串联技术在基因工程表达小分子肽研究中的应用

将一些小分子肽基因通过首尾连接、增加拷贝数、提高分子量,可以克服小分子肽体外表达量低的问题,并能优化蛋白质性质.该文主要介绍近年来串联表达技术在小分子肽表达中的应用情况、研究方法以及存在的问题.     

多拷贝策略在增强目的基因表达中的应用

作为基因工程领域增强目的基因表达的一种有效手段,多拷贝策略日益受到分子生物学研究者的青睐。该策略分为3种方式:表达盒多拷贝、目的基因多拷贝和启动子多拷贝,目前基因工程领域3种方式均有运用。实现多拷贝的方法有5种,分别是随机定向串联法、PCR扩增串联法、接头连接法、同尾酶法和化学合成法。多拷贝策略是通过分子生物学手段对基因工程菌目的蛋白质表达进行改良的一种方法,已有多项研究证明了该策略的可行性,该策略对基因工程起到了良好的补充作用,使得转基因产物产量更符合工业生产的需要。     

小肽多拷贝基因表达载体的构建及其高效表达(英文)

介绍一种快速、高效构建小肽多拷贝基因表达载体的策略 ,并构建了相应的表达载体pETE coT .用人工合成的编码 2 8个氨基酸残基的胸腺素α1基因为模型 ,采用限制酶EcoT14I识别序列CCAAGG为小肽基因两末端序列 ,利用其酶切后可产生非镜相对称粘性末端 ,一次连接反应就构建出一系列不同基因拷贝数的表达载体 ;在小肽基因两端分别引进编码FactorXa和羟胺蛋白切割位点的序列 ,表达出的融合蛋白可被FactorXa和羟胺剪切出不残留任何外源氨基酸的小肽 .不同拷贝数的小肽融合蛋白在大肠杆菌BL2 1(DE3)中均获得高效表达 .     

胰岛素原C肽多聚体基因在大肠杆菌中的表达及重组C肽在水溶液中的稳定性

合成了胰岛素原C肽三聚体的基因,并在大肠杆菌中得到高效表达。表达的融合蛋白质通过设计的特异性位点的酶切得到重组的人胰岛素原C肽单体。融合蛋白质以可溶性蛋白质的形式表达,表达量约为80mg／L。Ni—NTA亲和柱可有效地从细胞裂解的上清液中分离纯化融合蛋白质,得到纯度大于70％的融合蛋白质37．5mg／L。融合蛋白质可经胰蛋白酶/羧肽酶B双酶切有效释放天然的C肽。释放的C肽经氨基酸组成分析、与标准对照的RP—HPLC分析和免疫发光法定量证实与天然人C肽完全相同。C肽经RP-HPLC纯化后可得,总的收率为1．5mg/L,纯度大于95％。考察了C肽在冻干过程中及在水溶液中的稳定性。结果表明C肽在冻干过程中稳定,在水溶液中其稳定性受pH和温度的影响。在pH3和pH7．4缓冲液中,37℃或70℃下,C肽的降解符合一级动力学。C肽冻干品直接溶解于pH9的缓冲液中,立即降解,降解产物在37℃和70℃下基本稳定。C肽冻干品直接溶解于pH3的缓冲液中,立即发生降解反应,随后可观察到随温度升高和时间延长,降解反应的进行,37℃、10h,可观察到约80．3％的C肽保持,而在70℃、10h,只有43％C肽保持。C肽在pH7．4最稳定,37℃、6h或10g／L BSA存在下,70℃、3h,未观察到降解产物。PH7．4、37℃、10h,在有和没有10g／L BSA存在的情况下,C肽的保持率分别为99％和96％,从而显示了BSA的保护作用。     

恶性疟原虫pfs25多拷贝基因在毕赤酵母中的表达分析

目的:对恶性疟原虫pfs25的多拷贝基因重组菌株在毕赤酵母中进行表达分析,研究基因拷贝数对Pfs25蛋白表达量的影响。方法:构建重组质粒pAO815-αpfs25,利用BglⅡ-BamHⅠ同尾酶的特点,将基因表达框AOX1-αpfs25-AOX1(TT)插入到单拷贝表达质粒,依次重复,构建多拷贝重组质粒pAO815-(αpfs25)n,线性化后电转化毕赤酵母GS115,用MD平板筛选并进行表达分析。结果:构建得到1、2、3、4、5、6、7、8、10、12和14个pfs25基因拷贝的重组菌株,8拷贝pfs25基因的重组菌株表达量最高。结论:成功得到了pfs25基因的多拷贝表达重组菌株,经分析,多拷贝pfs25基因的目的蛋白表达量与其拷贝数并不呈线性正相关。     

