Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2015, Vol. 35 Issue (3): 18-24    DOI: 10.13523/j.cb.20150303
研究报告     
具有增强蛋白质表达活性序列ER3的筛选及其功能区域的初步鉴定
崔成成, 毕艳红, 王应明, 李攀, 杨思达, 黄芬, 曾韦锟, 井申荣
昆明理工大学医学院 昆明 650500
Screening of ER3 Sequences Enhanced Protein Expression Activity and Its Functional Regions Identification
CUI Cheng-cheng, BI Yan-hong, WANG Ying-ming, LI Pan, YANG Si-da, HUANG Fen, ZENG Wei-kun, JING Shen-rong
Medical Faculty, Kunming University of Science and Technology, Kunming 650500, China
 全文: PDF(905 KB)   HTML
摘要:

目的:利用构建的增强子样序列筛选载体,筛选在大肠杆菌中增强外源蛋白质表达的序列,并利用删除突变体初步鉴定其功能区域.方法:以氯霉素乙酰转移酶(CAT)基因序列与人乳头瘤病毒(HPV)主要衣壳蛋白基因 L1 的截短序列L11连接作为报告基因,从采集的样品中筛选增强子样序列,通过蛋白质表达来检测其增强活性,并通过构建删除突变体来初步鉴定其功能区域.结果: 成功筛选到一条增强子样序列,可使检测载体氯霉素抗体提高11倍,融合蛋白表达水平提高2.26倍,其功能区域主要集中在1~265bp.结论:从收集的样品中成功筛选出一个增强子序列,能提高外源基因在大肠杆菌中的表达.

关键词: 增强子样序列增强基因表达删除突变体功能区域    
Abstract:

Objective: Using a constructed enhancer sequence detecting vector, screening sequences that could enhanced heterologous protein expression in Escherichia coli, and preliminary identified its functional domain by deletion mutants. Methods: Chloramphenicol acetyltransferase (CAT) gene and truncated human papillomavirus (HPV) major capsid protein gene L1 (named L11) were fusion expressed as a report gene; to screen enhancer-like sequence from the collected samples, and detected the enhanced activity by protein expression. The functional regions were identified by construction of deletion mutants. Results: One enhancer-like sequence was successfully screened, which enhanced 11-folds of chloramphenicol resistance; fusion protein expression level increased 2.26-folds; the functional areas are located in the 1~265bp. Conclusion: An enhancer-like sequence were successfully screened, which can improve the expression of heterologous gene in Escherichia coli.

Key words: Enhancer-like sequence    Enhancer gene expression    Deletion mutants    Functional areas
收稿日期: 2014-12-11 出版日期: 2015-03-25
ZTFLH:  Q939.93  
基金资助:

国家自然科学基金(31360619,31160193),云南省应用基础研究面上项目(2010ZC055,2012FB135) 资助项目

通讯作者: 井申荣     E-mail: jingshenrong@163.com
服务  
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章  

引用本文:

崔成成, 毕艳红, 王应明, 李攀, 杨思达, 黄芬, 曾韦锟, 井申荣. 具有增强蛋白质表达活性序列ER3的筛选及其功能区域的初步鉴定[J]. 中国生物工程杂志, 2015, 35(3): 18-24.

CUI Cheng-cheng, BI Yan-hong, WANG Ying-ming, LI Pan, YANG Si-da, HUANG Fen, ZENG Wei-kun, JING Shen-rong. Screening of ER3 Sequences Enhanced Protein Expression Activity and Its Functional Regions Identification. China Biotechnology, 2015, 35(3): 18-24.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150303        https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I3/18


[1] Banerji J, Rusconi S, Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell, 1981, 27(2 Pt 1): 299-308.

[2] Geyer P K, Green M M, Corces V G. Tissue-specific transcriptional enhancers may act in trans on the gene located in the homologous chromosome: the molecular basis of transvection in Drosophila. EMBO J, 1990, 9(7): 2247-2256.

[3] Heintzman N D, Stuart R K, Hon G, et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet, 2007, 39(3): 311-318.

[4] Schnetz K, Rak B. IS5: A mobile enhancer of transcription in Escherichia coli. Proc Natl Acad Sci USA, 1992, 89(4):1244-1248.

