本文選題：肽抗生素 + hPAB-β　； 參考：《第三軍醫(yī)大學》2005年碩士論文

【摘要】： 肽抗生素(peptide antibiotics)是近年來發(fā)現(xiàn)的生物體基因編碼的具有抗微生物活性的小肽,通常是由12～60個氨基酸所組成,分子量㩳10kDa,是生物體天然免疫的重要組成部分。它作為一種新型的抗感染制劑,具有廣譜、高效的殺菌活性,在傳統(tǒng)抗生素耐藥性問題日益嚴重的今天,已成為抗生素研發(fā)中的一個重要熱點之一。 在過去五年,圍繞肽抗生素hPAB-β的研發(fā)工作中,我們已經(jīng)基本形成了一條完整的生產(chǎn)工藝路線。肽抗生素hPAB-β是用簡并引物從人皮膚角質(zhì)形成細胞中克隆到的一人β型肽抗生素。在獲得其堿基和氨基酸編碼序列后,通過化學合成并復性,證實了其抗菌活性。然后根據(jù)其特性,我們設計并篩選到了一個承載分子PaP3.30(16.7kDa),利用該分子能使hPAB-β融合蛋白在大腸埃希菌中的表達量達到菌體總蛋白的40%。然而,由于目標蛋白hPAB-β(4.3kDa)在整個融合蛋白分子(23.6kDa)中只占約1/6,其實際表達量占菌體總蛋白的比率(表達率)僅約7%,產(chǎn)量依然低下。為此,我們改用同尾酶法設計并構(gòu)建了hPAB-β多拷貝串聯(lián)體,并從得到的串聯(lián)體基因工程菌中篩選出3拷貝最佳工程菌phPAB-β(3)/JM109,對其發(fā)酵條件進行了初步優(yōu)化,并對表達產(chǎn)物進行純化,確定了肽抗生素hPAB-β制備的工藝路線:工程菌發(fā)酵→高壓勻漿破壁→包涵體提取→包涵體溶解→親和層析純化→羥胺裂解→反相層析純化→復性→分子篩層析純化→活性產(chǎn)物,最終得到了具有抗菌活性的目標肽抗生素hPAB-β。 在上述制備工藝路線中,仍然存在一些問題,需要進一步優(yōu)化和完善:①發(fā)酵工程菌phPAB-β(3)/JM109發(fā)酵后的細菌濕重最高只有39.2g/L,與理想的高密度發(fā)酵有一定差距。②在羥胺裂解反應中,存在著不同批次裂解產(chǎn)物的產(chǎn)量不穩(wěn)定現(xiàn)象,需要優(yōu)化,以實現(xiàn)目標蛋白產(chǎn)量最大化。此外,所制備的目的肽抗生素的抗菌活性如何?抗菌譜廣否?有沒有細胞毒作用?等等,也是我們所關(guān)心的。為此,我們開展了本課題的研究,主要研究內(nèi)容和結(jié)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:
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392

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