大肠杆菌双顺反子表达载体中各基因转录与表达水平的研究
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摘要
从20世纪70年代初期基因克隆技术建立至今,短短几十年的时间,基因克隆技术发展十分迅猛,迄今已有很多外源基因被克隆到不同的表达载体中,进行各种研究,以期造福人类[1]。构建多顺反子表达载体,研究原核生物多顺反子不同间隔区对外源基因表达效率的影响,将对多基因共表达具有一定的指导意义。
本研究从大肠杆菌基因组序列出发,对其多顺反子间隔区多种形式和序列进行分析。实验室已有以报告基因――β-半乳糖苷酶(GAL)和绿色荧光蛋白(GFP)基因组成的双顺反子表达载体pET28a-lacZ-gfpD(无间隔序列),pET28a-lacZ- gfpL (间隔序列为起始密码子与终止密码子重迭序列),pET28a-lacZ-gfpR (?-半乳糖苷酶与绿色荧光蛋白融合表达),pET28a-lacZ-gfp15 (间隔序列为pET-32a中T7启动子后的核糖体结合序列)及pET28a-lacZ-gfp51 (间隔序列为lacZ和lacY之间天然间隔区),在此基础上,进一步构建双顺反子表达载体pET28a-lacZ’-gfpL (lacZ加入RBS序列)和pET28a-lacZ’(lacZ加入RBS序列)。将上述质粒和实验室保存的对照质粒pET28a-gfp、pET28a-lacZ和pET28a转化至大肠杆菌Tuner(DE3)中,加入IPTG进行诱导表达,分别以GFP的荧光强度衡量GFP的表达水平,以GAL的酶活力衡量GAL的表达水平,最终用蛋白质电泳进行验证。结果显示,双顺反子表达载体中不同的间隔序列不仅对位于下游的顺反子gfp的表达水平有影响,对位于上游的顺反子lacZ的表达水平可能也有一定的影响,RBS位点有利于下游的顺反子gfp的表达。同时选取pET28a-gfp为研究对象,同一IPTG诱导表达条件下,按时间梯度半定量RT-PCR测定其mRNA水平,RNA转录水平随诱导时间的延长没有明显变化。我们设计的这种双顺反子的表达规律是否适用于其他外源基因,还有待于更多的应用来证实,但至少对于双顺反子载体的构建提供了一个借鉴。
Since 70th in twenty century when the technology of gene cloning was built, it has been greatly improved. Now, many exogenous genes have been cloned in to different expression vectors for kinds of research. The construction of polycistron expression vector and research on exogenous gene expression efficiency affected by different spacers of polycistrons in prokaryotes will be important to study on multigene cooperative expression.
This study intends to analyse the spacer sequences of polycistrons of genome in E. coli. A series of recombinant plasmids with a bicistron of green fluorescent protein (GFP) and GAL had been constructed in E. coli, including bicistron expression plasmid pET28a-lacZ-gfpD (no spacer between gfp and lacZ), pET28a-lacZ-gfpL (the star codon of gfp overlaps the stop codon of lacZ), pET28a-lacZ-gfpR (gfp and lacZ are fused), pET28a-lacZ-gfp15 (spacer is the sequence between RBS and the start codon of pET-28a), and pET28a-lacZ-gfp51(spacer is the crude one between lacZ and lacY). On this basis, I constructed two new bicistron expression plasmids pET28a-lacZ’(RBS sequence intergrate into lacZ), pET28a-lacZ’-gfpL (RBS sequence intergrate into lacZ in pET28a-lacZ-gfpL). These recombinant plasmids and the control plasmids, pET28a-gfp, pET28a-lacZ and pET28a, were transformed into Tuner(DE3), and induced gene expression with IPTG. The expression level of green fluorescence protein was measured by the relative fluorescence intensive ratio, the expression level ofβ-galactose was measured by the enzyme activity, and ultimately these were verified by protein electrophoresis. The results showed that not only the second cistron gfp was affected by the spacer sequences, but also the first cistron lacZ expression level might be changed due to the different spacer sequences, amd the RBS improved the expression level of gfp. Moreover, I selected the pET28a-gfp as the research object and determined its transcription levels by semi-quantitative RT-PCR with time under the same induce condition. The result showed the transcriptional level of gfp did not change significantly with time. We designed this bicistronic expression of the law applicable to other foreign genes, yet to be applied more to prove, but at least for the bicistronic vector provides a reference.
