isoelectric point (pI) and total average hydrophobicity (GRAVY) were 33.95,8.85 and -0.190 respectively,which was a stable hydrophilic basic protein;The secondary structure of the protein was mainly random coil (42.48%) and alpha helix (32.68%).This results provided a reference for the further study of molecular mechanism of Burkholderia pseudomallei BPSL1467 gene. |
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| Keywords: | Burkholderia pseudomallei BPSL1467 gene cloning prokaryotic expression bioinformatics analysis |
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