hBD3-BPI融合蛋白在巴斯德毕赤酵母系统中的表达及其产物的纯化和生物学活性研究
发布时间：2018-10-29 08:36
【摘要】： 大面积烧伤后皮肤和粘膜组织常合并有感染，因继发感染导致的脓毒症是造成烧伤患者多脏器衰竭和死亡的主要原因之一。近年来，细菌耐药性菌株的不断出现，使得传统抗生素治疗面临着严峻的挑战，因感染而造成的烧伤病人死亡率一直居高不下。事实上，不仅是烧伤学科，整个医学界都在为细菌的耐药性问题所困扰，因此，寻找新型的、具有不同抗菌机制或无细菌耐药性的抗生素极其重要。近年研究发现，真核生物能够产生大量的抗微生物肽，广泛参与抵制和杀灭外源性致病微生物的侵袭，构成机体宿主防御的一个重要途径。由于这些蛋白在机体内天然存在，不产生耐药性，因此有希望开发为新型的抗生素，解决临床上因传统抗生素耐药而出现的被动局面。 防御素是一类机体内广泛存在的抗微生物肽，对细菌、真菌等病原微生物具有广谱的杀伤作用，且不产生耐药性。其中人β-防御素3(human beta-defensin 3，hBD3)对革兰氏阴性、阳性细菌以及真菌等多种病原微生物具有杀伤作用，尤其是对包括金黄色葡萄球菌在内的革兰氏阳性菌作用最强。低浓度条件下，，hBD3还有免疫调节作用。最新的研究表明，hBD3还具有抗病毒活性和肿瘤细胞杀伤毒性。此外，hBD3还具有热稳定性和酸碱环境稳定性，且在高盐条件下仍能保持很高的抗菌活性。hBD3的这些物理、化学和生物学活性特征均表明其具有广阔的开发和临床应用前景。另外，大肠杆菌表达的hBD3与天然的以及合成的hBD3蛋白具有相同的生物学活性，也进一步提示该蛋白有望开发成为一种新型的肽类抗生素，解决临床上常见而棘手的传统抗生素细菌耐药性问题。 由于hBD3分子富含正电荷，因此既往的hBD3基因工程研究多采用与带负电荷的蛋白分子融合的方式来表达，以中和hBD3的正电荷，降低其宿主毒性。但由于采用原核表达系统，融合蛋白产物仍以包涵体形式存在，因而使得后续处理非常复杂，包括包涵体溶解、融合蛋白切割、纯化、复性、再纯化等，导致产量大大降低，难以满足开发需要。本研究借鉴以往在大肠杆菌表达系统表达hBD3和在酵母表达系统表达其他防御素的经验，将具有强烈的革兰氏阳性菌抗菌活性的hBD3和另一种具有强烈的革兰氏阴性菌抗菌活性的抗菌肽——细菌膜穿透增加蛋白(bactericidal/permeability increasing protein，BPI)成熟肽第35-211位氨基酸的活性区段串联起来，利用巴斯德毕赤酵母表达系统基因组容量大、分泌表达、产量高、容易纯化的特点，在该系统中进行融合表达。 将hBD3基因与BPI基因串联后，克隆于巴斯德毕赤酵母表达载体pPICZαB中，电转导入X-33巴斯德毕赤酵母菌，经重组酵母基因组PCR和表型鉴定获得阳性克隆，对阳性克隆进行甲醇诱导表达，上清通过疏水层析和阳离子交换层析进行纯化。结果成功构建了pICZαB-hBD3-BPI酵母表达载体，重组X-33/pICZαB-hBD3-BPI克隆经甲醇诱导96小时后上清中有目的蛋白表达，Western blot分析表明重组蛋白抗人hBD3和BPI均阳性。rhBD3-BPI占表达上清总蛋白的15.6％，最终产量为5.48mg/L，回收率为68.5％，纯度为89％。 生物学活性分析表明，rhBD3-BPI对革兰氏阳性和革兰氏阴性细菌，包括标准菌株和临床分离的多重耐药菌株均表现出强烈的抗菌活性，其最小抑菌浓度(Minimal Inhibitory Concentration，MIC)在1-8μg/ml之间，最小杀菌浓度(Minimal Bactehcide Concentration，MBC)在4-16μg/ml之间。同时，鲎试验结果显示rhBD3-BPI和BPI蛋白一样具有内毒素中和活性。此外，重组蛋白还表现出和天然hBD3一致的耐高盐能力，在150mmol/L-200mmol/L的高盐环境下仍能保持杀菌活性。 本研究首次采用巴斯德毕赤酵母表达系统对hBD3进行了基因工程表达，产物以分泌形式进入培养基中，纯化后具有明显抗菌活性、高盐环境不敏感性和内毒素中和活性，表明应用毕赤酵母系统融合表达hBD3-BPI不仅是可行的，而且拓展了hBD3的抗菌谱和活性范围，提高了hBD3的应用潜能，为hBD3的基因工程开发和临床应用奠定了基础。
[Abstract]:Sepsis caused by secondary infection is one of the main causes of multiple organ failure and death in burn patients after large-area burn. In recent years, the emergence of bacterial drug-resistant strains leads to severe challenges for traditional antibiotic therapy, and the mortality of burn patients caused by infection has remained high. In fact, it is not only a burn subject, but the whole medical community is plagued by the problem of drug resistance of bacteria, and therefore, it is extremely important to find novel antibiotics with different antibacterial mechanisms or non-bacterial resistance. In recent years, it has been found that the true nuclear organisms can produce a large number of anti-microbial peptides, which are widely involved in resisting and killing exogenous pathogenic microorganisms and constitute an important way of host defense. Since these proteins are naturally present in the body and no drug resistance is generated, it is desirable to develop new antibiotics to address the passive situation that occurs clinically as a result of resistance to traditional antibiotics. Defensin is a kind of anti-microbial peptide which is widely present in organism, and has broad-spectrum killing bacteria, fungi and other pathogenic microorganisms. Human OPG-Defensin 3 (hBD3) has a killing effect on various pathogenic microorganisms such as Gram-negative, positive bacteria and fungi, especially for Staphylococcus aureus. Gram-positive bacteria have the strongest effect. Under low-concentration conditions, hB The latest