布鲁氏菌外膜蛋白2b基因的克隆、原核表达及蛋白生物信息学分析

中国畜牧兽医 ›› 2014, Vol. 41 ›› Issue (9): 11-14.

布鲁氏菌外膜蛋白2b基因的克隆、原核表达及蛋白生物信息学分析

赵天靖, 贾晓晓, 焦寒伟, 朱华培, 徐开莲, 郭莳雨, 史巧芸, 荣辉, 成鹰, 张珈宁, 庞峰, 杜丽, 王凤阳

海南大学农学院, 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, 海南海口 570228

收稿日期:2014-04-24 出版日期:2014-09-20 发布日期:2014-09-24
通讯作者: 王凤阳 E-mail:fywang68@163.com
作者简介:赵天靖（1991- ），女，河南人，硕士生，研究方向：动物功能基因组学。
基金资助:
“863”计划（2011AA100302、2013AA102524）。

Cloning, Prokaryotic Expression and Bioinformatics Analysis of Omp2b Gene of Brucella melitensi

ZHAO Tian-jing, JIA Xiao-xiao, JIAO Han-wei, ZHU Hua-pei, XU Kai-lian, GUO Shi-yu, SHI Qiao-yun, RONG Hui, CHENG Ying, ZHANG Jia-ning, PANG Feng, 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, College of Agriculture, Hainan University, Haikou 570228, China

Received:2014-04-24 Online:2014-09-20 Published:2014-09-24

摘要/Abstract

摘要： 本试验旨在克隆布鲁氏菌外膜蛋白2b（Omp2b）基因并进行原核表达和蛋白的生物信息学分析。根据布鲁氏菌M5-90株外膜蛋白Omp2b基因序列设计引物，以布鲁氏菌基因组为模板，通过PCR技术扩增得到Omp2b基因片段，回收纯化后，将此片段连接入pMD20-T质粒，将该重组质粒转化E.coli DH5α感受态细胞，挑取阳性克隆菌提取质粒后，送公司测序。将该片段亚克隆入pET28a载体，构建pET28a-Omp2b表达载体，转化E.coli BL21（DE3）菌株，IPTG诱导其表达，用SDS-PAGE和Western blotting分析鉴定此蛋白。运用DNAMAN、BioEdit等各种工具软件对Omp2b基因编码的氨基酸序列进行分析。结果显示，成功克隆了Omp2b基因，其开放阅读框为1041 bp，编码347个氨基酸；构建了pET28a-Omp2b原核表达载体，并在E.coli BL21（DE3）中成功表达了Omp2b基因，表达蛋白约38 ku；Omp2b蛋白二级结构中α-螺旋、伸展链、β-折叠和无规卷曲分别占20.17%、26.22%、5.76%和47.84%。

关键词: 布鲁氏菌; Omp2b; 克隆; 原核表达; 生物信息学分析

Abstract: The study was aimed at cloning, expressing the gene of outer membrance protein 2b (Omp2b) and analyzing the bioinformatics of Omp2b, according to the nucleotide sequence of Omp2b gene of Brucella melitensis M5-90, a pair of primers was designed, and Omp2b gene fragment was amplified from Brucella genome by PCR.The fragment was identified and cloned into prokaryotic expression vector pMD20-T. After sequencing, Omp2b fragment was ligated into pET28a to construct the recombinant expression plasmid pET28a-Omp2b, and then transformed into the E.coli BL21 (DE3) strains. IPTG was used to induce expression of Omp2b protein,and the protein was detected by SDS-PAGE and Western blotting. DNAMAN and BioEdit softwares were used to analyze the sequence of amino acids encoded by Omp2b gene. The results showed that Omp2b gene was cloned successfully; the ORF was 1041 bp, which encoded 347 amino acids; the expression vector pET28a-Omp2b was constructed; the molecular weight of the expression protein was about 38 ku; Omp2b protein secondary structure of alpha helix, extended strand, β turn and random coil were accounted for 20.17%, 26.22%, 5.76% and 47.84%, respectively.

Key words: Brucella; Omp2b; cloning; prokaryotic expression; bioinformatics analysis

中图分类号:

S852.61

赵天靖, 贾晓晓, 焦寒伟, 朱华培, 徐开莲, 郭莳雨, 史巧芸, 荣辉, 成鹰, 张珈宁, 庞峰, 杜丽, 王凤阳. 布鲁氏菌外膜蛋白2b基因的克隆、原核表达及蛋白生物信息学分析[J]. 中国畜牧兽医, 2014, 41(9): 11-14.

ZHAO Tian-jing, JIA Xiao-xiao, JIAO Han-wei, ZHU Hua-pei, XU Kai-lian, GUO Shi-yu, SHI Qiao-yun, RONG Hui, CHENG Ying, ZHANG Jia-ning, PANG Feng, DU Li, WANG Feng-yang. Cloning, Prokaryotic Expression and Bioinformatics Analysis of Omp2b Gene of Brucella melitensi[J]. , 2014, 41(9): 11-14.

参考文献

1 何亮,韩先干,胡青海,等. 牛型布鲁氏菌A19株外膜蛋白OMP2b的表达及鉴定[J]. 中国兽医科学,2010,40(9): 925～928.

2 张蕾,张亮,于澜,等. 羊布鲁氏菌外膜蛋白omp2b的克隆,原核表达及纯化[J]. 科学技术与工程,2011,11(35): 8705～8709.

3 贾晓晓,焦寒伟,郭莳雨,等. 布鲁氏菌外膜蛋白16基因的克隆机表达[J]. 中国畜牧兽医,2013,40(9): 51～54.

4 Cloeckaert A, Vizcaino N, Paquet J Y, et al. Major outer membrane proteins of Brucella spp.: Past, present and future[J]. Veterinary Microbiology, 2002, 90(1～4): 229～247.

