融合蛋白基因工程应用及研究

通过重组ＤＮＡ技术将不同的基因或基因片段融合在一起,经表达后可以得到由不同的功能蛋白拼合在一起而形成的新型多结构域的人工蛋白,这是一种极具潜在应用价值与理论意义的方法。目前这种方法已被广泛应用于诜我领域的研究中,并已显示出较高的价值,本文尝试对融合蛋白研究的几个领域的发展情况进行综述。     

单链抗体融合蛋白的构建及应用

通过重组DNA技术将单链抗体(scFv)基因与其他效应蛋白基因融合在一起,经表达后可以得到具有scFv特性和所融合的效应蛋白活性的scFv融合蛋白。这种融合蛋白已应用于许多领域的研究中,并已显示出较高的价值。本就scFv融合蛋白的构建和应用做一综述。     

全球融合蛋白药物研发态势分析

<正>1引言融合蛋白(fusion protein)是指将不同来源的基因或基因片段链接而表达的新型蛋白,其不仅能够提高功能蛋白的稳定性、延长在体内的代谢时间,同时又能融合一个或多个功能片段,形成高效靶向药物。目前融合蛋白已成为国际药学界研究的热点之一。本文的研究对象为利用重组DNA技术构建、以治疗为目的重组融合蛋白,也包括一些用于疫苗开发的病毒融合蛋白。     

草原兔尾鼠卵透明带3基因原核表达条件的优化

目的：通过大肠杆菌原核表达系统制备草原兔尾鼠卵透明带3（LZP3）融合蛋白,作为草原兔尾鼠疫苗免疫抗生育研究的抗原物质。方法：将LZP3基因的核心片段构建到融合表达载体pGEX-4T-1K中GST的3’端,形成GST-LZP3融合基因,经本科切鉴定和序列分析证实基因序列的正确性。在不同温度,不同时间和不同IPTG浓度进行诱导后,用SDS-PAGE检测融合蛋白的表达,发现有一条分子量约63kD的新增蛋白条带。结果：研究表明在37℃,25℃的不同温度条件下均能诱导出LZP3蛋白,经SDS-PAGE和间接ELISA法检测,初步证明了LZP3基因能够在大肠杆菌中有效表达可溶性的融合蛋白。结论：获得了LZP3融合蛋白表达的最佳诱导条件,为大量诱导产生LZP3融合蛋白奠定了理论基础。     

霍乱毒素B亚单位与谷氨酸脱羧酶抗原表位肽融合蛋白的表达及鉴定

目的:在大肠杆菌中表达霍乱毒素B单位(CTB)与谷氨酸脱羧酶(GAD)抗原表位肽段(531～545)的融合蛋白。方法:通过PCR技术将CTB基因与GAD基因融合在一起,插入pET22b载体,转化大肠杆菌BL21(DE3)后经诱导获得融合基因的表达;对表达产物进行包涵体复性后,纯化得到融合蛋白分子;对该融合蛋白分子进行了Western印迹和GM1-ELISA分析。结果:表达的融合蛋白的相对分子质量约为14000,Western印迹表明该融合蛋白具有霍乱毒素抗原性;GM1-ELISA实验表明该融合蛋白能够特异性地结合神经节苷酯GM1,表明该蛋白具有与CTB相似的五聚体结构。结论:融合蛋白CTB-GAD的成功表达,为后续动物实验提供了充足的抗原。     

嗜水气单胞菌外毒素和外膜蛋白双基因融合表达载体的构建和高效表达

用基因融合方式 ,以嗜水气单胞菌基因组DNA为模板 ,设计引物通过聚合酶链式反应 (PCR)把去除部分毒性活性编码区的细胞毒肠毒素基因 (act)与去除信号肽的外膜蛋白基因 (OmpTS)连接一起 ,两基因之间插入一个link er(Gly4Ser) 3 经BamHⅠ和HindⅢ双酶切 ,得到 2 .1kb的双基因融合片段 ,克隆于表达质粒pQE 30中 ,构建了双基因重组表达载体pQE30 /act GS ompTS ,转化大肠杆菌M15 (pREP4 ) ,经IPTG诱导 ,表达出预期大小 (81.0kD)的融合蛋白Act GS OmpTS ,此蛋白占菌体总蛋白的 4 2 %。Westernblot检测结果显示 ,该蛋白与抗Act兔血清和抗OmpTS兔血清都呈阳性反应 ,表明融合蛋白保留了外毒素和外膜蛋白的反应原性 ,为进一步研究此融合蛋白作为疫苗候选成分提供了理论依据     

