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
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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。
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 enhancerlike 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 ~ 265 bp. Conclusion: An enhancer-like sequence were successfully screened,which can improve the expression of heterologous gene in Escherichia coli.
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 enhancerlike 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 ~ 265 bp. Conclusion: An enhancer-like sequence were successfully screened,which can improve the expression of heterologous gene in Escherichia coli.
引文
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[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 type16 major structural gene L1 for DNA vaccination purposes.J Virol,2001,75(19):9201-9209.
[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 gln A 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:transcriptiontranslation 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 p AD1-encoded sex pheromone response in Enterococcus faecalis:expression of the positive regulator Tra E1.J Bacteriol,1993,175(4):1008-1018.
[12]Hovey A K,Frank D W.Analyses of the DNA-binding and transcriptional activation properties of Exs A,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 pho D 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 transacting elements involved in regulation of ani A,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 Nar L 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 s CMV 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 HindⅢB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 type16 major structural gene L1 for DNA vaccination purposes.J Virol,2001,75(19):9201-9209.