昆虫杆状病毒表达系统的研究进展与应用

昆虫杆状病毒表达载体系统具有安全性好、重组蛋白表达量高、能同时表达多个基因、重组蛋白翻译后加工完整等特点,因而得到了广泛的应用。随着重组杆状病毒构建技术的不断发展,昆虫杆状病毒表达载体系统的操作在逐渐简化,重组杆状病毒获得的效率也在不断提高。昆虫细胞培养技术的改进和转基因昆虫细胞系的发展,进一步推动了昆虫杆状病毒表达载体系统在商品化药物、治疗性抗体、生物农药研发和基因治疗中的应用。尽管仍存在着重组蛋白降解的问题,但随着分子生物学技术的发展,对杆状病毒载体的研究与改造也会更加深入,未来昆虫杆状病毒表达载体系统的应用将更为广泛。     

外源基因在昆虫杆状病毒表达系统中的表达

随着杆状病毒载体和筛选方法的不断改进,通过Bac-to-Bac方法可以使杆状病毒最大重组率达到100%,缩短了构建重组载体的时间,极大提高了工作效率。另外,研究者开发了一些新的宿主域扩大的昆虫杆状病毒载体,能够在家蚕或蛹内进行高水平表达重组蛋白。昆虫杆状病毒表达系统具有完备的翻译后加工修饰功能和高效表达外源蛋白的能力等特点,是一种非常理想的真核表达系统。利用该表达系统现已成功表达了约千种外源蛋白。以重组杆状病毒为载体的昆虫表达系统、外源基因在该表达系统中的表达情况及在农业领域中的应用进行了介绍。     

杆状病毒载体及其在昆虫细胞中表达外源基因的研究进展

本文介绍了杆状病毒载体在昆虫细胞中表达外源基因的基本策略和发展趋势。杆状病毒载体系统近年来已被人们广泛用来表达人类、动物和植物等的一些重要蛋白质分子,在医学、农业等领域的基因工程研究中发挥了越来越大的作用。杆状病毒载体系统表达外源基因的效率高,表达产物的结构和活性与天然产物一致,为当今基因工程研究中最有发展前途的病毒载体表达系统。     

昆虫杆状病毒表达系统的研究与应用进展

昆虫杆状病毒表达系统 (BEVS)因具有完备的翻译后加工修饰系统和高效表达外源基因的能力等特点 ,现已成功表达了近千种高价值蛋白。随着杆状病毒载体的不断改进 ,该系统获得重组病毒的几率已从最初的 0 1 %～ 1 %提高到现在的 80 %～ 90 %以上 ,并且出现了一些新的宿主域扩大的昆虫杆状病毒载体和高水平表达重组蛋白的昆虫细胞系。杆状病毒载体将在未来药物研发、疫苗生产、基因治疗、重组杆状病毒杀虫剂等领域得到广泛应用。但存在的一些问题如杆状病毒的基因组学研究相对薄弱 ,有关病毒晚期基因的高表达和调控机制等还不十分清楚 ,表达产物的纯化比较困难 ,多元表达等方面的技术还不够成熟等 ,均有待进一步解决。     

重组杆状病毒：一种新型哺乳动物细胞基因转移载体

昆虫杆状病毒表达载体系统已广泛应用于表达重组蛋白。近年来研究显示,含有哺乳动物细胞启动子元件的重组杆状病毒可有效地转导多种哺乳动物原代和传代细胞。借助于杆状病毒载体,已成功实现了外源基因在哺乳动物细胞内的瞬时或稳定表达;而在体内,杆状病毒可被血清中的补体成份所灭活,从而抑制了转导效率,但是通过对杆状病毒进行修饰（如伪型杆状病毒）,可以抵抗补体的灭活作用。研究人员对杆状病毒转导机制进行了探索,但是至今尚未完全弄清。杆状病毒基因转移系统最大特点是,杆状病毒能在昆虫细胞内大量繁殖,而不能在哺乳动物细胞内复制,因而具有很高的生物安全性;同时,此系统还具有操作简便、插入外源基因容量大等优点,使得杆状病毒作为哺乳动物细胞的基因传递载体,具有广泛的应用前景。     

杆状病毒表达系统——有效的VLP构建工具

杆状病毒表达系统是以杆状病毒为外源基因载体,昆虫细胞或活体昆虫为受体的真核表达系统。相对于其他表达系统,杆状病毒表达系统具有特殊的优势:杆状病毒基因组作为表达载体可以容纳更多外源基因;杆状病毒极晚期启动子能有效调控外源蛋白的表达;昆虫细胞作为受体能够对外源蛋白进行加工修饰;杆状病毒通常只感染节肢动物,不会对人畜构成危害。因此,该系统越来越受到人们的重视,并已应用于亚单位疫苗的研发与生产,特别其对于构建病毒样颗粒,即由一种或多种病毒结构蛋白自行装配而成且不含病毒基因组的蛋白颗粒,具有不可比拟的优势。对此做详细评述并展望病毒样颗粒疫苗的发展趋势。     

昆虫杆状病毒应用于哺乳动物基因治疗的研究进展

杆状病毒是一类宿主特异性的昆虫病毒。昆虫杆状病毒表达系统是一个高效的真核表达系统,被广泛用于在昆虫细胞或昆虫幼虫中生产外源蛋白质。杆状病毒不能感染哺乳动物,却可以进入不同物种和组织来源的多种哺乳动物细胞,并在合适的哺乳动物启动子控制下表达外源基因。杆状病毒在哺乳动物细胞中不能复制,对细胞没有毒性,加上杆状病毒本身具有基因组大、可操作性好等优点,作为哺乳动物基因治疗的载体,将治疗基因传递给哺乳动物细胞已受到了广泛关注。在此就杆状病毒作为基因治疗载体的最新研究进展进行了阐述并探讨其发展趋势。     