戊型肝炎病毒多拷贝重组质粒的构建及在毕赤酵母中的表达

目的体外构建戊型肝炎病毒(HEV)ORF2128-660多拷贝重组表达质粒,以提高HEVORF2128-660在毕赤酵母中的表达水平。方法采用PCR技术扩增目的基因ORF2128-660(E),然后将ORF2128-660(E)同义点突变为ORF2128-660,构建重组表达质粒ORF212 8-660/p AO815(单拷贝),Bam HⅠ和BglⅡ双酶切获得的目的基因表达盒(AOX-ORF2128-660)插入去磷酸化的质粒ORF2128-660/p AO815,得到2(AOX-ORF2128-660)/p AO815(2拷贝)质粒。重复上述方法依次构建3(AOX-ORF2128-660)/p AO815(3拷贝)、4(AOX-ORF2128-660)/p AO815(4拷贝)等多拷贝重组质粒。得到的多拷贝重组质粒电转化毕赤酵母菌GS115,甲醇诱导表达,SDS-PAGE分析比较不同拷贝数转化子的表达产量,ELISA检测表达产物的生物活性。结果构建了戊型肝炎病毒(HEV)ORF2128-660多拷贝重组表达质粒,表达的目的蛋白相对分子质量约为59 000,4拷贝重组质粒表达水平高于其他拷贝数重组质粒表达水平。结论成功构建了HEVORF2128-660多拷贝表达质粒,提高了其在毕赤酵母中的表达水平。     

一种多拷贝毕赤酵母表达载体的构建及人脑源性神经营养因子的表达

利用PCR技术扩增出pPIC9K载体的HIS4 Kan序列片段 ,与pPICZα重组 ,构建结合了两个载体特点的适合体外构建多拷贝基因表达盒的毕赤酵母表达载体pPICZα1,改造后的载体含HIS4 Kan序列 ,能整合到酵母染色体上 ,具有筛选方便、外源基因多拷贝组建迅速、蛋白产物分泌表达和易纯化等优点。将人脑源性神经营养因子(hBDNF)的cDNA(35 7bp)克隆入载体pPICZα1,利用同尾酶BglⅡ、BamHⅠ不可逆连接方式 ,分别构建含有 1、2、3、6个拷贝hBDNF表达盒的重组表达载体 ,电击法转化毕赤酵母GS115菌株 ,用G4 18和Zeocin筛选转化子 ,筛选到的阳性转化子用 0 5 %甲醇诱导 ,获得分泌型表达。表达产物类似于天然神经营养因子单体大小、分子量约 14kD。多拷贝hBDNF表达盒的表达水平亦高于单拷贝hBDNF表达盒 ,ELISA和Westernblot检测表明 :表达的蛋白能与鸡抗人脑源性神经营养因子抗体特异结合 ,证实该表达蛋白具有hBDNF的免疫原性     

FAD依赖的葡萄糖脱氢酶多拷贝毕赤酵母菌株的构建及高效表达

通过体外多拷贝构建实现FAD依赖的葡萄糖脱氢酶(FAD-GDH)在毕赤酵母(Pichia pastoris)X33菌株中的高效表达。将前期构建的经密码子偏好性优化FAD-GDH基因插入到pPICZαA质粒中，通过同尾酶法酶切酶连构建含1～4个表达盒的重组表达质粒，分别电转至毕赤酵母X33中，成功筛选到各种重组菌株。qRT-PCR测定结果表明，载体所含的表达盒数目与嵌合进毕赤酵母基因组中的GDH基因拷贝数之间存在正相关关系。重组菌在试管水平用甲醇诱导72 h,酶活达到最高，其中4拷贝的转化子表达水平最高；选择1拷贝和4拷贝转化子进行10 L发酵罐扩大培养，1拷贝菌株诱导108 h酶活达到最高697.125 U/mL,4拷贝菌株诱导132 h酶活达到最高1 063.279 U/mL,比1拷贝酶活提高52.52%。结果表明通过增加目的基因拷贝数策略有助于提高FAD-GDH的表达量，为其进一步扩大生产提供参考。     

抗真菌肽CGA-N46基因多顺反子表达载体的构建与鉴定

目的:为提高抗真菌肽CGA-N46表达量,对该基因多顺反子表达进行研究.方法:以pEASY-Blunt为克隆栽体,以“pET-30a rbs序列-起始密码子-CGA-N46编码序列-终止密码子”为外源片段,利用同尾酶Nhe I、Spe I和Xba I,构建了含有上述外源片段1、3、5、8拷贝的重组载体pT-CAN46、...     

