羊源类鼻疽伯克霍尔德菌BPSS1512基因克隆、原核表达及其蛋白的生物信息学分析

doi:10.16431/j.cnki.1671-7236.2017.08.003

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (8): 2234-2240.doi: 10.16431/j.cnki.1671-7236.2017.08.003

羊源类鼻疽伯克霍尔德菌BPSS1512基因克隆、原核表达及其蛋白的生物信息学分析

曹瑞勇, 聂鑫, 李宝宝, 张振兴, 黄海峰, 李亚颖, 彭冬梅, 李国华, 朱姝, 杨小健, 杜丽, 王凤阳

海南大学热带农林学院, 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

收稿日期:2017-04-13 出版日期:2017-08-20 发布日期:2017-08-18
通讯作者: 王凤阳 E-mail:fywang68@163.com
作者简介:曹瑞勇(1991-),男,内蒙古赤峰人,硕士生,研究方向:动物疾病防控,E-mail:2217497682@qq.com
基金资助:
国家肉羊产业技术体系（CARS-38）；海南省重大科技计划项目（ZDKJ2016017-01）

Cloning,Prokaryotic Expression of BPSS1512 Gene in Goat Burkholderia pseudomallei and Bioinformatics Analysis of its Proteins

CAO Rui-yong, NIE Xin, LI Bao-bao, ZHANG Zhen-xing, HUANG Hai-feng, LI Ya-ying, PENG Dong-mei, LI Guo-hua, ZHU Shu, YANG Xiao-jian, DU Li, WANG Feng-yang

Key Laboratory of Animal Genetic Engineering of Haikou City, Key Laboratory of Tropical Animal Reproduction & Breeding and Epidemic Disease Research of Hainan Province, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China

Received:2017-04-13 Online:2017-08-20 Published:2017-08-18

摘要/Abstract

摘要：

为克隆羊源类鼻疽伯克霍尔德菌BPSS1512基因，并对其编码的蛋白进行生物信息学分析，以类鼻疽伯克霍尔德菌基因组为模板，参照GenBank中Burkholderia pseudomallei K96243株基因组DNA序列（登录号：NC_006351.1）设计引物，PCR扩增BPSS1512基因，构建重组质粒，SDS-PAGE和Western blotting分析其蛋白表达，DNAMAN等软件对BPSS1512基因编码的氨基酸序列进行分析。结果显示，PCR扩增成功得到1 425 bp的特异性条带，BamHⅠ和Hind Ⅲ双酶切后得到约为5 000和1 500 bp的条带，表明重组质粒pET-28a-BPSS1512构建成功，IPTG浓度为10 mmol/L，诱导时间8 h为最适宜的诱导条件。BPSS1512基因编码的蛋白质分子质量为53 ku，在包涵体中表达；在BPSS1512蛋白二级结构中，α-螺旋、延伸链和无规卷曲分别占24.05%、14.77%、61.18%，并且疏水性区域分布在-2.0~+2.4之间，说明BPSS1512蛋白具有较强的疏水性，本试验结果可为类鼻疽病的防制提供参考依据。

关键词: 类鼻疽伯克霍尔德菌; BPSS1512基因; 克隆; 原核表达; 生物信息学分析

Abstract:

The experiment was aimed to study the clone and prokaryotic expression of BPSS1512 gene in goat Burkholderia pseudomallei and analyzed its proteins by bioinformatics. The geneome of Burkholderia pseudomallei was used as the template,and the primers were designed by DNAMAN software referring to genomic DNA sequence of Burkholoderia pseudomallei K96243 strain in GenBank (NC_006351.1).The BPSS1512 gene was amplified by PCR and the recombinant plasmid was constructed. Then the expressed protein was analyzed by SDS-PAGE and Western blotting, and the amino acid sequence encoded by BPSS1512 gene was analyzed by softwares such as DNAMAN.The results showed that the BPSS1512 gene was successfully cloned with the length of 1 425 bp,and the recombinant plasmid pET-28a-BPSS1512 was constructed. The optimum conditions for induction was that the IPTG was 10 mmol/L and 8 h for induction.The molecular weight of the protein was 53 ku,it was expressed as the form of inclusion body.In the secondary structure of BPSS15122 protein,alpha-helix,extended strand,and random coil were 24.05%,14.77% and 61.18%, respectively,and the hydrophobic core was distributed between -2.0 and +2.4 which indicated that the BPSS1512 protein was strong hydrophobicity.

Key words: Burkholderia pseudomallei; BPSS1512 gene; clone; prokaryotic expression; bioinformatics analysis

中图分类号:

Q786

曹瑞勇, 聂鑫, 李宝宝, 张振兴, 黄海峰, 李亚颖, 彭冬梅, 李国华, 朱姝, 杨小健, 杜丽, 王凤阳. 羊源类鼻疽伯克霍尔德菌BPSS1512基因克隆、原核表达及其蛋白的生物信息学分析[J]. 《中国畜牧兽医》, 2017, 44(8): 2234-2240.

CAO Rui-yong, NIE Xin, LI Bao-bao, ZHANG Zhen-xing, HUANG Hai-feng, LI Ya-ying, PENG Dong-mei, LI Guo-hua, ZHU Shu, YANG Xiao-jian, DU Li, WANG Feng-yang. Cloning,Prokaryotic Expression of BPSS1512 Gene in Goat Burkholderia pseudomallei and Bioinformatics Analysis of its Proteins[J]. , 2017, 44(8): 2234-2240.

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羊源类鼻疽伯克霍尔德菌BPSS1512基因克隆、原核表达及其蛋白的生物信息学分析

Cloning,Prokaryotic Expression of BPSS1512 Gene in Goat Burkholderia pseudomallei and Bioinformatics Analysis of its Proteins

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