study shows that hBD3 also has antiviral activity. in addition, hBD3 also has thermal stability and acid-base environmental stability, These physical, chemical, and biological activities of hBD3 show that they are broad In addition, the hBD3 expressed by E. coli has the same biological activity as natural and synthetic hBD3 proteins, and further suggests that the protein is expected to be developed into a novel peptide chemotherapeutic agent to solve the clinically common and difficult traditions. Since hBD3 molecules are rich in positive charges, previous hBD3 gene engineering studies have been carried out in a way that combines negatively charged protein molecules In the prokaryotic expression system, the fusion protein product still exists in inclusion body, so that the subsequent treatment is very complicated, including inclusion body lysis, fusion protein cleavage, purification, renaturation, Purification, etc., leads to a significant reduction in yield and is difficult to meet the development needs. The study has been used for the expression of h in Escherichia coli expression system. BD3 and the experience of expressing other defensins in a yeast expression system, hBD3 having a strong antibacterial activity against gram-positive bacteria and another antibacterial peptide-bacterial membrane penetration enhancer (BPI) having a strong antibacterial activity against gram-negative bacteria are mature. The active region of the 35-211 amino acid of the peptide is connected in series, the genome capacity of the Pichia pastoris expression system is large, the secretion expression and the yield are produced, the hBD3 gene is connected in series with the BPI gene, the hBD3 gene and the BPI gene are connected in series, and are cloned into the Pichia pastoris expression vector pPICZ-B, Positive clones were obtained from mother genome PCR and phenotypic identification, and positive clones were obtained. The expression vector of pICZ-B-hBD3-BPI yeast was successfully constructed, and the recombinant X-33/ pICZ-B-hBD3-BPI clone was purified by methanol for 96 hours. Western blot analysis showed that recombinant protein anti-human hBD3 and BPI were both positive. rhBD3-BPI accounted for 15. 6 of the total protein. The results showed that rhBD3-BPI exhibited a strong antibacterial activity against gram-positive and gram-negative bacteria, including standard strains and clinical isolates. The minimum inhibitory concentration (MIC) of rhBD3-BPI was 1-8. m/ ml, minimum bactericidal concentration (Mini imal Bactehcide Concentration,MBC)鍦
本文编号：2297185
[Abstract]:Sepsis caused by secondary infection is one of the main causes of multiple organ failure and death in burn patients after large-area burn. In recent years, the emergence of bacterial drug-resistant strains leads to severe challenges for traditional antibiotic therapy, and the mortality of burn patients caused by infection has remained high. In fact, it is not only a burn subject, but the whole medical community is plagued by the problem of drug resistance of bacteria, and therefore, it is extremely important to find novel antibiotics with different antibacterial mechanisms or non-bacterial resistance. In recent years, it has been found that the true nuclear organisms can produce a large number of anti-microbial peptides, which are widely involved in resisting and killing exogenous pathogenic