【作者】 庹晓晔；

【导师】 柴家科；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学烧伤， 2007， 博士

【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。更多还原

【Abstract】 Infection is a common complication of massive burns and the sepsis arises from these infection is one of the major reasons for multi-organ failure or even death. In recent years, treatment of infections with traditional antibiotics has been challenged by the increasingly severe problem of antibiotic-resistant bacterial strains, which has, at least partly, resulted in a constantly high mortality in burned patients. In fact, the drug-resistant strains have entangled not only burns surgeons but literally the whole medical science. Thus, it’s of great importance to find new type antibiotics that illicit no drug-resistance. Previous studies have demonstrated that antimicrobial peptides, as part of the host defense system, display extensive microorganism killing activities. Natural proteins and eliciting no drug-resistance, these peptides are potential candidate to be developed into new type antibiotics.Defensins are a cluster of antimicrobial peptides extensively present in our body with a broad spectrum of antimicrobial activities but will not cause drug-resistance. Amongst is human beta-defensin 3 (hBD3), which demonstrates antimicrobial activity against a vast variety of microorganisms like both Gram-positive and Gram-negative bacteria and even funges. It’s especially potent to Gram-positive strains including Staphylococcus aureus. In addition to the well-recognized antimicrobial properties, recent reports picture this protein as potent immunomodulators, virus and tumor cell killer. In addition to high bioactivity stability like other β-defensins in high temperature and extreme pH environments, hBD3 is salt-insensitive. These favorable physical and chemical properties, together with its attractive biological activities, make hBD3 promising target for drug development. Finally, it’s worth mention that gene-engineered hBD3 in E.coli shares the same biological activities with its natural or syntheticcounterparts. This further indicates that hBD3 may carry great hope for development of a new type of antibiotic--peptide antibiotic, and provide a powerful solution for current bacterial antibiotic-resistance problem.Since hBD3 is positively charged, protein fusion strategy is very popular for engineering of hBD3 in previous reports. In this way scholars aim to to neutralize the positive charges and thus attenuate its host toxicity. However, in these studies, the expression systems are unexceptionally prokaryotic, and the fusion protein is produced in the form of inclusion bodies, which makes the subsequent processes more complicated. As a result, this will come to a low productivity, unable to meet to the cost-product balance for drug development.Based on our experience in hBD3 gene engineering in E.coli and other groups’ experience in gene engineering of other defensins in yeast expression system, the present study adapts a new strategy for hBD3 gene engineering by fusing the potent Gram-positive bacteria killing peptide hBD3 with the bioactive 35-211 aa of a potent Gram-negative protein killing peptide, bactericidal/permeability increasing protein (BPI) , and express the fusion protein in pastoris picha yeast expression system. This system has a larger genome capacity and generally gives higher productivity. The foreign protein is more likely to be expressed in soluble form and secreted into the supernatants and thus facilitates the subsequent purification procedures.We successfully constructed the yeast expression vector pICZαB-hBD3-BPI, and the recombinant X-33/pICZαB-hBD3 -BPI clones demonstrated target fusion protein expression in supernatants after methanol induction for 96 hours. Western blot analysis confirmed that the recombinant hBD3-BPI (rhBD3-BPI) protein product was positive for both hBD3 and BPI. rhBD3-BPI accounts for 15.6% of the whole protein in supernatant. After purification by hydrophobic chromatography and cation exchange chromatography, the final productivity was 5.48mg/L, with a recollection rate of 68.5% and a purity of 89%.Bioactivity analysis indicates that rhBD3-BPI displayed potent antimicrobial activity against a wide spectrum of bacteria, including both Gram-positive andGram-negative strains, despite of standard or clinically isolated wild-type, antibiotic-resistant or not. Its minimal inhibitory concentration(MIC )is l-8μg/ml, minimal bactericidal concentration (MBC ) is 4-16μg/ml. In the meanwhile, like BPI, the rhBD3-BPI protein can also neutralize LPS activity. Additionally, like hBD3, rhBD3-BPI is also salt-insensitive. No significant discont of antimicrobial activity was observed at 150mmol/L-200mmol/L NaCl concentrations.The present study for the first time expressed hBD3 in pastoris picha yeast expression system. And also for the first time, hBD3 was fused with BPI for fusion expression. The fusion protein was secreted into the supernatant after methanol induction culture and the purified rhBD3-BPI demonstrated potent antimicrobial activity against both Gram-positive and Gram-negative strains. It’s also salt-insensitive and can neutralize LPS activity. So, in summary, the fusion expression of hBD3-BPI in pastoris picha yeast expression system is feasible and the fusion strategy has extended the biological activities in addition to antimicrobial spectrum, thus potentially increased the potential clinical applications and provided useful clues for gene engineering and drug development of hBD3.更多还原