本研究从大肠杆菌基因组序列出发,对其多顺反子间隔区多种形式和序列进行分析。实验室已有以报告基因――β-半乳糖苷酶(GAL)和绿色荧光蛋白(GFP)基因组成的双顺反子表达载体pET28a-lacZ-gfpD(无间隔序列),pET28a-lacZ- gfpL (间隔序列为起始密码子与终止密码子重迭序列),pET28a-lacZ-gfpR (?-半乳糖苷酶与绿色荧光蛋白融合表达),pET28a-lacZ-gfp15 (间隔序列为pET-32a中T7启动子后的核糖体结合序列)及pET28a-lacZ-gfp51 (间隔序列为lacZ和lacY之间天然间隔区),在此基础上,进一步构建双顺反子表达载体pET28a-lacZ’-gfpL (lacZ加入RBS序列)和pET28a-lacZ’(lacZ加入RBS序列)。将上述质粒和实验室保存的对照质粒pET28a-gfp、pET28a-lacZ和pET28a转化至大肠杆菌Tuner(DE3)中,加入IPTG进行诱导表达,分别以GFP的荧光强度衡量GFP的表达水平,以GAL的酶活力衡量GAL的表达水平,最终用蛋白质电泳进行验证。结果显示,双顺反子表达载体中不同的间隔序列不仅对位于下游的顺反子gfp的表达水平有影响,对位于上游的顺反子lacZ的表达水平可能也有一定的影响,RBS位点有利于下游的顺反子gfp的表达。同时选取pET28a-gfp为研究对象,同一IPTG诱导表达条件下,按时间梯度半定量RT-PCR测定其mRNA水平,RNA转录水平随诱导时间的延长没有明显变化。我们设计的这种双顺反子的表达规律是否适用于其他外源基因,还有待于更多的应用来证实,但至少对于双顺反子载体的构建提供了一个借鉴。
Since 70th in twenty century when the technology of gene cloning was built, it has been greatly improved. Now, many exogenous genes have been cloned in to different expression vectors for kinds of research. The construction of polycistron expression vector and research on exogenous gene expression efficiency affected by different spacers of polycistrons in prokaryotes will be important to study on multigene cooperative expression.
This study intends to analyse the spacer sequences of polycistrons of genome in E. coli. A series of recombinant plasmids with a bicistron of green fluorescent protein (GFP) and GAL had been constructed in E. coli, including bicistron expression plasmid pET28a-lacZ-gfpD (no spacer between gfp and lacZ), pET28a-lacZ-gfpL (the star codon of gfp overlaps the stop codon of lacZ), pET28a-lacZ-gfpR (gfp and lacZ are fused), pET28a-lacZ-gfp15 (spacer is the sequence between RBS and the start codon of pET-28a), and pET28a-lacZ-gfp51(spacer is the crude one between lacZ and lacY). On this basis, I constructed two new bicistron expression plasmids pET28a-lacZ’(RBS sequence intergrate into lacZ), pET28a-lacZ’-gfpL (RBS sequence intergrate into lacZ in pET28a-lacZ-gfpL). These recombinant plasmids and the control plasmids, pET28a-gfp, pET28a-lacZ and pET28a, were transformed into Tuner(DE3), and induced gene expression with IPTG. The expression level of green fluorescence protein was measured by the relative fluorescence intensive ratio, the expression level ofβ-galactose was measured by the enzyme activity, and ultimately these were verified by protein electrophoresis. The results showed that not only the second cistron gfp was affected by the spacer sequences, but also the first cistron lacZ expression level might be changed due to the different spacer sequences, amd the RBS improved the expression level of gfp. Moreover, I selected the pET28a-gfp as the research object and determined its transcription levels by semi-quantitative RT-PCR with time under the same induce condition. The result showed the transcriptional level of gfp did not change significantly with time. We designed this bicistronic expression of the law applicable to other foreign genes, yet to be applied more to prove, but at least for the bicistronic vector provides a reference.
