【作者】 胡乔；

【导师】 唐克轩；

【作者基本信息】 上海交通大学 ， 植物学， 2008， 硕士

【摘要】 溶菌酶(lysozyme, LYZ)是自然界普遍存在的一种酶,因其能溶解细菌细胞壁具有溶菌作用而得名。溶菌酶根据分子结构、分子量和来源的不同可分为六类:植物溶菌酶、微生物溶菌酶、噬菌体溶菌酶、c型、g型和I型3种动物溶菌酶,其中c型、g型和噬菌体溶菌酶最常见。溶菌酶是人和动物体液和组织中的一种防御因子,在非特异性免疫中具有重要作用,具有抗菌、消炎、促进组织修复和增强免疫力等多种药理作用,因而广泛应用于食品、饲料工业、科研和医学临床中。人溶菌酶(human lysozyme,hLYZ)属于c型溶菌酶,由130个氨基酸组成,分子量为14.4kD,含有4个二硫键;与其它溶菌酶相比,其一级结构差异较大,但高级结构非常相似。人溶菌酶与目前临床上应用最多的鸡蛋清溶菌酶相比具有以下优势:人溶菌酶是人体内的蛋白,具有天然的相容性,没有毒性和副作用;人溶菌酶比鸡蛋清溶菌酶的杀菌活性高3倍,而且热稳定性也要远高于鸡蛋清溶菌酶;人溶菌酶还有多种特有的与其自身的催化作用无关的理化作用,如抗病毒和抗肿瘤。随着人们对人溶菌酶功能的深入了解和需求的增加,其大规模的生产和应用越来越受到重视。目前应用的人溶菌酶大部分是从人乳和胎盘中提取,由于受到来源和规模的限制,存在着人溶菌酶产量不高、质量不稳定且生产成本昂贵等问题。利用生物反应器生产人溶菌酶不仅对上述问题的解决具有积极作用,也为人溶菌酶的生产提供了一条可选择的途径。本研究尝试利用生物反应器来表达重组人溶菌酶,所用的生物反应器包括大肠杆菌、毕赤酵母和植物油体三种表达系统,取得了如下研究结果:①分别按照大肠杆菌和植物偏爱密码子设计并合成了人溶菌酶基因,为了便于后期分离纯化,设计时在基因中引入了his-tag和肠激酶酶切位点;②成功构建了大肠杆菌表达载体pET32a-hLYZe,并转化入大肠杆菌表达型菌株BL21,获得了含有人溶菌酶基因的工程菌株,并通过IPTG诱导表达出了重组人溶菌酶蛋白,表达产物以包涵体形式存在,经过包涵体变性、溶解、复性后,通过SDS-PAGE和Western blot检测,证明重组蛋白在大肠杆菌中成功表达,且具有免疫原性,经过镍亲和层析柱纯化了重组蛋白,通过比浊法确认了其具有生物学活性;③成功构建了毕赤酵母分泌型表达载体pPICZ?-hLYZ,转化毕赤酵母GS115获得了工程菌株,利用甲醇诱导表达了重组人溶菌酶蛋白,通过SDS-PAGE和Western blot检测,证明重组蛋白得到表达,且具有免疫原性,重组蛋白经过镍亲和层析柱纯化后,得到了初步纯化的人溶菌酶蛋白,用Bradford法测得其含量为324mg/L,通过酶标仪快速比浊法测得毕赤酵母表达的重组人溶菌酶蛋白活性为4473U/mL,比活性约为13800U/mg,高于通过人乳提取等常规方法获得的人溶菌酶蛋白;④成功构建了含“twin T-DNAs”的植物双元表达载体OP::(his)-OG-hLYZ,转化入根癌农杆菌( Agrobacterium tumefaciens)GV3101中获得了基因工程菌,通过农杆菌介导法转化拟南芥,共获得了27株卡那霉素抗性植株。更多还原

【Abstract】 Lysozyme (LYZ) is a ubiquitous enzyme in the nature. It can kill bacterium by lysing the bacterial cell wall. Lysozymes are classified into six types according to their molecular structures, molecular weights and sources: plant type lysozyme, bacteria type lysozyme, phage lysozyme, chicken egg-white lysozyme (c-type, clyz), goose egg-white lysozyme (g-tpye, glyz) and invertebrate type lysozyme (i-type). The most common ones in the nature are c-type, g-type and phage lysozymes. Lysozyme is an important defensive factor presented in the humor body liquid and tissues of human and other animals and plays a significant role in the non-specific immunity. It also has many other pharmacological functions, such as anti-bacterium, anti-inflammation, promoting organism recovery, so it is widely used in food and feed industries, scientific research and medical clinical treatments.Human lysozyme (hLYZ) is a 130-animo acid c-type lysozyme with 4 disulfide bonds, and its molecular weight is 14.4kD. Comparing with other kinds of lysozyme, hLYZ has a much different primary structure, but has a very similar tertiary structure. Comparing with chicken egg-white lysozyme which is widely used in clinic, hLYZ has many advantages: it is a natural protein in human body, so it is safe and harmless when used as a drug; its bioactivity to kill bacterium is three times higher than chicken egg-white lysozyme, and its thermal stability is much higher, too; it also has many other specific important functions which are not related with its catalysis, such as anti-virus and anti-tumor and improving immunization. With the in-depth understanding of hLYZ functions and the increasing demand in the market, more and more attention is paid in the large-scale production and application of hLYZ.Currently, most hLYZ is extracted from human milk and placenta. Limited by the source and scale, the problems for production of hLYZ are low yield, unstable quality and high cost. Utilizing bioreactor to produce hLYZ can make positive contribution to solve these problems, and can also provide an alternative way to hLYZ.The present study tried to use bioreactors to express recombinant hLYZ in three different expression systems: Escherichia coli, Pichia pastoris and plant oil body. The major achievements in this study were as follows:①two different hLYZ genes were designed and synthesized according to the plant and E.coli codon bias usages, respectively, and in both genes a his-tag and a enterokinase cleavage site were introduced to simplify the purification procedures later on.②an E. coli expression vector pET32a-hLYZe was successfully constructed, which was subsequently transferred into E. coli strain BL21. After induction by IPTG, the engineered strain expressed recombinant hLYZ with specific immunogenicity, which existed as insoluble inclusion bodies, which was confirmed by SDS-PAGE and Western blot analyses. The recombinant protein was purified by Ni+ affinity chromatography after a series of denaturation, solution and renaturation procedures, and then analyzed for the hLYZ bioactivity by turbidimetric method. The result showed that the recombinant hLYZ expressed by E. coli was bioactive.③a P. pastoris secreton expression vector pPICZ?-hLYZ was successfully constructed, which was then transferred into P. pastoris strain GS115. After induction by methanol, the engineered strain expressed recombinant hLYZ with specific immunogenicity, which was confirmed by SDS-PAGE and Western blot analyses. The recombinant protein was purified by Ni+ affinity chromatography and measured for hLYZ concentration by Bradford assay, and then analyzed for the hLYZ bioactivity by turbidimetric method. The result indicated that the concentration of expressed hLYZ was 324mg/L, the bioactivity was 4473U/mL, and the specific activity was approximately 13800U/mg, which was higher than conventional hLYZ extracted from human milk.④a“Twin T-DNAs”-containing plant expression vector p2300-OP-(his)-OG-hLYZ was successfully constructed, which was then transferred into Agrobacterium tumefaciens strain GV3101. Twenty-seven kanamycin-resistant Arabidopsis plant were regenerated by Agrobacterium-mediated transformation.更多还原