大肠杆菌无细胞系统合成人β-防御素和艾滋病病毒蛋白的研究

发布时间：2018-04-05 08:48

本文选题：无细胞蛋白质合成系统　切入点：抗菌肽　出处：《浙江大学》2006年博士论文

【摘要】：近年来一种以外源mRNA或DNA为模板,通过在细胞抽提物的酶系中补充底物和能量物质来合成蛋白质的体外蛋白质合成系统越来越引起重视。与传统的体内系统相比,无细胞系统具有众多的优越性:1)反应简便,只需加入抽提物、DNA模板和各种必需的反应底物,就可以表达目的蛋白;2)可用于表达在体内系统中难于表达的蛋白质,如对细胞有毒害作用的抗菌肽和膜蛋白等;3)能够直接以PCR产物作为线性模板在微孔板上同时平行合成多种蛋白质,能够满足高通量药物筛选和蛋白质组学研究等的需要。人β-防御素是近年来发现的具有广谱抗菌活性并在机体抵御外来微生物入侵中起防御作用的一类阳离子抗菌肽。有望成为解决致病菌耐药性问题的新型内源性抗生素,并被证明具有抑制艾滋病病毒复制的作用。分别对从人体炎症皮肤组织中通过RT-PCR方法克隆获得的人β-防御素-2(HBD-2)人源基因、经密码子优化后的HBD-2人工合成基因构建了HBD-2的表达载体,得到了一系列不同密码子来源或带有不同融合标签的HBD-2的体外表达载体。将不同密码子来源的HBD-2的表达载体在大肠杆菌无细胞蛋白质合成系统中进行表达,对比实验结果表明,密码子优化后合成基因的蛋白质表达量与人源基因表达量相当。进一步将带有不同融合标签(如硫氧化还原蛋白,trxA和绿色荧光蛋白,GFP)的HBD-2表达载体在大肠杆菌无细胞蛋白质合成系统中进行表达研究。结果表明,融合标签的添加明显提高了HBD-2的表达量,其中trxA对HBD-2表达的促进作用最为明显。添加GFP融合标签的HBD-2基因的表达水平虽然略低于添加trxA融合标签的,但GFP融合标签有利于实时检测该融合蛋白的表达。在无细胞蛋白质合成系统中表达的融合蛋白可溶性高,避免了在大肠杆菌表达抗菌肽时易形成包涵体的难题。比较了反应操作模式对无细胞系统中目标蛋白质表达水平的影响。研究表明,与间歇式操作(Batch)相比,连续交换式(CECF,Continuous Exchange Cell-free)无细胞反应方式能够明显提高目标蛋白质的表达水平,目标蛋白的表达量最高达到了2毫克/毫升。这主要是因为在CECF模式下,能够不断补充底物和能量,并同时移走抑制性副产物,从而大大延长了反应时间,提高了产物产量。对无细胞系统表达的目标蛋白质(trxA-HBD-2)的分离纯化进行了研究,建立了一条高效分离目标蛋白质的纯化工艺,总收率达到50%,目标蛋白质纯度达到95.2%。所得产物经溴化氰(CNBr)消化后,采用固体平板扩散法进行抑菌活性的测定,测得结果具有明显抑菌活性。
[Abstract]:In recent years, an in vitro protein synthesis system using exogenous mRNA or DNA as template, which can synthesize proteins by adding substrate and energy to the enzyme system of extracellular extracts, has attracted more and more attention.Compared with the traditional in vivo system, the cell-free system has many advantages: 1) the reaction is simple and simple, only by adding the extract DNA template and all kinds of necessary reaction substrates.It can be used to express proteins that are difficult to express in the body system.For example, antimicrobial peptides and membrane proteins, which are toxic to cells, can directly use PCR products as linear templates to parallel synthesize a variety of proteins on microporous plates at the same time, and can meet the needs of high-throughput drug screening and proteomics research.Human 尾 -defensin is a kind of cationic antimicrobial peptide which has broad spectrum antibacterial activity and plays a defensive role in the body against the invasion of foreign microorganisms.It is expected to be a new endogenous antibiotic to solve the drug resistance of pathogenic bacteria, and has been proved to inhibit the replication of HIV.The expression vector of human 尾 -defensin-2 (HBD-2) gene, which was cloned from human inflammatory skin tissue by RT-PCR method, was constructed by codon optimized HBD-2 synthetic gene.A series of HBD-2 expression vectors with different codon sources or different fusion tags were obtained.The expression vector of HBD-2 from different codon sources was expressed in the acellular protein synthesis system of E. coli. The results of comparative experiments showed that the protein expression of the synthesized gene after codon optimization was equal to that of human gene.Furthermore, HBD-2 expression vectors with different fusion tags (such as sulfur redox protein trxA and green fluorescent protein GFP) were expressed in acellular protein synthesis system of Escherichia coli.The results showed that the expression of HBD-2 was significantly increased with the addition of fusion tags, and the effect of trxA on the expression of HBD-2 was the most obvious.Although the expression level of HBD-2 gene with GFP fusion tag was slightly lower than that with trxA fusion tag, GFP fusion tag was helpful to detect the expression of the fusion protein in real time.The fusion protein expressed in acellular protein synthesis system is highly soluble and avoids the difficulty of forming inclusion bodies when expressing antibacterial peptides in Escherichia coli.The effect of reaction mode on the expression of target protein in cell-free system was compared.The results showed that the cell-free reaction mode of continuous Exchange Cell-freewas significantly higher than that of batch operation, and the expression of target protein was up to 2 mg / ml.This is mainly because in the CECF mode, the substrate and energy can be continuously replenished and the inhibitory byproducts can be removed at the same time, thus greatly prolonging the reaction time and increasing the yield of the products.The purification of the target protein, trxA-HBD-2, expressed by cell-free system, was studied. An efficient purification process was established. The total yield and purity of the target protein were 50 and 95.2, respectively.The antibacterial activity of the product was determined by solid plate diffusion method after digested by CNBr. the results showed that the antibacterial activity was obvious.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：Q78;R346

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