杆状病毒在昆虫中的持续感染

杆状病毒（Baculovirus）是一类特异性感染节肢动物的环状双链DNA病毒,是野外控制害虫种群的重要生物因子,并已被开发为一种生物杀虫剂加以应用。杆状病毒感染昆虫宿主并不一定导致昆虫死亡,其持续感染（persistentinfection）在昆虫种群中普遍存在,且在某些刺激条件下,持续感染可被激活为增殖性感染并引发病毒流行病爆发。因此,杆状病毒持续感染对昆虫种群动力学以及病毒流行病学的研究具有重要意义。     

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。     

无血清培养昆虫细胞(BTI-Tn-5B1-4)的适应过程

昆虫细胞培养是近年来迅速发展起来的动物细胞培养工程中的一个新领域。人们可以利用杆状病毒在昆虫细胞内的感染、复制,来大量生产昆虫病毒作为生物杀虫剂[1]。而昆虫细胞杆状病毒表达载体系统的建立,则可通过昆虫细胞的体外培养大量表达病毒携带的外源基因。实践证明,这…     

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。     

SA脂质体介导DNA转染昆虫细胞的研究

ＳＡ脂质体介导ＤＮＡ转染昆虫细胞的研究张传溪（浙江农业大学应用昆虫学研究所杭州３１００２９）吴祥甫（中国科学院上海生物化学研究所上海２０００３１）杆状病毒昆虫表达系统是８０年代发展起来的高效真核表达系统,具有表达量高,表达产物后加工较完全等优点,因...     

昆虫杆状病毒若干基因的研究进展

昆虫杆状病毒(Insect baculovirus)对鳞翅目、双翅目和膜翅目等昆虫具有病原性,是一 种开发应用较广、高效的生物杀虫剂,具有杀虫专一、效果好、有流行传播作用等优点.为 了更好地利用昆虫杆状病毒为防治农林害虫服务,加快昆虫杆状病毒杀虫剂研究的步伐,本文归纳了近几年来有关昆虫杆状病毒基因研究进展供从事研究的人员参考.     

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。     

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。     

BJ46a基因在昆虫细胞的转染、蛋白表达及超微结构观察

目的研究蛇毒金属蛋白酶抑制剂BJ46a在杆状病毒表达系统的表达及宿主细胞Sf9超微结构的变化。方法将构建的杆状病毒重组穿梭载体Bacmid BJ46a,经Cellfectin脂质体介导转染Sf9昆虫细胞,获重组杆状病毒颗粒;以高滴度病毒感染Sf9昆虫细胞,行Western blot观察BJ46a融合蛋白的表达。在病毒扩增、蛋白表达过程应用透射电镜和超薄切片技术观察Sf9细胞超微结构的变化。结果Western blot分析表明：在转染病毒的Sf9细胞出现BJ46a融合蛋白表达条带。电镜观察表明：Sf9细胞在病毒扩增过程,细胞核增大,病毒发生基质形成,杆状病毒在其周围装配。蛋白表达期间,杆状病毒量剧增,细胞核内外存在大量丝状纤维。结论BJ46a基因可在杆状病毒表达系统成功表达,并伴随着宿主细胞超微结构的显著变化,其结果为杆状病毒表达系统的研究奠定坚实的基础。     

杆状病毒经口感染及经口感染因子研究进展

杆状病毒(Baculovirus)是一类专一性感染节肢动物的病原微生物,其宿主主要为鳞翅目、双翅目及膜翅目昆虫。在自然界中,杆状病毒通过经口感染的方式在其宿主中建立感染,其中杆状病毒经口感染因子发挥着重要的作用。本文回顾了杆状病毒的基本特性,阐述杆状病毒建立经口感染在进化上的必要性以及在此过程中的主要事件,包括杆状病毒经口感染方式的进化、病毒粒子与昆虫中肠微绒毛细胞的结合和融合等,并着重介绍杆状病毒经口感染因子的鉴定和功能研究。这些研究对人类了解和利用杆状病毒进行生物防治和外源基因表达具有重要的指导意义。     

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。     

Establishment of a soaking RNA interference and Bombyx mori nucleopolyhedrovirus (BmNPV)-hypersensitive cell line using Bme21 cell

The double-stranded RNA (dsRNA) mediated RNA interference (RNAi) is widely employed in silkworm and its tissue-derived cell lines for gene function analysis. Baculovirus expression vector system (BEVS) has an advantage for large-scale protein expression. Previously, combining these useful tools, we improved traditional AcMNPV-Sf9 BEVS to produce modified target glycoproteins, where the ectopic expression of Caenorhabditis elegans systemic RNAi defective-1 (SID-1) was found to be valuable for soaking RNAi. In current study, we applied CeSID-1 protein to a Bombyx mori NPV (BmNPV)-hypersensitive Bme21 cell line and investigated its properties both in soaking RNAi ability and recombinant protein expression. The soaking RNAi-mediated suppression in the Bme21 cell enables us to produce modified glycoproteins of interest in BmNPV–Bme21 BEVS.     

Catalase effect on cell death for the improvement of recombinant protein production in baculovirus-insect cell system

Baculovirus expression vector system (BEVS) in host insect cells is a powerful technology to produce recombinant proteins, as well as virus-like particles (VLP). However, BEVS is based on baculovirus infection, which limits the recombinant protein production by inducing insect cell death. Herein a new strategy to enhance cell life span and to increase recombinant protein production was developed. As baculovirus infection induces cellular oxidative stress, the ability of several antioxidants to inhibit cell death was tested during infection. The production of rotavirus structural proteins was used as model to analyse this new strategy. We found that only catalase is able to partially prevent cell death triggered by baculovirus infection and to inhibit lipid peroxidation. An increase in recombinant protein production was coupled with the partial cell death inhibition. In summary, the addition of catalase is a promising strategy to improve recombinant protein production in BEVS, by delaying insect cell death.     

