【作者】 魏魏；

【导师】 张部昌；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 红霉素(erythromycin)是糖多孢红霉菌(Saccharopolyspora erythraea)合成的次生代谢产物，为一类广谱大环内酯类抗生素，在临床上具有广泛的应用。红霉素的发展已经经历了三代。第三代红霉素-酮内酯类抗生素(ketolide)，不仅可以增强红霉素的酸稳定性，而且对许多耐药性细菌具有杀死或抑制作用。当前第三代红霉素也是通过化学方法对红霉素结构进行修饰完成的，现在人们正在用基因工程方法探索合成第三代红霉素，以避开烦琐的化学修饰步骤。红霉素的生物合成包括两个部分：红霉素母环6-脱氧-红霉内酯B(6-deoxy-erythronolide B，6-dEB)的合成和6-dEB的后修饰。对糖多孢红霉菌染色体上红霉素生物合成基因进行改造，已经合成了多种红霉素类似物。为了对红霉素类似物进行结构修饰研究，需要能够在糖多孢红霉菌中有效表达外源片段的载体。本实验室利用现有的质粒和遗传材料，构建了糖多孢红霉菌染色体整合型表达载体pZMW和游离型多拷贝表达载体pZM。因大多数链霉菌质粒的启动子在糖多孢红霉菌中都不能发挥作用，故利用糖多孢红霉菌染色体上红霉素抗性基因PermE启动子作表达载体启动子，并利用pSET152质粒上链霉菌染色体整合位点(attP)和氨朴霉素抗性基因，构建了表达载体pZMW。该载体带有红霉素抗性基因PermE启动子、fd终止子、多克隆位点(MCS)、氨苄青霉素和氨朴霉素抗性基因、链霉菌染色体整合位点(attP)，载体大小为10260bp。游离型多拷贝表达载体pZM是可以在大肠杆菌、链霉菌和糖多孢红霉菌中扩增的穿梭型质粒，带有PermE启动子、fd终止子、多克隆位点、硫链丝菌肽和氨苄青霉素抗性基因以及在大肠杆菌和糖多孢红霉菌中复制的ColE1 ori和pJV1 ori复制子，载体大小9880bp。表达载体pZMW和pZM最重要的功能就是表达外源基因，所以其稳定性就显得尤为重要。为此我们对pZMW和pZM进行稳定性研究。小量提取表达载体pZMW质粒，制备糖多孢红霉菌A226原生质体。利用PEG介导表达载体pZMW转化。对转化菌株进行传代，共传5-10代。小量提取pZMW转化菌株的总DNA进行稳定性鉴定。提取pZM质粒，转化糖多孢红霉菌A226原生质体，对转化菌株进行传代，共传5-10代。小量提取pZM转化菌株的质粒进行PCR鉴定。对表达载体进行稳定性研究表明pZMW和pZM可以在糖多孢红霉菌中稳定存在。染色体整合型表达载体pZMW具有两个大肠杆菌复制子，导致质粒在大肠杆菌中不稳定性，同时具有两种抗性基因，增大了表达载体的大小，不利于外源基因的表达。为了解决上述问题，我们通过PCR扩增3’端BamHⅠ位点突变的红霉素抗性基因PermE启动子，连接到pET-22b(+)上，再将pKA质粒上fd终止子与之相连后插入多克隆位点，最后连接至EcoRⅠ／EcoRⅤ位点突变的pSET152质粒上，构建了糖多孢红霉菌整合型表达载体pZW。该载体具有一个大肠杆菌复制子，一种抗性基因，比pZMW载体小2530bp。在糖多孢红霉菌中，pZW可以表达硫链丝菌肽抗性基因(thio)和绿色荧光蛋白基因(EGFP)，是更适合在糖多孢红霉菌中稳定表达外源基因的整合型表达载体。更多还原

【Abstract】 Erythromycin is the secondary metabolite of Saccharopolyspora erythraea. It is a broad spectrum macrolide antibiotic and clinically used widely. Erythromycin has already developed three generations. Ketolide, the third-generation erythromycin can not only strengthen the acidity stability, but also kill or restrain many drug-resistant bacteria. At present, the third generation is also completed by modifying the structures of erythromycin by chemical means. In order to avoid complicated chemical steps, now genetic engineering methods are used to synthesize the third-generation erythromycin.The synthesis process includes two parts: the synthesis and post-modification of 6-deoxy-erythronolide B, 6-dEB. Based on changing erythromycin biosynthesis genes in S. erythraea chromosome, many erythromycin analogs had been synthesized. To modify the structures of erythromycin analogs in S. erythraea, stable expression vectors need to be constructed.Existing plasmids and inherited materials are used to construct expression vector pZMW which could be integrated into S. erythraea chromosome and multi-copy expression vector pZM. As most Streptomyces promoters are invalid in S. erythraea, the promoter of erythromycin-resistant gene, ermE, and Streptomyces chromosomic integration site, attP, and apramycin-resistant gene, apr from plasmid pSET152 are utilized to construct the expression vector pZMW. The vector is 10260bp containing PermE promoter, fd terminator, multiple clone site, aminobenzylpenicillin and apramycin resistance genes, Streptomyces chromosomic integration site, attP.The multi-copy expression vector pZM is a shuttle plasmid which can replicate in Escherichia coli, Streptomyces and S. erythraea. It contains PermE promoter, fd terminator, multiple clone site, thiostrepton and ampicillin resistance genes, and ColE1 ori and pJV1 ori replicons which make the vector replicate in E. coli and S. erythraea. This vector is 9880bp.The most important function of expression vectors pZMW and pZM is to express exogenous genes. As their stabilities stand more important positions, stability research on pZMW and pZM would be done. The plasmid pZMW was isolated from E. coli. S. erythraea A226 protoplast was prepared and transformed with plasmid pZMW by PEG-mediation. The A226 transformant culture was transferred 5 to 10 generations in TSB media without antibiotics. Total DNA of pZMW transformants was extracted on a small scale in order to PCR identification. And S. erythraea A226 protoplast was prepared and transformed with plasmid pZM. The A226 transformant culture was transferred 5 to 10 generations in TSB media without antibiotics and plasmid DNA of pZM transformants was prepared. Stability research on expression vector indicates that pZMW and pZM are stable expression vectors in S. erythraea.But pZMW, the chromosome integrated expression vector, has two E. coli replicons. The result is that the plasmid is unstable in E. coli. Because of two resistance genes, it increases the length of expression vector and it is not suitable for the expression of exogenous genes. In order to solve the problems mentioned above, the promoter of erythromycin-resistant gene where the site BamH I was inactivated was amplified by PCR from genomic DNA of S. erythraea, and ligated to plasmid pET-22b(+). Fd terminator from plasmid pKA was utilized, then multiple clone site was inserted. Finally, plasmid pSET152 where the sites EcoR I and EcoR V were inactivated was utilized to construct the chromosome integrated expression vector pZW. The pZW contains one E. coli replicon and one resistance gene. The length is reduced by 2530bp. In S. erythraea, pZW could express reporter genes thiostrepton resistance gene and enhanced green fluorescin gene. And it is more suitable to express exogenous genes in S. erythraea stably.更多还原