昆虫杆状病毒载体系统研究为我国基因工程疫苗的研制开辟了新的途径

中山大学昆虫研究所王珣章博士(教授)和谢伟东博士(副教授)等近年来,在著名昆虫学家、中国科学院学部委员蒲蛰龙教授的支持下,开展对昆虫杆状病毒基因工程的研究工作,经过几年的艰苦努力,在杆状病毒载体系统的研究与开发上取得了重大突破,构建出了系列的高效、多功能的杆状病毒载体系统。建立了两个新的、高效的昆虫杆状病毒载体和表达系统,即粉纹夜蛾核型多角体病毒——草地夜蛾细胞和粉纹夜蛾幼虫系统以及粉纹夜蛾核型多     

杆状病毒表达系统研究进展

介绍了杆状病毒表达系统的构建策略,载体发展情况及其表达外源基因的影响因素.杆状病毒表达系统在基因工程、药物开发、疫苗生产等方面发挥了越来越重要的作用,其表达效率高,表达产物与天然产物有相似的结构和活性,且对人畜无害,为当今基因工程研究中最有发展前途的表达系统.     

杆状病毒基因工程研究进展

综述了杆状病毒基因工程研究的最新进展和研究成果,归纳了近年来（１９９１年以后）通过杆状病毒载体系统表达的外源基因,并提出了杆状病毒基因工程研究发展的方向。     

Optimizing the baculovirus expression vector system

Baculoviruses have a unique bi-phasic life cycle and powerful promoters, which greatly facilitates their use for recombinant protein expression in insect cells. We have developed an expression system that utilizes homologous recombination in insect cells between a transfer plasmid containing a gene to be expressed and a replication-deficient virus (bacmid). Only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses using robotic liquid handlers. The bacmid has also been genetically optimized for improved protein expression and stability. We describe the application of this system for high level production of recombinant proteins.     

丝氨酸蛋白酶的重组表达

丝氨酸蛋白酶是蛋白酶家族中的一大家族 ,广泛分布于细菌、病毒、真菌、植物、动物中 ,并参与生命的各种反应。随着丝氨酸蛋白酶研究的深入 ,重组表达获得大量的目的蛋白酶已成为研究该类蛋白酶的一大基础技术。本文重点综述了丝氨酸蛋白酶在不同表达系统中重组表达的优缺点及最新进展。     

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.     

Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system

The baculovirus-silkworm expression system is widely used as a mass production system for recombinant secretory proteins. However, the final yields of some recombinant proteins are not sufficient for industrial use. In this study, we focused on the signal peptide as a key factor for improving the efficiency of protein production. Endoplasmic reticulum (ER) translocation of newly synthesized proteins is the first stage of the secretion pathway; therefore, the selection of an efficient signal peptide would lead to the efficient secretion of recombinant proteins. The Drosophila Bip and honeybee melittin signal peptides have often been used in this system, but to the best of our knowledge, there has been no study comparing secretion efficiency between exogenous and endogenous signal peptides. In this study we employed signal peptides from 30K Da and SP2 proteins as endogenous signals, and compared secretion efficiency with those of exogenous or synthetic origins. We have found that the endogenous secretory signal from the 30K Da protein is the most efficient for recombinant secretory protein production in the baculovirus-silkworm expression system.     

Human cells: new platform for recombinant therapeutic protein production

The demand for recombinant therapeutic proteins is significantly increasing. There is a constant need to improve the existing expression systems, and also developing novel approaches to face the therapeutic proteins demands. Human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because this expression system is expected to produce recombinant proteins with post translation modifications more similar to their natural counterpart and reduce the potential immunogenic reactions against nonhuman epitopes. Currently, little information about the cultivation of human cells for the production of biopharmaceuticals is available. These cells have shown efficient production in laboratory scale and represent an important tool for the pharmaceutical industry. This review presents the cell lines available for large-scale recombinant proteins production and evaluates critically the advantages of this expression system in comparison with other expression systems for recombinant therapeutic protein production.     

The recombinant limb as a model for the study of limb patterning, and its application to muscle development

The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning. Received: 31 August 1998 / Accepted: 29 September 1998     

Expression, characterization, and biochemical properties of recombinant human salivary amylase

Human salivary amylase, a major component of human salivary secretions, possesses multiple functions in the oral cavity. It is the only enzyme in saliva capable of degrading oligosaccharides, which are used by the oral microflora for nutritional purposes. In order to understand its role in disease processes such as caries, we have undertaken the structure-function analyses of amylase. In this regard, the nonglycosylated human salivary amylase was expressed in a baculovirus expression system. The native and the recombinant amylases exhibit similar biochemical as well as biophysical properties. Unlike recombinant human pancreatic amylase, recombinant human salivary amylase is not glycosylated when expressed in a baculovirus system as determined from the crystal structure determination of the recombinant enzyme. Therefore, this system is suitable for further structure-function work without resorting to enzymatic removal of the carbohydrate chain. Details of the expression, purification, and biophysical properties will be presented.     

Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.     

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.     

A protocol for rapid generation of recombinant adenoviruses using the AdEasy system

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We have developed an approach that simplifies the generation and production of such viruses called the AdEasy system. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, employing homologous recombination in bacteria rather than in eukaryotic cells. After transfection of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of GFP encoded by a gene incorporated into the viral backbone. This system has expedited the process of generating and testing recombinant adenoviruses for a variety of purposes. In this protocol, we describe the practical aspects of using the AdEasy system for generating recombinant adenoviruses. The full protocol usually takes 4-5 weeks to complete.