【作者】 李慧；

【导师】 刘成；

【作者基本信息】 天津大学 ， 生物化学与分子生物学， 2009， 硕士

【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。更多还原

【Abstract】 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.更多还原