玉米赤霉烯酮降解酶多拷贝毕赤酵母菌株的构建及高效表达

通过密码子优化、体外多拷贝构建实现玉米赤霉烯酮(Zearalenone,ZEN)降解酶基因(zlhy-6)在毕赤酵母GS115菌株中的高效表达。按酵母密码子偏好性优化zlhy-6基因的密码子,与α因子信号肽编码序列一起合成,插入到pAO815质粒中,通过酶切酶连构建含1–6个表达盒的表达质粒,将其转入毕赤酵母GS115菌中,获得ZEN降解酶重组菌株。重组蛋白分子量为28.9 kDa,与预期一致。重组菌用甲醇诱导3 d,蛋白浓度达最高,之后下降;在pH 5.0、4.5条件下诱导培养,表达量最高;每天添加0.8%的甲醇、接种量10%表达水平最高;4拷贝的转化子表达水平最高,三角瓶发酵3 d,酶活性达到10 U/mL。在1 g玉米渣中添加0.1–0.5 mL发酵上清液,水解24 h,玉米渣中ZEN的降解率为44.08%–75.51%。研究结果为ZEN降解酶工业生产及在食品饲料中的应用奠定了基础。     

Smart tools for antimicrobial peptides expression and application: The elastic perspective

In recent years, antimicrobial peptides (AMPs) have become a promising alternative to the use of conventional and chemically synthesized antibiotics, especially after the emergence of multidrug-resistant organisms. Thus, this review aims to provide an updated overview of the state-of-the-art for producing antimicrobial peptides fused or conjugated with the elastin-like (ELP) peculiar carriers, and that are mostly intended for biomedical application. The elastin-like biopolymers are thermosensitive proteins with unique properties. Due to the flexibility of their modular structure, their features can be tuned and customized to improve the production of the antimicrobial domain while reducing their toxic effects on the host cells. Both fields of research faced a huge rise in interest in the last decade, as witnessed by the increasing number of publications on these topics, and several recombinant fusion proteins made of these two domains have been already described but they still present a limited variability. Herein, the approaches described to recombinantly fuse and chemically conjugate diverse AMPs with ELPs are reviewed, and the nature of the AMPs and the ELPs used, as well as the main features of the expression and production systems are summarized.     

抗菌肽融合表达研究进展

抗菌肽抗菌谱广、活性稳定,且具有与抗生素不同的抗菌机制,在抑杀病原微生物的同时不易产生耐药性,因而在食品、饲料、医药等领域具有重要的应用价值。基因工程技术是降低抗菌肽生产成本的主要方式,其中融合表达在提高抗菌肽产量方面起到了重要作用。文中综述了抗菌肽融合表达的国内外研究进展,探讨了部分融合标签用于抗菌肽表达的策略,并对今后的发展提出了自己的看法。     

丝状真菌纤维素酶合成诱导及转录调控

利用廉价可再生木质纤维素资源水解产生的可发酵糖生产生物能源和生物基化学品是近年来国内外研究的热点。纤维素酶酶解是木质纤维素原料生物降解的重要手段,但目前纤维素酶生产成本过高,限制了纤维素生物转化和生物炼制的工业化应用。对丝状真菌纤维素酶基因表达和调控进行研究,有利于进一步选育纤维素酶高产菌株,降低纤维素酶生产成本。随着高通量测序及丝状真菌遗传操作等技术的进步,对丝状真菌纤维素酶诱导和基因表达调控机理有了更深入的认识。本文综述了近年来丝状真菌纤维素酶诱导和纤维素酶基因表达调控的最新进展,重点论述糖转运蛋白、转录因子和染色质重塑对纤维素酶表达调控的影响,并对利用人工锌指蛋白进行丝状真菌纤维素酶诱导调控研究进行了展望。     

Tachykinin-related peptides in invertebrates: a review

Peptides with sequence similarities to members of the tachykinin family have been identified in a number of invertebrates belonging to the mollusca, echiuridea, insecta and crustacea. These peptides have been designated tachykinin-related peptides (TRPs) and are characterized by the preserved C-terminal pentapeptide FX1GX2Ramide (X1 and X2 are variable residues). All invertebrate TRPs are myostimulatory on insect hindgut muscle, but also have a variety of additional actions: they can induce contractions in cockroach foregut and oviduct and in moth heart muscle, trigger a motor rhythm in the crab stomatogastric ganglion, depolarize or hyperpolarize identified interneurons of locust and the snail Helix and induce release of adipokinetic hormone from the locust corpora cardiaca. Two putative TRP receptors have been cloned from Drosophila; both are G-protein coupled and expressed in the nervous system. The invertebrate TRPs are distributed in interneurons of the CNS of Limulus, crustaceans and insects. In the latter two groups TRPs are also present in the stomatogastric nervous system and in insects endocrine cells of the midgut display TRP-immunoreactivity. In arthropods the distribution of TRPs in neuronal processes of the brain displays similar patterns. Also in coelenterates, flatworms and molluscs TRPs have been demonstrated in neurons. The activity of different TRPs has been explored in several assays and it appears that an amidated C-terminal hexapeptide (or longer) is required for bioactivity. In many invertebrate assays the first generation substance P antagonist spantide I is a potent antagonist of invertebrate TRPs and substance P. Locustatachykinins stimulate adenylate cyclase in locust interneurons and glandular cells of the corpora cardiaca, but in other tissues the putative second messenger systems have not yet been identified. The heterologously expressed Drosophila TRP receptors coupled to the phospholipase C pathway and could induce elevations of inositol triphosphate. The structures, distributions and actions of TRPs in various invertebrates are compared and it is concluded that the TRPs are multifunctional peptides with targets both in the central and peripheral nervous system and other tissues, similar to vertebrate tachykinins. Invertebrate TRPs may also be involved in developmental processes.     