5 Connolly J P, Comerci D, Alefantis T G, et al. Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development[J]. Proteomics, 2006, 6(13): 3767～3780.

6 Marquis H, Ficht T A. The omp2 gene locus of Brucella abortus encodes two homologous outer membrane proteins with proteins with properties characteristic of bacterial porins[J]. Infection and Immunity, 1993, 61(9): 3785～3790.

7 Mehmet D, Bilgehan A. Human brucellosis: An overview[J]. International Journal of Infectious Diseases, 2003, 7(3): 173～182.

8 Mobasheri H, Ficht T A, Marquis H, et al. Brucella Omp2a and Omp2b porins: Single channel measurements and topology prediction[J]. FEMS Microbiology Letters, 1997, 155(1): 23～30.

9 Oliveira S C, Soeurt N, Splitter G. Molecular and cellular interactions between Brucella abortus antigens and host immune responses[J]. Veterinary Microbiology, 2002, 90(1～4): 417～424.

10 Rajesh C, Mandeep S, Katoch R C, et al. Brucellosis outbreak in an organized dairy farm involving cows and in contact human beings[J]. Veterinarski Arhiv, 2003, 73(2): 95～102.

11 Salhi I, Boigegrain R A, Machold J, et al. Characterization of new members of the group 3 outer membrane protein family of Brucella spp.[J]. Infection and Immunity, 2003, 71(8): 4326～4332.

12 Schijns V E. Immunological concepts of vaccine adjuvant activity[J]. Curr Opin Immunol, 2000, 12(4): 456～463.

13 Selleem M N, Boyle S M, Sriranganathan N. Brucella: A pathogen without classic virulence genes[J]. Veterinary Microbiology, 2008, 129(1～2): 1～14.

14 Shang D Q. Progress in the study of prevention and control of Brucellosis in China in last 50 years[J]. Chinese Journal of Epidemiology, 2000, 21(1): 55～57.

[1]	焦琳琳, 成玉婷, 吴庆国, 吴双, 吴植, 朱善元, 钱莺娟. 鸭坦布苏病毒Capsid蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2395-2402.
[2]	李灿, 李楚楚, 曾维, 张宁, 纪春晓, 陈韬. 猪RegⅢγ蛋白的重组表达及功能研究[J]. 中国畜牧兽医, 2023, 50(6): 2414-2426.
[3]	宋越, 苏胜杰, 张帆, 白帆, 戴伶俐, 王娜, 王大伟, 杨茜雯, 赵世华, 张月梅. 溶血性曼氏杆菌截短型白细胞毒素的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2479-2486.
[4]	欧云文, 潘琴, 汪洋, 代军飞, 任绍科, 张洋, 张杰. 猪细小病毒6型ORF2基因的截短表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2487-2495.
[5]	刘佳隆, 张译文, 张悦然, 孙奇娟, 陈建, 何雷, 贾艳艳, 丁轲, 余祖华. 鸡ELAVL1基因克隆、原核表达及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(5): 1807-1817.
[6]	卓娜, 朱忠武, 李静, 胡世享, 王建昌, 林华, 陈朝林, 韩佃刚, 艾军, 陈培富. 猪源戊型肝炎病毒ORF2蛋白生物信息学分析及真核表达研究[J]. 中国畜牧兽医, 2023, 50(5): 1959-1970.
[7]	张芳源, 蔺雅婷, 杨大为, 陈虎, 李桂梅, 单虎. 非洲猪瘟病毒P30蛋白原核表达及间接ELISA检测方法建立[J]. 中国畜牧兽医, 2023, 50(5): 2012-2022.
[8]	王雪平, 张孝俊, 李晓月, 王国栋, 张福良, 宋玉伟, 张明亮, 马磊. 高致病性禽腺病毒血清4型Hexon蛋白的原核表达及多克隆抗体的制备[J]. 中国畜牧兽医, 2023, 50(5): 2053-2060.
[9]	范冰峰, 李文, 刘理想, 赵向远, 邵静, 林乐丰, 孙慧敏, 张旭林, 许保增. 水貂AANAT基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(4): 1307-1318.
[10]	刘红润, 朱思燃, 奉玲丽, 张坤, 颜港, 王钰滨, 张帅, 江山, 许迪, 兰干球, 梁晶. 大白猪CDO1基因克隆、生物信息学分析及组织表达定位[J]. 中国畜牧兽医, 2023, 50(4): 1352-1363.
[11]	胡乐玉, 胡欣瑶, 李颖, 赵盛淼, 沈凤臻, 王长法, 李亮亮, 王彤彤, 任慧英. 德州驴HO-1基因生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1499-1510.
[12]	王文婕, 茹鹏辉, 郁川, 杜付熙, 陈松彪, 尚珂, 张春杰, 程相朝. 猫细小病毒洛阳分离株VP2蛋白原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(4): 1511-1521.
[13]	刘家兴, 韩雪英, 张鑫茹, 邓嘉昕, 姚伦广, 刘阳坤. 携带猪流行性腹泻病毒抗原表位的铁蛋白纳米颗粒制备与免疫原性分析[J]. 中国畜牧兽医, 2023, 50(4): 1567-1574.
[14]	贾新月, 马静, 张艳艳, 马勋, 王正荣, 薄新文. 细粒棘球绦虫EGR-07243和EGR-07244基因生物信息学分析及原核表达[J]. 中国畜牧兽医, 2023, 50(3): 847-858.
[15]	张永杰, 隋志远, 张志帅, 王晨光, 李孝君, 邢凤. 多浪羊NPTX1基因克隆、生物信息学分析及在初情期的表达研究[J]. 中国畜牧兽医, 2023, 50(3): 1048-1058.