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。     

昆虫杆状病毒表达系统研究进展

本文概括了昆虫杆状病毒的生物学、基因组结构和调控方面取得的进展,并对该载体系统的构建及外源基因的表达等研究作了评述。     

昆虫杆状病毒表达载体系统与疫苗研制的30年回顾

1983年12月,一篇关于用昆虫杆状病毒感染昆虫细胞表达重组人β干扰素文章的发表,标示了杆状病毒表达载体系统(Baculovirus expression vector system,BEVS)的诞生。迄今为止,已采用BEVS表达生产了数千种重组蛋白,其中7种已被成功用于商品化人用或兽用疫苗的抗原。仅对30年来基于BEVS的疫苗研制作一论述。     

昆虫表达系统及其在基因工程抗体中的应用

昆虫表达系统是一类应用广泛的真核表达系统,该系统具有与其他高等真核表达系统相似的翻译后修饰、加工及转移外源蛋白的能力。本文介绍了昆虫表达系统的构建过程,并以基因工程抗体为主讨论了外源蛋白在昆虫表达体系中的表达特征。     

昆虫杆状病毒系统表达外源蛋白的糖基化

昆虫表达系统作为一类应用广泛的真核表达系统 ,具有与多数高等真核生物相类似的翻译后修饰的过程。但其生产的重组糖蛋白一般仅具有高甘露糖或寡甘露糖型糖链 ,难以生成复杂构型糖链成为该系统的缺陷之一。综述了目前昆虫杆状病毒系统表达外源蛋白的糖基化研究进展。     

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。     

蚕杆状病毒载体表达系统及其

就杆状病毒的分子生物学特性及其作为表达载体以蚕体为表达外源基因的特点和优点进行综述,并对该表达系统在动物疫苗生产中的应用作一简要概述.     

昆虫杆状病毒表达载体系统在疫苗研究中的应用进展

昆虫杆状病毒表达载体系统(Baculovirus expression vector system,BEVS)已成功应用于多种蛋白的表达,并为疫苗开发提供了充足的原材料。相比其他表达系统,BEVS具有许多优势:杆状病毒专一寄生于无脊椎动物,安全性高;重组蛋白表达水平高;可对重组蛋白进行正确折叠和翻译后修饰,获得具有生物活性的蛋白;适应于多基因表达如病毒样颗粒(Virus-like particle)的复杂设计;适用于大规模无血清培养等。为了更好地理解BEVS在疫苗研究中的应用前景,文中将从BEVS的发展及其在疫苗研究中的应用等方面进行综述。     

杆状病毒表达载体的应用现状

杆状病毒是一类具有囊膜的双链环状DNA病毒,主要感染无脊椎动物,在病毒生活周期中会产生两种不同形态的病毒粒子:出芽型病毒粒子(BV),主要负责细胞之间的感染;包埋型病毒粒子(ODV),主要负责虫体之间的感染。随着对杆状病毒研究的不断深入,人们对杆状病毒的应用也越来越广泛,通过对病毒基因组的改造使其成为一种新颖的真核表达载体,现已被广泛应用于蛋白生产中;其次,由于杆状病毒特有的形态,可将靶蛋白展示在病毒粒子表面,进而被应用于诸如医药、临床和生物等多个研究领域中;此外,杆状病毒不能在哺乳动物细胞中进行复制,也不会在这类细胞中增殖和扩散。因此,杆状病毒不会刺激哺乳动物产生强烈的免疫应答,也不会对它们造成功能性的损伤,这些特性使其成为一种极具应用前景的基因治疗载体,给肿瘤治疗、组织再生和靶向给药等民生领域带来福音。本文就杆状病毒在蛋白表达、表面展示和基因治疗方面的研究进行综述,为杆状病毒的分子改造和临床应用提供依据。     

A new Trichoplusia ni cell line for membrane protein expression using a baculovirus expression vector system

A new cell line, MSU-TnT4 (TnT4), was established from Trichoplusia ni embryos for use with baculovirus expression vectors and evaluated for its potential for membrane protein production. To evaluate membrane protein synthesis, recombinant baculoviruses were constructed to express the human neurotensin receptor 1 as an enhanced green fluorescent protein (GFP) fusion. TnT4 cells had a doubling time of 21 h and expressed the membrane-GFP fusion protein at approximately twice the level as Sf21 cells from the p10 promoter, as evaluated by GFP intensity. Expression of secreted alkaline phosphatase (SEAP) was similar to that of Sf21 cells. Expression of membrane-GFP fusion proteins in recombinant baculoviruses provides a rapid method for evaluating the potential of new cell lines for the production of membrane proteins using a baculovirus expression vector system (BEVS).     

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.     

