原核增强子样序列筛选及其功能区域鉴定
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摘要
目的从污水和土壤中富集的微生物菌体中筛选原核增强子样序列,构建包含增强子样序列的表达载体,并通过构建缺失突变体鉴定其功能区域。方法将人乳头瘤病毒(human papilloma virus,HPV)主要衣壳蛋白基因L1截短序列L11与氯霉素乙酰转移酶基因(chloramphenicol acetyltransferase,CAT)连接,作为报告基因,从污水和土壤富集的微生物菌体中筛选具有增强子活性的序列,构建包含增强子样序列的表达载体,表达HPV L11蛋白,检测其增强活性;通过构建ER1的缺失突变体,测定不同突变体对L11蛋白表达的作用情况,确定其功能区域。结果从样品基因组DNA中筛选出1条具有一定增强蛋白表达功能的增强子样序列,可使检测菌株氯霉素抗性提高5倍,HPV L11蛋白表达水平提高1.78倍。其增强活性主要位于117~317 bp区域。结论从污水和土壤中成功筛选到1条增强子样序列,携带有增强子样序列的表达载体可提高目的蛋白表达水平。
Objective To screen prokaryotic enhancer-like sequences from the microorgans enriched in collected sewage and soil samples to construct expression vectors, and identify its domains by construction of depletion mutant. Methods The major capsid protein gene L1(named as L11)of truncated human papillomavirus(HPV)was linked to chloramphenicol acetyltransferase(CAT) gene, and used as a reporter gene for screening of sequences with activity of enhancer from the mi croorgans enriched in collected sewage and soil samples, based on which the expression vector containing enhancer-like sequences was constructed, and the expressed HPV L11 protein was determined for enhancing activity. The depletion mutant of ER1 was constructed, by which the effect of various mutants on expression of L11 protein was evaluated to de termine the domains. Results An enhancer-like sequence was screened from the samples, which increased the resistance of bacterial strain to chloramphenicol by 5 folds; and the expression level of HPV L11 protein by 1. 78 folds. The activity-enhancing region was located in 117 ~ 317 bp. Conclusion An enhancer-like sequences was successfully screened from the collected sewage and soil samples, and the expression vectors carrying the sequences increased the ex-pression level of target protein.
Objective To screen prokaryotic enhancer-like sequences from the microorgans enriched in collected sewage and soil samples to construct expression vectors, and identify its domains by construction of depletion mutant. Methods The major capsid protein gene L1(named as L11)of truncated human papillomavirus(HPV)was linked to chloramphenicol acetyltransferase(CAT) gene, and used as a reporter gene for screening of sequences with activity of enhancer from the mi croorgans enriched in collected sewage and soil samples, based on which the expression vector containing enhancer-like sequences was constructed, and the expressed HPV L11 protein was determined for enhancing activity. The depletion mutant of ER1 was constructed, by which the effect of various mutants on expression of L11 protein was evaluated to de termine the domains. Results An enhancer-like sequence was screened from the samples, which increased the resistance of bacterial strain to chloramphenicol by 5 folds; and the expression level of HPV L11 protein by 1. 78 folds. The activity-enhancing region was located in 117 ~ 317 bp. Conclusion An enhancer-like sequences was successfully screened from the collected sewage and soil samples, and the expression vectors carrying the sequences increased the ex-pression level of target protein.
引文
[1]Banerji J,Rusconi S,Schaffner W.Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences[J].Cell,1981,27(2 Pt 1):299-308.
[2]Heintzman ND,Stuart RK,Hon G,et al.Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[J].Nat Genet,2007,39(3):311-318.
[3]Zhang F,Tanasa B,Merkurjev D,et al.Enhancer-bound LDB1regulates a corticotrope promoter-pausing repression program[J].Proc Natl Acad Sci USA,2015,112(5):1380-1385.
[4]Yoo EJ,Brown CD,Tsai YC,et al.Autonomous actions of the human growth hormone long-range enhancer[J].Nucleic Acids Res,2015,43(4):2091-2101.
[5]Ortega JL,Wils on OL,Sengupta-Gopalan C.The 5'untranslated region of the soybean cytosolic glutamine synthetase beta(1)gene contains prokaryotic translation initiation signals and acts as a translational enhancer in plants[J].Mol Genet Genomics,2012,287(11-12):881-893.
[6]Hannig G,Makrides SC.Strategies for optimizing heterologous protein expression in Escherichia coli[J].Trends Biotechnol,1998,16(2):54-60.
[7]Han F,Bi XL,Cao R,et al.Screening and application of enhancer-like sequences from vaccinia virus[J].Chin J Exp Clin Virol,2007,21(4):301-303.(in Chinese)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[J].中华实验和临床病毒学杂志,2007,21(4):301-303.
[8]Han F,He JX,Liao XH,et al.Screening and application of prokaryotic enhancer-like sequences 3A[J].Chin J Exp Clin Virol,2010,24(3):175-177.(in Chinese)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[J].中华实验和临床病毒学杂志,2010,24(3):175-177.
[9]Fan CY,Feng LX,Fan JL,et al.Recent advances on the expression systems for recombinant protein production[J].Biotechnology,2012,22(2):76-80.(in Chinese)范翠英,冯利兴,樊金玲,等.重组蛋白表达系统的研究进展[J].生物技术,2012,22(2):76-80.
