【摘要】： 大面積燒傷后皮膚和粘膜組織常合并有感染，因繼發(fā)感染導致的膿毒癥是造成燒傷患者多臟器衰竭和死亡的主要原因之一。近年來，細菌耐藥性菌株的不斷出現(xiàn)，使得傳統(tǒng)抗生素治療面臨著嚴峻的挑戰(zhàn)，因感染而造成的燒傷病人死亡率一直居高不下。事實上，不僅是燒傷學科，整個醫(yī)學界都在為細菌的耐藥性問題所困擾，因此，尋找新型的、具有不同抗菌機制或無細菌耐藥性的抗生素極其重要。近年研究發(fā)現(xiàn)，真核生物能夠產(chǎn)生大量的抗微生物肽，廣泛參與抵制和殺滅外源性致病微生物的侵襲，構成機體宿主防御的一個重要途徑。由于這些蛋白在機體內(nèi)天然存在，不產(chǎn)生耐藥性，因此有希望開發(fā)為新型的抗生素，解決臨床上因傳統(tǒng)抗生素耐藥而出現(xiàn)的被動局面。 防御素是一類機體內(nèi)廣泛存在的抗微生物肽，對細菌、真菌等病原微生物具有廣譜的殺傷作用，且不產(chǎn)生耐藥性。其中人β-防御素3(human beta-defensin 3，hBD3)對革蘭氏陰性、陽性細菌以及真菌等多種病原微生物具有殺傷作用，尤其是對包括金黃色葡萄球菌在內(nèi)的革蘭氏陽性菌作用最強。低濃度條件下，，hBD3還有免疫調(diào)節(jié)作用。最新的研究表明，hBD3還具有抗病毒活性和腫瘤細胞殺傷毒性。此外，hBD3還具有熱穩(wěn)定性和酸堿環(huán)境穩(wěn)定性，且在高鹽條件下仍能保持很高的抗菌活性。hBD3的這些物理、化學和生物學活性特征均表明其具有廣闊的開發(fā)和臨床應用前景。另外，大腸桿菌表達的hBD3與天然的以及合成的hBD3蛋白具有相同的生物學活性，也進一步提示該蛋白有望開發(fā)成為一種新型的肽類抗生素，解決臨床上常見而棘手的傳統(tǒng)抗生素細菌耐藥性問題。 由于hBD3分子富含正電荷，因此既往的hBD3基因工程研究多采用與帶負電荷的蛋白分子融合的方式來表達，以中和hBD3的正電荷，降低其宿主毒性。但由于采用原核表達系統(tǒng)，融合蛋白產(chǎn)物仍以包涵體形式存在，因而使得后續(xù)處理非常復雜，包括包涵體溶解、融合蛋白切割、純化、復性、再純化等，導致產(chǎn)量大大降低，難以滿足開發(fā)需要。本研究借鑒以往在大腸桿菌表達系統(tǒng)表達hBD3和在酵母表達系統(tǒng)表達其他防御素的經(jīng)驗，將具有強烈的革蘭氏陽性菌抗菌活性的hBD3和另一種具有強烈的革蘭氏陰性菌抗菌活性的抗菌肽——細菌膜穿透增加蛋白(bactericidal/permeability increasing protein，BPI)成熟肽第35-211位氨基酸的活性區(qū)段串聯(lián)起來，利用巴斯德畢赤酵母表達系統(tǒng)基因組容量大、分泌表達、產(chǎn)量高、容易純化的特點，在該系統(tǒng)中進行融合表達。 將hBD3基因與BPI基因串聯(lián)后，克隆于巴斯德畢赤酵母表達載體pPICZαB中，電轉導入X-33巴斯德畢赤酵母菌，經(jīng)重組酵母基因組PCR和表型鑒定獲得陽性克隆，對陽性克隆進行甲醇誘導表達，上清通過疏水層析和陽離子交換層析進行純化。結果成功構建了pICZαB-hBD3-BPI酵母表達載體，重組X-33/pICZαB-hBD3-BPI克隆經(jīng)甲醇誘導96小時后上清中有目的蛋白表達，Western blot分析表明重組蛋白抗人hBD3和BPI均陽性。rhBD3-BPI占表達上清總蛋白的15.6％，最終產(chǎn)量為5.48mg/L，回收率為68.5％，純度為89％。 生物學活性分析表明，rhBD3-BPI對革蘭氏陽性和革蘭氏陰性細菌，包括標準菌株和臨床分離的多重耐藥菌株均表現(xiàn)出強烈的抗菌活性，其最小抑菌濃度(Minimal Inhibitory Concentration，MIC)在1-8μg/ml之間，最小殺菌濃度(Minimal Bactehcide Concentration，MBC)在4-16μg/ml之間。同時，鱟試驗結果顯示rhBD3-BPI和BPI蛋白一樣具有內(nèi)毒素中和活性。此外，重組蛋白還表現(xiàn)出和天然hBD3一致的耐高鹽能力，在150mmol/L-200mmol/L的高鹽環(huán)境下仍能保持殺菌活性。 本研究首次采用巴斯德畢赤酵母表達系統(tǒng)對hBD3進行了基因工程表達，產(chǎn)物以分泌形式進入培養(yǎng)基中，純化后具有明顯抗菌活性、高鹽環(huán)境不敏感性和內(nèi)毒素中和活性，表明應用畢赤酵母系統(tǒng)融合表達hBD3-BPI不僅是可行的，而且拓展了hBD3的抗菌譜和活性范圍，提高了hBD3的應用潛能，為hBD3的基因工程開發(fā)和臨床應用奠定了基礎。
[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

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