抗菌肽在大肠杆菌中的融合表达

抗菌肽作为新一代抗生素的潜在应用价值使其备受关注,大量高纯度的抗菌肽是开展基础及临床实验的关键。天然来源的抗菌肽资源有限、纯化困难,化学合成抗菌肽成本高、活性不稳定,因此通过基因重组表达得到大量抗菌肽是低成本、高效益的方法。目前采用大肠杆菌表达系统获得抗菌肽已成为研究者的首选,通常以形成融合蛋白的方式表达,这不仅可避免抗菌肽对宿主的杀伤作用,也保护了抗菌肽免受蛋白酶降解。文章结合课题组的研究工作,综述了近年来抗菌肽在大肠杆菌中表达的融合载体、融合蛋白的裂解方法及表达条件优化的研究进展。     

Physical reasons for secondary structure stability: alpha-helices in short peptides

It was recently found that some short peptides (including C- and S-peptide fragments of RNase A) can have considerable helicity in solution, ^1–12 which was considered to be surprising. Does the observed helicity require a new explanation, or is it consistent with previous understanding? In this work we show that this helicity is consistent with the physical theory of secondary structure^12–19 based on an extension of the conventional Zimm-Bragg model.²⁰ Without any special modifications, this theory explains reasonably well almost all the experimentally observed dependencies of helicity on pH, temperature, and amino acid replacements. We conclude that the observed “general level” of helicity of C- and S-peptides (5–30% at room temperature and 10–50% near 0°C) is “normal” for short peptides consisting mainly of helix-forming and helix-indifferent residues. The helicity is modified by a multitude of weak specific side chain interactions, many of which are taken into account by the present theory;^13–19 some discrepancies between the theory and experiment can be explained by weak side-chain-side chain interactions that were neglected. A reasonable coincidence of the theory with experiment suggests that it had been used to investigate the role of local interactions in the formation of α-helical “embryos” in unfolded protein chains.     

Central genomic regulation of the expression of oestrous behaviour in dairy cows: a review

The expression of oestrous behaviour in Holstein Friesian dairy cows has progressively decreased over the past 50 years. Reduced oestrus expression is one of the factors contributing to the current suboptimal reproductive efficiency in dairy farming. Variation between and within cows in the expression of oestrous behaviour is associated with variation in peripheral blood oestradiol concentrations during oestrus. In addition, there is evidence for a priming role of progesterone for the full display of oestrous behaviour. A higher rate of metabolic clearance of ovarian steroids could be one of the factors leading to lower peripheral blood concentrations of oestradiol and progesterone in high-producing dairy cows. Oestradiol acts on the brain by genomic, non-genomic and growth factor-dependent mechanisms. A firm base of understanding of the ovarian steroid-driven central genomic regulation of female sexual behaviour has been obtained from studies on rodents. These studies have resulted in the definition of five modules of oestradiol-activated genes in the brain, referred to as the GAPPS modules. In a recent series of studies, gene expression in the anterior pituitary and four brain areas (amygdala, hippocampus, dorsal hypothalamus and ventral hypothalamus) in oestrous and luteal phase cows, respectively, has been measured, and the relation with oestrous behaviour of these cows was analysed. These studies identified a number of genes of which the expression was associated with the intensity of oestrous behaviour. These genes could be grouped according to the GAPPS modules, suggesting close similarity of the regulation of oestrous behaviour in cows and female sexual behaviour in rodents. A better understanding of the central genomic regulation of the expression of oestrous behaviour in dairy cows may in due time contribute to improved (genomic) selection strategies for appropriate oestrus expression in high-producing dairy cows.     

一种新的食品酵母表达系统:产朊假丝酵母

由于其安全性及高效性,产朊假丝酵母表达系统受到越来越广泛的重视。该酵母具有以下特点：严格好气的条件下生长不会产生乙醇,发酵密度高,在廉价的糖蜜中能生长,本详细介绍了该系统的生物学特点,转化方法和应用前景。