A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells

Since the number of potential drug targets identified has significantly increased in the past decade, rapid expression of recombinant proteins in sufficient amounts for structure determination and modern drug discovery is one of the major challenges in pharmaceutical research. As a result of its capacity for insertion of large DNA fragments, its high yield of recombinant protein and its high probability of success compared to protein expression in Escherichia coli, the baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. For some targets, however, expression of the recombinant protein with the BEVS in insect cells fails and mammalian expression systems have to be used to achieve proper post-translational processing of the nascent polypeptide. We now introduce a modified BEVS as a very useful tool for simultaneously testing the expression of target proteins in both insect and mammalian cells by using baculovirus infection of both host systems. The expression yields in insect cells are comparable to those obtained with state-of-the-art baculovirus vectors, such as the Bac-to-Bac system. Using the same virus, we can transduce mammalian cells to quickly assess target gene expression feasibility and optimize expression conditions, eliminating additional cloning steps into mammalian expression vectors. This reduces time and effort for finding appropriate expression conditions in various hosts.     

Modeling and optimization of the baculovirus expression vector system in batch suspension culture

A mathematical model has been developed that predicts the cell population dynamics and production of recombinant protein and infective extracellular virus progeny by insect cells after infection with baculovirus in batch suspension culture. Infection in the model is based on the rate of virus attachment to suspended insect cells under culture conditions. The model links the events following infection with the sequence of gene expression in the baculovirus replicative cycle. Substrate depletion is used to account for the decrease in product yield observed when infecting at high cell densities. Model parameters were determined in shaker flasks for two media: serum-supplemented IPL-41 medium and serum free Sf900II medium. There was good agreement between model predictions and the results from an independent series of experiments performed to validate the mode. The model predicted: (1) the optimal time of infection at high multiplicity of infection: (2) the timing and magnitude of recombinant protein production in a 2-L bioreactor; and (3) the timing and magnitude of recombinant protein production at multiplicities of infection from 0.01 to 100 plaque-forming units per cell. Through its ability to predict optimal infection strategies in batch suspension culture, the model has use in the design and optimization of large-scale systems for the production of recombinant products using the baculovirus expression vector system. (c) 1994 John Wiley & Sons, Inc.     

Optimization of protein-production by the baculovirus expression vector system in shake flasks

Summary Shake flasks were successfully employed for the cultivation of Spodoptera frugiperda (Sf-9) insect cells and for the production of \-galactosidase, a recombinant model protein, utilizing the baculovirus expression vector system. The culture doubling time and maximal cell density were 20 h and 5 × 10⁶ cells/ml respectively. The optimal liquid volumes for flasks rotating at 100 rpm were 25–40% of the flask total volume. Enzyme production (about 600 mg/l) was best at a multiplicity of infection of between 1 and 20 and at a cell density at time of infection of 0.7 × 10⁶ cells/ml. At a rotation speed of 100 rpm, Pluronic F-68 had no effect on growth and enzyme production. Offprint requests to: Y. Shoham     

Overexpression of plant histidinol dehydrogenase using a baculovirus expression vector system.

A cDNA encoding cabbage histidinol dehydrogenase, including the chloroplast transit peptide sequence, was overexpressed using a baculovirus expression vector system. The maximum level of the expression of histidinol dehydrogenase was reached 5 days after infection of the insect cells. Two forms of recombinant histidinol dehydrogenase with molecular masses of 53 and 52 kDa, respectively, were obtained by a one-step purification from the cell homogenate. Compared with the 52-kDa form, the 53-kDa form contained 10 additional amino acids at the N-terminus derived from the transit peptide. By incubating the cell homogenate for 2 h at 30 degrees C, the 53-kDa form could be completely converted to the 52-kDa form. This conversion was blocked by leupeptin. Eighty percent of the converted 52-kDa form had Cys at position 31 at the N-terminal amino acid and the rest had Met 33. Kinetic properties of the recombinant enzyme were virtually identical to those of histidinol dehydrogenase isolated from cabbage plants. The overexpression of recombinant cabbage histidinol dehydrogenase in insect cells, the proteolytic processing of the preprotein next to the N-terminus (compared to the mature cabbage enzyme), and its easy purification allow the preparation of large amounts of the active enzyme for structural and functional studies.     

Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system

Fed-batch culture can offer significant improvement in recombinant protein production compared to batch culture in the baculovirus expression vector system (BEVS), as shown by Nguyen et al. (1993) and Bedard et al. (1994) among others. However, a thorough analysis of fed-batch culture to determine its limits in improving recombinant protein production over batch culture has yet to be performed. In this work, this issue is addressed by the optimisation of single-addition fed-batch culture. This type of fed-batch culture involves the manual addition of a multi-component nutrient feed to batch culture before infection with the baculovirus. The nutrient feed consists of yeastolate ultrafiltrate, lipids, amino acids, vitamins, trace elements, and glucose, which were added to batch cultures of Spodoptera frugiperda (Sf9) cells before infection with a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) expressing beta-galactosidase (beta-Gal). The fed-batch production of beta-Gal was optimised using response surface methods (RSM). The optimisation was performed in two stages, starting with a screening procedure to determine the most important variables and ending with a central-composite experiment to obtain a response surface model of volumetric beta-Gal production. The predicted optimum volumetric yield of beta-Gal in fed-batch culture was 2.4-fold that of the best yields in batch culture. This result was confirmed by a statistical analysis of the best fed-batch and batch data (with average beta-Gal yields of 1.2 and 0.5 g/L, respectively) obtained from this laboratory. The response surface model generated can be used to design a more economical fed-batch operation, in which nutrient feed volumes are minimised while maintaining acceptable improvements in beta-Gal yield.