【作者】 马波；

【导师】 孙晖；

【作者基本信息】 东北师范大学 ， 遗传学， 2005， 硕士

【摘要】 当温度突然降低时,原核生物和真核生物都会表现出冷激反应,它们会通过合成冷激蛋白(cold shock proteins,CSPs)来克服冷激的不利影响。CspA 是大肠杆菌中一种主要的冷激蛋白,近来研究人员对其结构、功能和在转录、翻译水平的调控以及 mRNA稳定性等方面作了大量研究。但对于 CspA 基因上游调控元件存在的真实性、5’-UTR 3’端的作用,以及两个 ATGAAA 序列基元的作用机制仍然存在很大的争议。 本实验旨在构建包含 CspA 冷激启动子的低温表达载体,探讨 cspA 启动子及其上下游区域的表达调控机制,并通过报告基因 cecropinB 测定其转录表达活性,同时期望获得 CecropinB 的高效融合表达载体。本论文所使用的 CecropinB 是黑腹果蝇的一种抗菌肽的前体,含有 63 个氨基酸序列,其中 1-23 aa 为信号肽序列。抗菌肽能抵抗果蝇正常生活环境中的各种病原微生物的感染,是自然免疫系统的一部分,可以导致大肠杆菌的死亡。 以 E. coli 基因组 DNA 为模板扩增了 9 段具有 CspA 冷激启动子及其他不同调节元件的片段,用其替换掉 pET28a(+)载体上的 T7 启动子。提取果蝇的总 RNA,通过体外反转录获得 cDNA,以其为模板,PCR 扩增出 cecropinB 的全长 cDNA 序列,构建了四个含有冷激启动子以CecropinB为报告基因的表达载体pMBC系列(pMBC2、pMBC3、pMBC4 和 pMBC5),以及含有 T7 启动子的 cecropinB 的表达质粒 pCEC。 pMBC 系列载体、pCEC 质粒诱导表达,通过宿主菌大肠杆菌 BL21 生长曲线的测试,报告基因 cecropinB 融合蛋白的 SDS-PAGE 分析以及 cecropinB mRNA 的 RT-PCR 结果分析,表明 pMBC2 在低温下的表达效率与 pCEC 在 37℃ IPTG 诱导表达效率相同,pMBC3、pMBC4、pMBC5 的低温诱导表达效率略低于 pMBC2,pCEC 在低温条件下的诱导表达效率极低。 另外,缺失实验分析显示 cspA mRNA 的 5’-UTR 的 3’端对于外源蛋白的表达没有明显的作用,截短的 5’-UTR 与野生型的同样获得了较高的表达效率。cspA 启动子上游调控元件的缺失也同样不影响外源蛋白的表达。更多还原

【Abstract】 Both prokaryotes and eukaryotes exhibit a cold-shock response upon an abrupttemperature downshift. About 26-27 cold-shock proteins are synthesized to overcome thedeleterious effects of low temperature stress. CspA, the major cold-shock protein ofEscherichia coli, has recently been studied with respect to its structure, function andregulation at the level of transcription, translation and mRNA stability, especially the functionof 5’-UTR of its mRNA. Overexpression of proteins in E. coli at low temperature improves their solubility andstability. We apply the unique features of the cspA gene to develop a series of low temperatureinduced expression vectors, termed pMBC vectors. To detect the level of expression,cecropinB was choosed as the report gene. CecropinB was used in this case is a precusor ofcecropinB, a kind of Drosophila melanogaster cecropins, which were tested againstpathogens including E.coli from the normal enviroment of Drosophila as part of the innateimmune system. Nine sequences with cspA promoter and different regulatory motifs have been amplifiedfrom the E.coli genome by utilizing PCR. The products have been ligated into the pET28a(+)vector plasmids instead of the T7 promoter. The total RNA has been isolated from theDrosophila, and the cDNA sequence of cecropinB has been amplified from thesingle-stranded cDNA reverse transcripted from the total RNA. We obtained 4 lowtemperature induced expression vectors successfully (pMBC2、pMBC3、pMBC4 and pMBC5respectively), as well as a IPTG-induced expression plasmid pCEC that cecropinB reportergene was inserted in the multiple clone sites of the pET28a(+) vector. The low temperature-induced expression vectors and pCEC vector were induced in thiscase. The growth curve of the host cells showed the expressed fusion protein was deleteriousto the host cells; SDS-PAGE assay and RT-PCR result of cecropinB showed pMBC2epression efficiency was identical to pCEC, compared to pMBC2, pMBC3、pMBC4 andpMBC5 expression level of the reporter gene cecropinB was lower. Additionally, deletion experiment proved that the 3’ terminal of 5’-UTR was no effectto the expression, as well as the deletion of the up regulate element was adiaphorous.更多还原