microorganisms and constitute an important way of host defense. Since these proteins are naturally present in the body and no drug resistance is generated, it is desirable to develop new antibiotics to address the passive situation that occurs clinically as a result of resistance to traditional antibiotics. Defensin is a kind of anti-microbial peptide which is widely present in organism, and has broad-spectrum killing bacteria, fungi and other pathogenic microorganisms. Human OPG-Defensin 3 (hBD3) has a killing effect on various pathogenic microorganisms such as Gram-negative, positive bacteria and fungi, especially for Staphylococcus aureus. Gram-positive bacteria have the strongest effect. Under low-concentration conditions, hB The latest study shows that hBD3 also has antiviral activity. in addition, hBD3 also has thermal stability and acid-base environmental stability, These physical, chemical, and biological activities of hBD3 show that they are broad In addition, the hBD3 expressed by E. coli has the same biological activity as natural and synthetic hBD3 proteins, and further suggests that the protein is expected to be developed into a novel peptide chemotherapeutic agent to solve the clinically common and difficult traditions. Since hBD3 molecules are rich in positive charges, previous hBD3 gene engineering studies have been carried out in a way that combines negatively charged protein molecules In the prokaryotic expression system, the fusion protein product still exists in inclusion body, so that the subsequent treatment is very complicated, including inclusion body lysis, fusion protein cleavage, purification, renaturation, Purification, etc., leads to a significant reduction in yield and is difficult to meet the development needs. The study has been used for the expression of h in Escherichia coli expression system. BD3 and the experience of expressing other defensins in a yeast expression system, hBD3 having a strong antibacterial activity against gram-positive bacteria and another antibacterial peptide-bacterial membrane penetration enhancer (BPI) having a strong antibacterial activity against gram-negative bacteria are mature. The active region of the 35-211 amino acid of the peptide is connected in series, the genome capacity of the Pichia pastoris expression system is large, the secretion expression and the yield are produced, the hBD3 gene is connected in series with the BPI gene, the hBD3 gene and the BPI gene are connected in series, and are cloned into the Pichia pastoris expression vector pPICZ-B, Positive clones were obtained from mother genome PCR and phenotypic identification, and positive clones were obtained. The expression vector of pICZ-B-hBD3-BPI yeast was successfully constructed, and the recombinant X-33/ pICZ-B-hBD3-BPI clone was purified by methanol for 96 hours. Western blot analysis showed that recombinant protein anti-human hBD3 and BPI were both positive. rhBD3-BPI accounted for 15. 6 of the total protein. The results showed that rhBD3-BPI exhibited a strong antibacterial activity against gram-positive and gram-negative bacteria, including standard strains and clinical isolates. The minimum inhibitory concentration (MIC) of rhBD3-BPI was 1-8. m/ ml, minimum bactericidal concentration (Mini imal Bactehcide Concentration,MBC)鍦
本文编号：2297185
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