[10]Kim GY,Moon JM,Han JH,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[J].Biotechnol Lett,2011,33(7):1319-1326.
[11]Hou YD,Cui H,Duan SM.Enhancing effect of SV40 DNA HindⅢB fragments on expression of human interferonαD gene in prokaryotic cells[J].Chin J Virol,1985,1(3):278-281.(in Chinese)侯云德,崔宏,段淑敏.SV40 DNA HindⅢB片段对人αD型干扰素基因在原核细胞中表达的增强作用[J].病毒学报,1985,1(3):278-281.
[12]Wu SH,Zhang LL,Zhang XZ,et al.Enhancing effect of the SV40 DNA HindⅢB fragments on the expression of the humanβinterferon gene in Escherichia coli[J].Chin J Virol,1985,1(4):385-388.(in Chinese)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[J].病毒学报,1985,1(4):385-388.
[13]Li ZF,Gao LJ.A study progress on prokaryotic enhancer-like element[J].J Biol,2001,18(6):4-6.(in Chinese)李植峰,高丽杰.原核增强子样序列研究进展[J].生物学杂志,2001,18(6):4-6.
[14]Park MA,Kim HJ,Kim HJ.Optimum conditions for production and purification of human papillomavirus type 16 L1 protein from Saccharomyces cerevisiae[J].Protein Expr Purif,2008,59(1):175-181.
[15]Kim SN,Jeong HS,Park SN,et al.Purification and immunogenicity study of human papillomavirus type 16 L1protein in Saccharomyces cerevisiae[J].J Virol Methods,2007,139(1):24-30.
[16]Leder C,Kleinschmidt JA,Wiethe C,et al.Enhancement of capsid gene expression:preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes[J].J Virol,2001,75(19):9201-9209.
[2]Heintzman ND,Stuart RK,Hon G,et al.Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[J].Nat Genet,2007,39(3):311-318.
[3]Zhang F,Tanasa B,Merkurjev D,et al.Enhancer-bound LDB1regulates a corticotrope promoter-pausing repression program[J].Proc Natl Acad Sci USA,2015,112(5):1380-1385.
[4]Yoo EJ,Brown CD,Tsai YC,et al.Autonomous actions of the human growth hormone long-range enhancer[J].Nucleic Acids Res,2015,43(4):2091-2101.
[5]Ortega JL,Wils on OL,Sengupta-Gopalan C.The 5'untranslated region of the soybean cytosolic glutamine synthetase beta(1)gene contains prokaryotic translation initiation signals and acts as a translational enhancer in plants[J].Mol Genet Genomics,2012,287(11-12):881-893.
[6]Hannig G,Makrides SC.Strategies for optimizing heterologous protein expression in Escherichia coli[J].Trends Biotechnol,1998,16(2):54-60.
[7]Han F,Bi XL,Cao R,et al.Screening and application of enhancer-like sequences from vaccinia virus[J].Chin J Exp Clin Virol,2007,21(4):301-303.(in Chinese)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[J].中华实验和临床病毒学杂志,2007,21(4):301-303.
[8]Han F,He JX,Liao XH,et al.Screening and application of prokaryotic enhancer-like sequences 3A[J].Chin J Exp Clin Virol,2010,24(3):175-177.(in Chinese)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[J].中华实验和临床病毒学杂志,2010,24(3):175-177.
[9]Fan CY,Feng LX,Fan JL,et al.Recent advances on the expression systems for recombinant protein production[J].Biotechnology,2012,22(2):76-80.(in Chinese)范翠英,冯利兴,樊金玲,等.重组蛋白表达系统的研究进展[J].生物技术,2012,22(2):76-80.
[10]Kim GY,Moon JM,Han JH,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[J].Biotechnol Lett,2011,33(7):1319-1326.
[11]Hou YD,Cui H,Duan SM.Enhancing effect of SV40 DNA HindⅢB fragments on expression of human interferonαD gene in prokaryotic cells[J].Chin J Virol,1985,1(3):278-281.(in Chinese)侯云德,崔宏,段淑敏.SV40 DNA HindⅢB片段对人αD型干扰素基因在原核细胞中表达的增强作用[J].病毒学报,1985,1(3):278-281.
[12]Wu SH,Zhang LL,Zhang XZ,et al.Enhancing effect of the SV40 DNA HindⅢB fragments on the expression of the humanβinterferon gene in Escherichia coli[J].Chin J Virol,1985,1(4):385-388.(in Chinese)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[J].病毒学报,1985,1(4):385-388.
[13]Li ZF,Gao LJ.A study progress on prokaryotic enhancer-like element[J].J Biol,2001,18(6):4-6.(in Chinese)李植峰,高丽杰.原核增强子样序列研究进展[J].生物学杂志,2001,18(6):4-6.
[14]Park MA,Kim HJ,Kim HJ.Optimum conditions for production and purification of human papillomavirus type 16 L1 protein from Saccharomyces cerevisiae[J].Protein Expr Purif,2008,59(1):175-181.
[15]Kim SN,Jeong HS,Park SN,et al.Purification and immunogenicity study of human papillomavirus type 16 L1protein in Saccharomyces cerevisiae[J].J Virol Methods,2007,139(1):24-30.
[16]Leder C,Kleinschmidt JA,Wiethe C,et al.Enhancement of capsid gene expression:preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes[J].J Virol,2001,75(19):9201-9209.