引文
[1]耿风廷,赵晓瑜,高珊,多基因在大肠杆菌中的共表达策略[J],生物技术通讯,2007,5(1):339
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[3] Naylor L.H. Repoter gene technology:the future looks bright. Biochem Pharmacol ,1999, 58:749-757
[4] Chalfie M., Tu Y, Euskirchen G., et al. Green fluorescent protein as a marker for gene expression. Science,1994. 263: 802-5
[5] Nuc P, Nuc K. Recombinant protein production in Escherichia coli. Postepy Biochem,2006;52(4):448-56
[6] Boothman D.A, Geller A.I, Pardee A.B. Expression of the E.coli. LacZ gene from a defective HSV-1 vector in various human normal, cancer-prone and humor cells[J].FEBS Letters, 1989, 258: 159-162
[7]曹诚,石成华,霍乱毒素B亚基融合蛋白表达系统的构建,生物化学,1994,10(5):579-583
[8]谢明,吴淑华,大肠杆菌M增强子样序列结构和功能的研究,中国科学,1997,27(2):179-185
[9]张敏,任慧霞,报告基因的应用研究进展,食品与药品,2007,9(9):45
[10] Shimomura O, Johnson F.H, Saiga Y. Extraction purification and properties of aequorin,abioluminescent protein from the luminous hydromedusan, Aequorea[J]. Cell Comp Physiol, 1962 ,59:223-239
[11] Johnson F.H,Shimomura O, Saiga Y ,et al. Quantum eficieny of Cypridinal uminescence,with a note on that of Aequorea[J]. Cell Comp Physiol, 1962,60: 85 -1 0 4
[12] Matz M.V, Fradkov F, Labas Y.A , et a1. Fluorescent proteins from non-bioluminescent Anthocoa species. Nature Biotcchnology, 1999, 17(10): 969-973
[13] Yanushcvich Y G, Staroverov D B, Savitsky A P, et a1. A strategy for the generation of non-aggregating mutants of Anthocoa fluorescent proteins. FEBS Lett, 2002, 5ll(13): 11-l4
[14] Markus R, Dussmann H, Janicke RU, et al. Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process[J]. Biol Chem, 2002, 277(27): 24506-24514
[15] Novy R, Berg G, Yaeger K, et a1. pET TRX fusion system for increased solubility of proteins expressed in E .coli[J]. Innovations, 1995, 3: 7-9
[16] LaValli ER, DiBlasio EA, Kovacic S, et al. A thloredoxin gene fusion expression system that circumvents inclusion body formation in the E.coli cytoplasm[J]. Biotechnology, 1993, 11: 187-193
[17] Shimomura O, Johnson FH, Saiga Y. Extraction purification and properties of aequorin, abioluminescent protein from the luminous hydromedusan, Aequorea[J]. Cell Comp Physiol, 1962, 59: 223-239
[18] Prasher D, Echenrode V, Wand W, et al. Primary structure of the Aequorea vctoria green fluorescent protein.Genetics, 1992, 111: 229-233
[19] Chalfre M., Tu Y,Euskirchen G,et al. Green fluorescent protein as a marker for gene expression. Science, 1994, 263, 802-805
[20] Ward W.W, Bokman S.H. Reversible denaturation of Aequorea green-fluorescent protein physicalseparation and characterization of the renatured protein. Biochemistry, 1982, 21: 4535-4540
[21] Heim R, Prasher D.C, Tsien R.Y. Wavelength mutations post-translation autoxidation of green fluorescent protein. Proc Natl Acad Sci USA, 1994, 91: 12501-12504
[22] Xianqiang Li, Guohong Zhang, Nhatanh Ngo, et al. Kain and Chiao-Chain Huang Deletion of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence. Biol Chem, 1997, 272: 28545-28549
[23] Heim R, Cubitt A.B, Tsien R.Y. Improved green fluorescence. Nature, 1995, 373, 663-664
[24] Roger Y.T. The green fluorescent protein. Annu. Rev. Biochem, 1998, 67: 509–44
[25] Zacharias D.A, Violin J.D, Newton A.C, et al. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science, 2002, 296: 913-916
[26] Cornea A, Janovick J.A, Maya-Nú?ez G, et al. Gonadotropin-releasing hormone receptor microaggregation. Rate monitored by fluorescence resonance energy transfer. J Biol Chem, 2001, 276(3): 2153-8
[27] Oliver Scholz, Anja Thiel, Wolfgang Hillen, et al. Quantitative analysis of gene expression with an improved green fluorescent protein. Eur. J. Biochem,2000, 267, 1565-1570