丙肝抗原决定簇与CTB的融合、表达及融合蛋白的纯化

通过固相化学合成法合成了编码丙型肝炎病毒(HCV)结构区和非结构区的4个抗原决定簇基因,这些抗原决定簇基因片段以不同方式串联后与ctxB基因融合,构建了12种表达不同嵌合蛋白的重组质粒,各重组质粒转化大肠杆菌后均能高效分泌性表达融合蛋白,表达产量在10～50μg/ml之间,随所融合的抗原决定簇不同而不同,表达水平主要与抗原决定簇的氨基酸组成有关,而与抗原决定簇的大小及串联次数关系不大。融合蛋白通过亲和层析纯化,达到了电泳纯,为进一步研究融合蛋白的抗原性及用作抗-HCV ELISA诊断试剂抗原打下了基础。     

《自然-方法学》：基因编码“温度计”可潜入细胞测温度

据物理学家组织网10月16日报道,日本京都大学科学家最近将绿色荧光蛋白和沙门氏菌体内感受热量的一种蛋白融合在一起。制造出一种能检测细胞内部不同细胞器温度波动的基因编码”温度计”,并将细胞器温度变化与细胞内部功能联系在一起。有助于人们进一步理解细胞行为。相关论文发表在最近的《自然-方法学》杂志上。     

猴Fertilinβ氨基端93肽基因的克隆及其在大肠杆菌中表达

Fertilin是一种异二聚体精子表面蛋白.业已证明,它在精卵反应中起着重要作用.成熟的Fertilinβ亚基的氨基端93肽含有与整联蛋白结合的整联蛋白配体区.该整联蛋白配体区与蛇毒整联蛋白配体区高度同源,它们能与细胞表面的整联蛋白相结合.在此我们报道猴成熟Fertilinβ氨基端93肽基因的克隆和在大肠杆菌中的表达.用RT-PCR方法获得mmFβNTP93基因,克隆入表达载体pT7-7/hCGβ的EcoRI和BamHI位点,该基因与hCGβ基因一起表达融合蛋白mFβNTP93-hCGβ,Western blotting结果显示,融合蛋白的表观分子量为33kDa,能与抗hCG抗体专一性结合.由于mFβNTP93基因与hCGβ基因相连,因此我们推测,mFβNTP93基因已获得表达.     

SARS亚基疫苗-突刺蛋白受体结合区RBD在烟草叶绿体中的高效表达

叶绿体表达系统为植物源重组药用蛋白和亚基疫苗的生产提供了一个有效的途径。为验证SARS亚基疫苗在叶绿体中表达的可行性,以及为植物源SARS亚基疫苗的生产提供一套高效、低成本的技术平台,本研究将人工优化合成的SARS-CoV突刺蛋白(S蛋白)受体结合区序列RBD与载体分子CTB融合基因导入烟草叶绿体基因组中。PCR和Southern杂交分析表明,外源融合基因已整合到烟草叶绿体基因组中,并获得同质化。Western杂交分析表明,重组融合蛋白CTB-RBD在叶绿体转基因烟草中获得表达,且主要以可溶性单体形式存在。ELISA分析表明,在不同生长阶段、不同生长部位和不同时间点烟草叶片中,重组融合蛋白CTB-RBD的表达水平呈现明显的变化。重组蛋白在成熟叶片中的表达水平最高可以达到10.2%TSP。本研究通过SARS亚基疫苗RBD在烟草叶绿体中的高效表达,有望为植物源SARS亚基疫苗的生产以及SARS血清抗体的检测提供一个有效的技术平台。     

A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins.     

Study of protein splicing and intein-mediated peptide bond cleavage under high-cell-density conditions

Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins.     

Acidic pH induces fusion of cells infected with baculovirus to form syncytia.

The enveloped baculovirus insect cell system has been used extensively for expression of recombinant proteins, including viral fusion proteins. We tested wild-type baculovirus for endogenous fusion protein activity. Syncytia formation, dye transfer, and capacitance changes were observed after incubating infected Spodoptera frugiperda cells in acidic media, consistent with fusion protein activity. Only a short acidic pulse of 10 s is needed to trigger syncytia formation. Identical results were obtained with recombinant baculovirus. This new system is convenient for studying pH activated cell-cell fusion. However, using this enveloped virus to study the mechanism of recombinant fusion proteins requires caution.     

Secretion of the antibacterial recombinant protein enbocin

The insect baculovirus expression vector system (BEVS) is useful for the production of biologically active recombinant proteins. However, the overexpression of foreign proteins in this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we have developed a versatile baculovirus expression and secretion system using the Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion improved the secretion and antibacterial activity of recombinant enbocin proteins. Thus, bPDI gene fusion is a useful addition to the BEVS for the large-scale production of bioactive recombinant proteins.     

Bombyx mori protein disulfide isomerase enhances the production of nuecin, an antibacterial protein

The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpressions of foreign proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.     

Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase

Prokaryotic expression of polypeptides as fusion proteins with glutathione-S-transferase has recently been reported as a one-step means of purifying recombinant protein. The usefulness of the glutathione-S-transferase/glutathioneagarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insuluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione-S-transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one-step purification utilizing this scheme requires proper folding of recombinant glutathione-S-transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione-agarose affinity purification. Low-temperature induction of fusion protein synthesis, but not incubation with anion-exchange resins, led to improved one-step purification of glutathione-S-transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity-purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione-S-transferase.     

Progress on Ig Fusion Proteins Application

Ig融合蛋白是指在基因水平将目的基因同Ig部分片段基因相连 ,克隆并表达的重组蛋白。该蛋白具有目的蛋白的功能 ,又获取Ig的新特性。本文综述了Fab融合蛋白及Fc融合蛋白在临床及科学研究应用方面的进展。     

Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli

Extracellular production of recombinant proteins in Escherichia coli has several advantages over cytoplasmic or periplasmic production. However, nonpathogenic laboratory strains of E. coli generally excrete only trace amounts of proteins into the culture medium under normal growth conditions. Here we report a systematic proteome-based approach for developing a system for high-level extracellular production of recombinant proteins in E. coli. First, we analyzed the extracellular proteome of an E. coli B strain, BL21(DE3), to identify naturally excreted proteins, assuming that these proteins may serve as potential fusion partners for the production of recombinant proteins in the medium. Next, overexpression and excretion studies were performed for the 20 selected fusion partners with molecular weights below 40 kDa. Twelve of them were found to allow fused proteins to excrete into the medium at considerable levels. The most efficient excreting fusion partner, OsmY, was used as a carrier protein to excrete heterologous proteins into the medium. E. coli alkaline phosphatase, Bacillus subtilis alpha-amylase, and human leptin used as model proteins could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. When only the signal peptide or the mature part of OsmY was used as a fusion partner, no such excretion was observed; this confirmed that these proteins were truly excreted rather than released by outer membrane leakage. The recombinant protein of interest could be recovered by cleaving off the fusion partner by enterokinase as demonstrated for alkaline phosphatase as an example. High cell density cultivation allowed production of these proteins to the levels of 250-700 mg/L in the culture medium, suggesting the good potential of this approach for the excretory production of recombinant proteins.     

Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves

The production of recombinant proteins in plants is an active area of research and many different high-value proteins have now been produced in plants. Tobacco leaves have many advantages for recombinant protein production particularly since they allow field production without seeds, flowers or pollen and therefore provide for contained production. Despite these biosafety advantages recombinant protein accumulation in leaves still needs to be improved. Elastin-like polypeptides are repeats of the amino acids “VPGXG” that undergo a temperature dependant phase transition and have utility in the purification of recombinant proteins but can also enhance the accumulation of recombinant proteins they are fused to. We have used a 11.3 kDa elastin-like polypeptide as a fusion partner for three different target proteins, human interleukin-10, murine interleukin-4 and the native major ampullate spidroin protein 2 gene from the spider Nephila clavipes. In both transient analyses and stable transformants the concentrations of the fusion proteins were at least an order of magnitude higher for all of the fusion proteins when compared to the target protein alone. Therefore, fusions with a small ELP tag can be used to significantly enhance the accumulation of a range of different recombinant proteins in plant leaves. An erratum to this article can be found at     

结核分枝杆菌ESXB-ESXA融合蛋白的表达及其作为检测抗原的初步应用

目的:探索一种大量表达结核分枝杆菌ESXB-ESXA融合蛋白的方法,分析其抗原性,评价其在结核抗体检测中的初步应用。方法:采用PCR方法从结核分枝杆菌基因组中分别扩增esxB和esxA基因,用Linker(G4S)3连接2条目的片段,连接pET-32a(+)载体,构建ESXB-ESXA融合蛋白表达载体;重组表达载体转化宿主细胞大肠杆菌BL21(DE3),IPTG诱导表达、纯化,Western印迹分析重组蛋白的抗原性;建立以融合重组蛋白为抗原的ELISA和胶体金检测方法,分析其在结核病抗体检测中的应用。结果:构建了ESXB-ESXA融合蛋白的表达载体,重组ESXB-ESXA在大肠杆菌中得以高效表达,表达量占菌体总蛋白的70%以上;Western印迹分析表明该重组蛋白具有较好的抗原性;ELISA和胶体金检测结果显示,用重组ESXB-ESXA抗原检测临床结核病患者有较好的特异性。结论:重组ESXB-ESXA融合蛋白表达量高并具有较好的抗原性,可作为结核病抗体检测的备选抗原。