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大肠杆菌和铜绿假单胞菌中蛋白表达监测系统的构建

中文摘要

20世纪90年代，“人类基因组计划”的推进和完成，标志着“后基因组时代”的到来，“功能基因组学”中蛋白质组学的研究提上了未来生命科学研究的日程。目前蛋白质研究的主要方法是通过2-DE配合质谱对蛋白质的分离鉴定。虽然对蛋白质的研究技术在不断地改进，但是目前的生物化学技术已不能满足研究蛋白质组所需要的高灵敏度、高通量以及可规模化的要求，对于蛋白质组学的研究期待着新型的技术方法的突破。

本课题以大肠杆菌和铜绿假单胞菌为研究对象，采用了将带有RBS的目的基因克隆至缺失自身RBS的报道基因luxABCDE上游，构建二者共用RBS的预期的蛋白表达监测系统；报道基因的表达量取决于克隆基因的转录及翻译水平，利用发光值读取仪检测CPS值衡量目的基因蛋白质的表达量。对该监测系统进行验证，并将两种菌的全基因组克隆至各自相应的监测系统中，建立反映蛋白质表达情况的荧光值数据库。

在大肠杆菌中，将重组载体pUCNot-lux中得到的报道基因luxABCDE克隆至蛋白表达载体pPROEX HTa、pPROEX HTb、pPROEX HTc，初步构建得到大肠杆菌的蛋白表达监测系统，命名为pPROEX HTa-lux、pPROEX HTb-lux、pPROEX HTc-lux。报道基因luxABCDE在载体pPROEX HTc-lux中翻译时能够正确阅读；并在IPTG梯度浓度条件下，测定含有载体pPROEX HTc-lux的细菌发光量，由结果可知克隆到蛋白表达载体中的缺失自身RBS的报道基因luxABCDE可以使用载体上游的trc启动子及其RBS，并且该报道基因的蛋白表达受到trc启动子调节因素的调节。测定预期的蛋白表达载体pPROEX HTc-lux中的报道基因luxABCDE两侧的序列，结果表明载体pPROEX HTc与报道基因luxABCDE连接处的碱基序列与已知碱基序列相同。

在铜绿假单胞菌中，将从已构建的大肠杆菌蛋白表达监测载体酶切后得到的luxABCDE（a）、luxABCDE（b）、luxABCDE（c）克隆至去除原有报道基因luxCDABE的pMS402剩余载体中，初步构建铜绿假单胞菌蛋白表达监测系统，命名为pMS402- luxABCDE（a）、pMS402- luxABCDE（b）、pMS402- luxABCDE（c）。

在大肠杆菌和铜绿假单胞菌中构建的蛋白表达监测方法可以实现高通量、高灵敏度、规模化以及对低丰度蛋白表达的监测；该监测方法不仅可用于蛋白质组学的基础理论性研究，而且可用于攻克重大疾病和开发生物医药等领域的实践应用性研究。

关键词：大肠杆菌，铜绿假单胞菌，蛋白表达监测系统

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Construction of Protein Expression Monitoring System in Escherichia coli K12 and Pseudomonas aeruginosa PAO1

Abstract

Genomics has been focused on by researchers during the 20th century. Then proteomics was being the main studying direction in the post-genomic era after the accomplishment of the “Human Genome Project”. Various methods like 2-DE technique with Mass Spectrometry were usually used for isolating and identifying proteins. However, those techniques still couldn’t meet the needs of proteome development. Therefore, exploring a high sensitive and high-throughput and large-scale research method is imperactive and being expected.

In this study, Escherichia coli (E.coli) and Pseudomonas aeruginosa PAO1 were selected as host cells for constructing a protein expression monitoring system, respectively, using the reporter gene without its self-RBS. Since the reporter gene could share the RBS of the cloned gene, the transcriptional and translational levels of the cloned gene were depended on the intensity of luminescence so that the expression of the cloned gene could be determined by detecting the CPS. To verify the monitoring system constructed above and further to establish the fluorescence database for the corresponding protein of the whole-gemone of E.coli and PAO1 were going to be studied in the following research.

The protein expression monitoring vectors were constructed in E.coli and been named pPROEX HTa-lux and pPROEX HTb-lux and pPROEX HTc-lux, repectively. pPROEX HTc-lux was the appreciate vector for the expression of luxABCDE in correct reading frame. The result of measuring the intensity of luminescence in E.coli containing pPROEX HTc-lux with IPTG showed the trc promoter could transcript the reporter gene luxABCDE, and the expression of the luxABCDE also could be induced by the regulating factor of trc promoter. Meanwhile, the region around the start codon of luxABCDE gene of pPROEX HTc-lux was sequenced to indicate that the sequencing region of pPROEX HTc-lux was matched with expected. Besides, the protein expression monitoring vectors were constructed in PAO1 which named pMS402-luxABCDE(a), pMS402-luxABCDE(b), pMS402- luxABCDE(c) as well.

The protein expression monitoring systems established in E.coli and PAO1 in this study were new techniques which could achieve high-throughput and high sensitive and large-scale detection. This new method could be used for not only proteomics theory study, but also the practical fields like biomedical investigation.

Key words: E.coli, Pseudomonas aeruginosa, Monitoring system of protein expression

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缩写对比表

PA

E.coli DH10B RBS CPS MCS PCR DNA Min Amp Tc Gm Kan Tmp EDTA lacZ Ori SDS TE HEPES RNAase DMSO

铜绿假单胞菌

大肠埃希氏菌DH10B菌株 核糖体结合位点 每秒钟化学发光 多克隆位点 聚合酶链式反应 脱氧核苷三磷酸 分钟 氨苄青霉素 四环素 庆大霉素 卡那霉素 甲氧苄氨嘧啶 乙二胺四乙酸 Β-半乳糖苷酶基因 复制起始位点 十二烷基硫酸钠 TE缓冲液

N-2-羟乙基哌嗪N-2-乙磺酸RNA酶 二甲基亚砜

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