Expression and biological activity of Baculovirus generated wild-type human slow alpha tropomyosin and the Met9Arg mutant responsible for a dominant form of nemaline myopathy

We have previously reported a Met9Arg mutation in the human skeletal muscle alpha tropomyosin gene (TPM3) associated with autosomal dominant nemaline myopathy [Nat. Genet. 9 (1995) 75]. We describe here the generation of wild-type (Wt-tpm3) and Met9Arg (M9R-tpm3) mutant human skeletal muscle slow alpha tropomyosin using the Baculovirus expression vector system (BEVS). This system produces correct posttranslationally modified recombinant tropomyosin proteins in insect cells. We show that the interactions of Wt-tpm3 with actin and tropomyosin are comparable to those of fast alpha tropomyosin isolated from chicken striated muscle. However, the recombinant M9R-tpm3 is at least 100 times less effective at binding actin than Wt-tpm3. This paper represents the first study of this mutation directly on the human isoform of tropomyosin that is involved in nemaline myopathy. It also represents the first time that human tpm3 has been produced using BEVS. This system can now be used to accurately demonstrate the effect of this (and other disease-associated tropomyosin mutations) on the interactions of tpm3 with the other protein components of the muscle thin filament, including those responsible for differing forms of nemaline myopathy.     

Development of a novel recombinant influenza vaccine in insect cells

Influenza is a highly contagious viral respiratory illness which is best prevented through vaccination. Currently, all US licensed influenza vaccines are produced in embryonated chicken eggs. The Baculovirus Expression Vector System (BEVS) technology offers several advantages over existing technology, including an exact match between the circulating virus and the antigen in the vaccine, speed, safety, versatility, and reliable scale-up. The expresSF+ insect cells are grown in the absence of serum and have been extensively qualified for safety according to ICH and US FDA guidance and for suitability for the production of recombinant proteins using BEVS. FluBlok^®, a recombinant hemagglutinin influenza vaccine, is composed of purified hemagglutinin protein produced using the BEVS technology. FluBlok has been shown to be safe, effective, and efficacious in human clinical studies.     

Chaperone and foldase coexpression in the baculovirus-insect cell expression system

Conclusions The BEVS has become widely utilized for production of recombinant proteins. However, protein aggregation and inefficient processing often limit yields, especially for secreted and membrane proteins. Since many proteins of pharmaceutical interest require similar posttranslational processing steps, engineering the folding, assembly, and secretion pathway may enhance the production of a wide variety of valuable complex proteins. Efforts should be undertaken to coexpress the relevant chaperones or foldases at low levels in concert with the final product to ensure the ideal folding and assembly environment. In the future, expression of oligosaccharide modifying enzymes and secretion factors may further improve secretion rates of assembled proteins and provide heterologous proteins with altered glycoforms. Also significant is the use of BEVS as an in vivo eucaryotic laboratory to study the fundamental roles of differnt chaperones, foldases, and secretion factors. The coexpression of chaperones and foldases will complement other approaches such as the development of alternative insect cell lines, promoters, and signal peptides to optimize the baculovirus-insect cell expression system for generating high yields of valuable proteins.Abbreviations BEVS Baculovirus expression vector system - BiP immunoglobulin heavy chain binding protein - ELISA Enzyme-linked immunosorbent assay - ER Endoplasmic reticulum - GRP Glucose regulated protein - Hsp Heat shock protein - IgG Immunoglobulin G - PDI Protein Disulfide Isomerase - PPI Peptidyl-prolyl cis-trans isomerase - Sf-9 Spodoptera frugeperda     

Production of human skeletal alpha-actin proteins by the baculovirus expression system

Mutations within the human skeletal muscle alpha-actin gene cause three different skeletal muscle diseases. Functional studies of the mutant proteins are necessary to better understand the pathogenesis of these diseases, however, no satisfactory system for the expression of mutant muscle actin proteins has been available. We investigated the baculovirus expression vector system (BEVS) for the abundant production of both normal and mutant skeletal muscle alpha-actin. We show that non-mutated actin produced in the BEVS behaves similarly to native actin, as shown by DNase I affinity purification, Western blotting, and consecutive cycles of polymerisation and depolymerisation. Additionally, we demonstrate the production of mutant actin proteins in the BEVS, without detriment to the insect cells in which they are expressed. The BEVS therefore is the method of choice for studying mutant actin proteins causing human diseases.     

Production and characterization of a recombinant beta‐1,4‐endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipes

Cell‐1 is a host‐derived beta‐1,4‐endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Here, we report on the heterologous production of Cell‐1 using eukaryotic (Baculovirus Expression Vector System; BEVS) and prokaryotic (E. coli) expression systems. The BEVS‐expressed enzyme was more readily obtained in solubilized form and more active than the E. coli–expressed enzyme. K_m and V_max values for BEVS‐expressed Cell‐1 against the model substrate CMC were 0.993% w/v and 1.056 µmol/min/mg. Additional characterization studies on the BEVS‐expressed enzyme revealed that it possesses activity comparable to the native enzyme, is optimally active around pH 6.5–7.5 and 50–60°C, is inhibited by EDTA, and displays enhanced activity up to 70°C in the presence of CaCl₂. These findings provide a foundation on which to begin subsequent investigations of collaborative digestion by coevolved host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1 beta glucosidases, phenol‐oxidizing laccases, and others. © 2010 Wiley Periodicals, Inc.     

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。