[28] Jacob F, Monod J. Genetic Regulatory Mechanism in the Synthesis of Protein. Journal of Molecular Biology, 1961, 3: 318-356
[29] Temim H.M, Mizutani S. RNA-Dependent DNA Polymerase in Virions of Rous Sarcoma Virus.Nature, 1970, 226: 1211-1213
[30]崔金全,石一复,周怀君等,葡萄胎和绒毛膜癌基因表达谱改变与滋养细胞增生的关系,中华肿瘤杂志,2004,12(26): 236-242
[31] Larsen A.B. Pedersen M.W. Stockhausen MT Activation of the EGFR gene target EphA2 inhibits epidermal growth factor-induced cancer cell motility. Mol CancerRes, 2007, 5(3): 283-293
[32] Lin YG, Han LY, Kamat AA, et al. EphA2 overexpression is associated with angiogenesis in ovarian cancer. Cancer, 2007, 109(2): 332-340
[33]高海波,张拴林,陈燕红等,半定量RT—PCR在基因表达方面的应用,生物技术通报,2008,2:226-230
[34] Gibson U.E,Heid C.A,Williams P.M. A novel method for real time quantitative RT-PCR. Genome Research,1996,6, 995-1001
[35]朱捷,杨成君,王军,荧光定量PCR技术及其在科研中的应用,生物技术通报,2009,2:206-210
[36] Bustin S.A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology,2000,25:169-193
[37] Schluger N.W, Condos R, Lewis S, et al. Amplification of DNA of Mycobacterium tuberculosis from peripheral blood of patients with pulmonary tuberculosis. Lancet, 1994, 344: 232-233
[38]王怡瑾,王宏,聂立波等,分子信标技术,化学通报,2004,12(67):205-210
[39] D A Stahl, B Flesher, H R Mansfield, et al. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl Environ Microbiol, 1988, 54(5): 1079–1084
[40]余利岩,徐平,姚天爵等,新型Actinobacteria荧光原位杂交(FISH)探针的设计和应用,中国抗生素杂志,2000,6:212-216
[41] Nathalie D , AxeUe D , V6mnique S , et al. Quantification of Human immunodeficiency virus type l proviral load by a TaqMan rea1-time PCR assay. J Clin Mierobiol, 200l , 39 :1303-1310
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[43]刘敬忠,闫梅,王立荣等,实时荧光聚合酶链反应结合融解曲线分析在α地中海贫血基因诊断中的价值,中华检验医学杂志,2005,28(8):858-861
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[2]于彦春,张光恒,郭龙彪等,CecropinB与Xa21基因共表达提高转基因水稻白叶枯病抗性[J],中国水稻科学,2006,20(1):105
[3] Naylor L.H. Repoter gene technology:the future looks bright. Biochem Pharmacol ,1999, 58:749-757
[4] Chalfie M., Tu Y, Euskirchen G., et al. Green fluorescent protein as a marker for gene expression. Science,1994. 263: 802-5
[5] Nuc P, Nuc K. Recombinant protein production in Escherichia coli. Postepy Biochem,2006;52(4):448-56
[6] Boothman D.A, Geller A.I, Pardee A.B. Expression of the E.coli. LacZ gene from a defective HSV-1 vector in various human normal, cancer-prone and humor cells[J].FEBS Letters, 1989, 258: 159-162
[7]曹诚,石成华,霍乱毒素B亚基融合蛋白表达系统的构建,生物化学,1994,10(5):579-583
[8]谢明,吴淑华,大肠杆菌M增强子样序列结构和功能的研究,中国科学,1997,27(2):179-185
[9]张敏,任慧霞,报告基因的应用研究进展,食品与药品,2007,9(9):45
[10] Shimomura O, Johnson F.H, Saiga Y. Extraction purification and properties of aequorin,abioluminescent protein from the luminous hydromedusan, Aequorea[J]. Cell Comp Physiol, 1962 ,59:223-239
[11] Johnson F.H,Shimomura O, Saiga Y ,et al. Quantum eficieny of Cypridinal uminescence,with a note on that of Aequorea[J]. Cell Comp Physiol, 1962,60: 85 -1 0 4
[12] Matz M.V, Fradkov F, Labas Y.A , et a1. Fluorescent proteins from non-bioluminescent Anthocoa species. Nature Biotcchnology, 1999, 17(10): 969-973
[13] Yanushcvich Y G, Staroverov D B, Savitsky A P, et a1. A strategy for the generation of non-aggregating mutants of Anthocoa fluorescent proteins. FEBS Lett, 2002, 5ll(13): 11-l4
[14] Markus R, Dussmann H, Janicke RU, et al. Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process[J]. Biol Chem, 2002, 277(27): 24506-24514
[15] Novy R, Berg G, Yaeger K, et a1. pET TRX fusion system for increased solubility of proteins expressed in E .coli[J]. Innovations, 1995, 3: 7-9
[16] LaValli ER, DiBlasio EA, Kovacic S, et al. A thloredoxin gene fusion expression system that circumvents inclusion body formation in the E.coli cytoplasm[J]. Biotechnology, 1993, 11: 187-193
