【作者】 陈海琴；

【导师】 岑沛霖； 徐志南； 徐乃正；

【作者基本信息】 浙江大学 ， 生物工程， 2006， 博士

【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。更多还原

【Abstract】 Recently there is an increasing interest to study and apply the cell-free protein synthesis system in biology and biotechnology. Cell-free protein synthesis system enables the direct in-vitro expression of protein from template DNA or mRNA. This has been achieved by combining a crude lysate from growing cells, which contains all the necessary enzymes and machinery for protein synthesis, with the exogenous supply of essential amino acids, nucleotides, salts and energy-generating factors. Cell-free protein synthesis offers several advantages over conventional cell-based protein expression methods. For example: without cell wall and plasma membrane, the cell-free protein synthesis system is of full ability in gene transcription and protein translation, therefore, is favorable to synthesize proteins with some antibiotic properties. Furthermore, cell-free protein synthesis system can produce proteins directly from PCR linear templates without the need of constructing expression plasmids, allowing it to be easily adapted for high throughput protein synthesis, and to create a selection of very powerful tools for proteomic applications.Human defensins are a family of cationic antibiotic peptides with broad antibacterial spectrum discovered in recent years, which plays important roles in the defense against microbial invasion. Human defensins are hopeful to become new kinds of endogenesis antibiotics without acquired microbial resistance and has proven the ability in inhibiting the replication of HIV virus.In this work, the expression performance in E. coli cell-free system was compared by using two different HBD-2 genes. One was cloned by RT-PCR from human skin, and the other was synthesized chemically with preferential codons of hosts. The results revealed that, the expression level of target protein from the synthesized gene was the same to the cloned one.The expression level of HBD-2 under different fusion tags (trxA and GFP) was compared. The results indicate that the expression of small peptide is stabilized with the Trx-tag and GFP-tag in the cell-free system. The expression level of HBD-2 fused with GFP is lower than that fused with trxA, whereas, the GFP moiety provides directly visuable and quantitative monitoring of the polypeptide synthesis. Furthermore, our results also showed that, almost all of the HBD-2 fusion protein was expressed in soluble form in the E. coli cell-free system, whereas cell-based methods yield insoluble aggregates for many antibiotic peptides synthesis.The expression level under two different cell-free reaction mode (Batch-mode and Continuous Exchange Cell-Free (CECF) mode) was evaluated. The results indicated that about five-fold improvement of productivity (ca.2.0 mg/ml soluble fusion protein) could be achieved by using a CECF system compared to that in batch system. With continuoussupply of substrates and continuous removal of low-molecular-weight products through a dialysis membrane, proteins can be synthesized continuously with longer period.A purification procedure was established to obtain the mature recombinant protein trxA-HBD-2 in a purity of 95.2% with a recovery of 50%. The fusion protein was cleaved by cyanogens bromide (CNBr), and the released mature HBD-2 had demonstrated strong inhibition on the growth of E. coli D31 at low concentration.The same expression strategy was used for the expression of HBD-3 and HBD-4 in E. coli cell-free system, respectively. This suggested that the cell-free system might be a preferable method for the production of proteins or polypeptides with antibacterial activity.In addition, we obtained 16 kinds of HIV target genes by PCR amplification, and constructed specific plasmids for the cell-free protein expression. Similarly, the PCR templates for cell-free protein expression were also constructed by using a two-step PCR amplification process. By targeting at one HIV protein-proteinase (P10) as a demonstrative protein, we used two expression templates (plasmid and PCR product) for P10 expression in the E. coli cell-free system. The results showed that the target protein P10 could be detected either by using plasmid template or by PCR template. This cell-free method can be used to express many HIV genes simulatenously, which will facilitate the construction of high-throughput screening models for new anti-HIV drugs in the future.Finally, the cell extraction from E. coli was prepared by ourselves. The cell growth phase, the pressure for cell disruption and the storage condition of cell extraction were optimized. In the meanwhile, the optimal substrate concentrations and the energy regeneration system were evaluated. Under the optimized conditions, the GFP reporter gene was efficiently expressed in the E. coli cell-free system.更多还原