摘要
阿维菌素(Avermectin)是由阿维链霉菌(Streptomyces avermitilis)产生的十六元大环内酯类抗生素。阿维菌素具有广谱、高效的抗寄生虫活性，对宿主不良反应极低，是目前理想的驱虫、杀虫药物。此外，以阿维菌素为原料，再进行化学合成可得到一系列的半合成阿维菌素。
本论文利用分子生物学的方法，通过对阿维菌素生物合成基因aveF基因的失活，得到了产生5-酮阿维菌素的基因工程菌株，为化学合成新的阿维菌素衍生物提供了可利用的原料来源。此外，本论文还对阿维菌素生物合成基因簇中aveC基因的功能进行了探索。
首先，根据文献报导的aveF基因及其上下游核苷酸序列，设计了PCR引物，并扩增出了含有aveF基因的1.8kb DNA片段。将该DNA片段连接到pMD18-T载体后，将1.5kb的安普霉素抗性基因插入到该片段aveF基因的SalI位点，然后再将该3.3kbDNA片段切出，插入到具有接合转移功能的链霉菌-大肠杆菌穿梭质粒pHJL401的多克隆位点，得到重组质粒pPC5。
将重组质粒pPC5通过接合转移导入到阿维链霉菌后，经过筛选对安普霉素具有抗性而对硫链丝菌素敏感的同源双交换菌株，得到了aveF基因失活了的基因工程菌株AV-F1。
对基因重组菌株AV-F1的发酵产物分析表明，该重组菌株产生两个主要组分，分别是5-酮阿维菌素1和5-酮阿维菌素2，此外，还产生一个小组分为阿维菌素B1a。
为了进一步探索aveC基因在阿维菌素生物合成过程中的作用，本论文采用插入抗性基因片段的方法将该基因失活，得到重组菌株AV-C24。该菌株只能产生少量的“2”组分，而不能产生“1”组分，表明aveC基因与“1”组分的生物合成有关，可能是参与了聚酮体链合成后的脱水闭环反应。
Avermectins are 16-membered macrocyclic polyketide antibiotics produced by Streptomyces avermitilis. They are known to be an excellent anthelmintic agents with a high efficiency and broad spectrum of activity against a variety of nematode and anthropod parasites, and with a low level of side-effects on the host. Furthermore, a series of semi-synthesized avermectins could be produced by chemical synthesis with avermectins as the raw materials.
In this paper, the molecular biological methods were used and a gene-engineered strain, which was able to produce 5-O-avermectins, was obtained by the gene disruption of aveF which was involved in the biosynthesis of avermectins. Besides, the function of aveC in the gene cluster involved in the biosynthesis of avermectins was also explored.
First, the PCR products of 1.8 kb DNA fragment containing aveF gene were obtained by PCR method with the primers designed according to the nucleotide sequence up and down stream of aveF gene reported by literature. Then the 1.5 kb DNA fragment of apramycin resistance gene was inserted into the SalI site in the aveF gene after the PCR product was ligated into the plasmid vector pMD18-T. Afterwards, the 3.3 kb DNA fragment was excided out, recovered and then ligated into the MCS site of plasmid pHJL401, a shuttle vector of Streptomyces-E. coli with the function of conjugal transfer. As a result, the recombinant disruption plasmid pPC5 was obtained.
After the plasmid pPC5 was transferred into the Streptomyces avermitilis by conjugal transfer, the recombinant strain AV-F1 with a disrupted aveF gene was obtained by selecting strains which were resistance to apramycin and sensitive to thiostreptone.
The analysis of fermentation products of strain AV-F1 showed that this strain produced mainly two components which were 5-O-avermectinl and 5-O-avermectin2 respectively. Moreover, it produced a small amount of avermectinB1a.
In order to explore the function of aveC gene in the biosynthesis of avermectins, the aveC gene was interrupted by inserting a DNA fragment of antibiotic resistance gene. As a result, a recombinant strain AV-C24 was obtained which produced only small amount "2" components without "1"components. This indicated that aveC gene was related to the biosynthesis of "1"components, it may involved in the dehydration-cyclization reaction of the polyketide chain which was released from polyketide synthase after the thioester bond was hydrolyzed.
引文
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