[17] Shimomura O, Johnson FH, Saiga Y. Extraction purification and properties of aequorin, abioluminescent protein from the luminous hydromedusan, Aequorea[J]. Cell Comp Physiol, 1962, 59: 223-239
[18] Prasher D, Echenrode V, Wand W, et al. Primary structure of the Aequorea vctoria green fluorescent protein.Genetics, 1992, 111: 229-233
[19] Chalfre M., Tu Y,Euskirchen G,et al. Green fluorescent protein as a marker for gene expression. Science, 1994, 263, 802-805
[20] Ward W.W, Bokman S.H. Reversible denaturation of Aequorea green-fluorescent protein physicalseparation and characterization of the renatured protein. Biochemistry, 1982, 21: 4535-4540
[21] Heim R, Prasher D.C, Tsien R.Y. Wavelength mutations post-translation autoxidation of green fluorescent protein. Proc Natl Acad Sci USA, 1994, 91: 12501-12504
[22] Xianqiang Li, Guohong Zhang, Nhatanh Ngo, et al. Kain and Chiao-Chain Huang Deletion of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence. Biol Chem, 1997, 272: 28545-28549
[23] Heim R, Cubitt A.B, Tsien R.Y. Improved green fluorescence. Nature, 1995, 373, 663-664
[24] Roger Y.T. The green fluorescent protein. Annu. Rev. Biochem, 1998, 67: 509–44
[25] Zacharias D.A, Violin J.D, Newton A.C, et al. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science, 2002, 296: 913-916
[26] Cornea A, Janovick J.A, Maya-Nú?ez G, et al. Gonadotropin-releasing hormone receptor microaggregation. Rate monitored by fluorescence resonance energy transfer. J Biol Chem, 2001, 276(3): 2153-8
[27] Oliver Scholz, Anja Thiel, Wolfgang Hillen, et al. Quantitative analysis of gene expression with an improved green fluorescent protein. Eur. J. Biochem,2000, 267, 1565-1570
[28] Jacob F, Monod J. Genetic Regulatory Mechanism in the Synthesis of Protein. Journal of Molecular Biology, 1961, 3: 318-356
[29] Temim H.M, Mizutani S. RNA-Dependent DNA Polymerase in Virions of Rous Sarcoma Virus.Nature, 1970, 226: 1211-1213
[30]崔金全,石一复,周怀君等,葡萄胎和绒毛膜癌基因表达谱改变与滋养细胞增生的关系,中华肿瘤杂志,2004,12(26): 236-242
[31] Larsen A.B. Pedersen M.W. Stockhausen MT Activation of the EGFR gene target EphA2 inhibits epidermal growth factor-induced cancer cell motility. Mol CancerRes, 2007, 5(3): 283-293
[32] Lin YG, Han LY, Kamat AA, et al. EphA2 overexpression is associated with angiogenesis in ovarian cancer. Cancer, 2007, 109(2): 332-340
[33]高海波,张拴林,陈燕红等,半定量RT—PCR在基因表达方面的应用,生物技术通报,2008,2:226-230
[34] Gibson U.E,Heid C.A,Williams P.M. A novel method for real time quantitative RT-PCR. Genome Research,1996,6, 995-1001
[35]朱捷,杨成君,王军,荧光定量PCR技术及其在科研中的应用,生物技术通报,2009,2:206-210
[36] Bustin S.A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology,2000,25:169-193
[37] Schluger N.W, Condos R, Lewis S, et al. Amplification of DNA of Mycobacterium tuberculosis from peripheral blood of patients with pulmonary tuberculosis. Lancet, 1994, 344: 232-233
[38]王怡瑾,王宏,聂立波等,分子信标技术,化学通报,2004,12(67):205-210
[39] D A Stahl, B Flesher, H R Mansfield, et al. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl Environ Microbiol, 1988, 54(5): 1079–1084
[40]余利岩,徐平,姚天爵等,新型Actinobacteria荧光原位杂交(FISH)探针的设计和应用,中国抗生素杂志,2000,6:212-216
[41] Nathalie D , AxeUe D , V6mnique S , et al. Quantification of Human immunodeficiency virus type l proviral load by a TaqMan rea1-time PCR assay. J Clin Mierobiol, 200l , 39 :1303-1310
[42] Hwa HL,Ko TM,Yen ML,et al. Fetal gender determination using real-time quantitative polymerase chain reaction analysis of maternal plasma.J Fonnos Mod As-soc,2004,l03(5):364-368
[43]刘敬忠,闫梅,王立荣等,实时荧光聚合酶链反应结合融解曲线分析在α地中海贫血基因诊断中的价值,中华检验医学杂志,2005,28(8):858-861
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