肽抗生素hPAB-β制备工艺优化及生物学活性初探

发布时间：2018-07-07 20:33

本文选题：肽抗生素 + hPAB-β　；参考：《第三军医大学》2005年硕士论文

【摘要】： 肽抗生素(peptide antibiotics)是近年来发现的生物体基因编码的具有抗微生物活性的小肽,通常是由12～60个氨基酸所组成,分子量㩳10kDa,是生物体天然免疫的重要组成部分。它作为一种新型的抗感染制剂,具有广谱、高效的杀菌活性,在传统抗生素耐药性问题日益严重的今天,已成为抗生素研发中的一个重要热点之一。在过去五年,围绕肽抗生素hPAB-β的研发工作中,我们已经基本形成了一条完整的生产工艺路线。肽抗生素hPAB-β是用简并引物从人皮肤角质形成细胞中克隆到的一人β型肽抗生素。在获得其碱基和氨基酸编码序列后,通过化学合成并复性,证实了其抗菌活性。然后根据其特性,我们设计并筛选到了一个承载分子PaP3.30(16.7kDa),利用该分子能使hPAB-β融合蛋白在大肠埃希菌中的表达量达到菌体总蛋白的40%。然而,由于目标蛋白hPAB-β(4.3kDa)在整个融合蛋白分子(23.6kDa)中只占约1/6,其实际表达量占菌体总蛋白的比率(表达率)仅约7%,产量依然低下。为此,我们改用同尾酶法设计并构建了hPAB-β多拷贝串联体,并从得到的串联体基因工程菌中筛选出3拷贝最佳工程菌phPAB-β(3)/JM109,对其发酵条件进行了初步优化,并对表达产物进行纯化,确定了肽抗生素hPAB-β制备的工艺路线:工程菌发酵→高压匀浆破壁→包涵体提取→包涵体溶解→亲和层析纯化→羟胺裂解→反相层析纯化→复性→分子筛层析纯化→活性产物,最终得到了具有抗菌活性的目标肽抗生素hPAB-β。在上述制备工艺路线中,仍然存在一些问题,需要进一步优化和完善:①发酵工程菌phPAB-β(3)/JM109发酵后的细菌湿重最高只有39.2g/L,与理想的高密度发酵有一定差距。②在羟胺裂解反应中,存在着不同批次裂解产物的产量不稳定现象,需要优化,以实现目标蛋白产量最大化。此外,所制备的目的肽抗生素的抗菌活性如何?抗菌谱广否?有没有细胞毒作用?等等,也是我们所关心的。为此,我们开展了本课题的研究,主要研究内容和结果如下:
[Abstract]:Peptide antibiotic (peptide antibiotics) is a small peptide with antimicrobial activity which is encoded by organism gene in recent years. It is usually composed of 12 ~ 60 amino acids and its molecular weight is 10 kDa. it is an important part of innate immunity of organism. As a new anti-infective agent, it has broad spectrum and efficient bactericidal activity. Nowadays, the problem of drug resistance of traditional antibiotics is becoming more and more serious, which has become one of the important hot spots in antibiotic research and development. In the past five years, we have basically formed a complete production process in the research and development of peptide antibiotic hPAB- 尾. The peptide antibiotic hPAB- 尾 was cloned from human skin keratinocytes with degenerate primers. The antimicrobial activity was confirmed by chemical synthesis and renaturation after the nucleotide and amino acid coding sequences were obtained. Then we designed and screened a carrier molecule PaP3.30 (16.7 kDa), which can make the expression of hPAB- 尾 fusion protein in Escherichia coli reach 40% of the total bacterial protein. However, because the target protein hPAB- 尾 (4.3 kDa) only accounted for about 1 / 6 of the fusion protein molecule (23.6kDa), the actual expression of hPAB- 尾 (4.3kDa) was only about 7% of the total cell protein, and the yield was still low. For this reason, we designed and constructed hPAB- 尾 multicopy tandem with the same tail enzyme method, and screened out 3 copies of the best engineered strain phPAB- 尾 (3) / JM109 from the genetically engineered bacteria. The fermentation conditions of hPAB- 尾 (3) / JM109 were preliminarily optimized, and the expressed product was purified. The technological route of preparation of peptide antibiotic hPAB- 尾 was determined as follows: the extraction of inclusion body from high pressure homogenate by engineering bacteria, the purification of hydroxy amine by reverse phase chromatography, and the purification of the active product by refolding molecular sieve chromatography. Finally, the target peptide antibiotic hPAB- 尾 with antibacterial activity was obtained. In the above preparation process, there are still some problems, which need to be further optimized and improved. The highest wet weight of bacteria after fermentation of the engineering strain phPAB- 尾 (3) / JM109 is only 39.2 g / L, which is less than that of the ideal high-density fermentation. There is instability in the yield of different batches of pyrolysis products, which needs to be optimized in order to maximize the yield of target protein. In addition, what is the antibacterial activity of the target peptide antibiotics prepared? Is the antibacterial spectrum widespread? Is there a cytotoxic effect? Wait, that's what we care about. Therefore, we have carried out the research of this subject, the main research contents and results are as follows:
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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