[5] Reitzer L J, Magasanik B. Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell, 1986, 45(6): 785-792.

[6] Cenatiempo Y. Prokaryotic gene expression in vitro: transcription-translation coupled systems. Biochimie, 1986, 68(4): 505-515.

[7] 韩峰, 何建新, 廖小慧,等. 原核增强子样序列的筛选及其应用研究. 中华实验和临床病毒学杂志, 2010, 24(3): 175-177. Han F, He J X, Liao X H, et al. Screening and application of enhancer-like sequences 3A. Chinese Journal of Experimental and Clinical Virology, 2010, 24 (3): 175-177.

[8] 韩峰, 秘晓林, 曹茹,等. 痘苗病毒增强子样序列的筛选及应用研究. 中华实验和临床病毒学杂志, 2007, 21(4): 301-303. Han F, Mi X L, Cao R, et al. Screening and application of enhancer-like sequences from vaccinia virus. Chinese Journal of Experimental and Clinical Virology, 2007, 21 (4): 301-303.

[9] Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene, 1984, 28(3): 351-359.

[10] 何建新, 廖小慧, 秘晓林,等. 原核增强子样序列3A的克隆及其结构与功能的研究. 中国生物化学与分子生物学报, 2001, 17(2): 189-193. He J X, Liao X H, Mi X L, et al. Cloning and structure-function of prokaryotic enhancer-like sequence 3A. Chinese Journal of Biochemistry and Molecular Biology, 2001, 17(2): 189-193.

[11] Tanimoto K, Clewell D B. Regulation of the pAD1-encoded sex pheromone response in Enterococcus faecalis: expression of the positive regulator TraE1. J Bacteriol, 1993, 175(4): 1008-1018.

[12] Hovey A K, Frank D W. Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon. J Bacteriol, 1995, 177(15): 4427-4436.

[13] Eder S, Liu W, Hulett F M. Mutational analysis of the phoD promoter in Bacillus subtilis: implications for Pho Pbinding and promoter activation of Pho regulon promoters. J Bacteriol, 1999, 181(7): 2017-2025.

[14] Householder T C, Belli W A, Lissenden S, et al. cis-and trans-acting elements involved in regulation of aniA, the gene encoding the major anaerobically induced outer membrane protein in Neisseria gonorrhoeae. J Bacteriol, 1999, 181(2): 541-551.

[15] Darwin A J, Tyson K L, Busby S J, et al. Differential regulation by the homologous response regulators NarL and Nar Pof Escherichia coli K-12 depends on DNA binding site arrangement. Mol Microbiol, 1997, 25(3): 583-595.

[16] Siegele D A, Hu J C, Walter W A, et al. Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase. J Mol Biol, 1989, 206(4): 591-603.

[17] Kim G Y, Moon J M, Han J H, et al. The sCMV IE enhancer/promoter system for high-level expression and efficient functional studies of target genes in mammalian cells and zebrafish. Biotechnol Lett, 2011, 33(7): 1319-1326.

[18] 吴淑华, 张丽兰, 张秀珍,等. SV40 DNA Hind Ⅲ B 片段增强人 β 干扰素基因在大肠杆菌中的表达. 病毒学报, 1985, 1(4):385-388. Wu S H, Zhang L L, Zhang X Z, et al. SV40 DNA Hind III B fragments increased the expression of human beta interferon gene in Escherichia coli. Chinese Journal of Virology, 1985,1(4):385-388.

[19] Park M A, Kim H J. Optimum conditions for production and purification of human papillomavirus type 16 L1 protein from Saccharomyces cerevisiae. Protein Expr Purif, 2008, 59(1): 175-181.

[20] Kim S N, Jeong H S, Park S N, et al. Purification and immunogenicity study of human papillomavirus type 16 L1 protein in Saccharomyces cerevisiae. J Virol Methods, 2007, 139(1): 24-30.

[21] Leder C, Kleinschmidt J A, Wiethe C, et al. Enhancement of capsid gene expression: preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes. J Virol, 2001, 75(19): 9201-9209.

[1] 高宗良, 谷元兴, 赵峰, 刘永生. 生物被膜主动分散机制研究进展[J]. 中国生物工程杂志, 2012, 32(02): 117-122.