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。     

昆虫细胞内N-糖基化途径及人源化糖蛋白表达

虽然昆虫杆状病毒表达系统在蛋白表达领域得到了广泛的应用, 但由于不能表达复杂的末端唾液酸化的N-糖链, 使得该系统在生物制药行业的应用受到了很大的限制。通过比较哺乳动物细胞和昆虫细胞内糖基化途径可知, 其起始步骤一致, 之后再发生分化, 主要表现为3方面, 即昆虫细胞内缺乏哺乳动物细胞所具备的N-乙酰葡萄糖氨转移酶II、 半乳糖基转移酶/N-乙酰氨基半乳糖转移酶、α-2,3-唾液酸转移酶和α-2,6-唾液酸转移酶等延长N-糖链的糖基转移酶; 另外, 昆虫细胞内具有能够特异性地将蛋白质末端的N-乙酰氨基葡萄糖残基从GlcNAcMan3GlcNAc(±α3/6-Fuc)GlcNAc上切除的N-乙酰氨基葡萄糖苷酶及核心α-1,3-岩藻糖基转移酶。本文从上述异同出发, 综述了克服昆虫细胞内不能表达人源化糖蛋白这一缺陷所进行的N-糖基化途径的改造研究--主要集中在昆虫细胞内GlcNAcase的抑制和昆虫细胞内GnT2, GalT/ GalNAcT, ST3及ST6等基因的导入等方面, 结果表明经改造的昆虫细胞可表达人源化糖蛋白, 这将极大地拓宽昆虫杆状病毒表达系统的应用领域。本文还探讨了选择特殊细胞系及特殊培养条件以在昆虫细胞内表达唾液酸化蛋白的可行性。     

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。     

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。     

农业昆虫气味受体功能研究进展

昆虫主要依靠嗅觉系统寻找食物、发现配偶、控制交配、选择产卵地、逃避天敌等,因此嗅觉系统对昆虫的繁殖和生存至关重要。气味受体(odorant receptor, OR)是嗅觉系统中的关键成分之一,可被信息化合物激活引发特定行为产生。随着测序技术的发展,大量的农业昆虫的基因组和转录组被测序,从测序数据中分析获得了它们的OR家族。现在OR功能研究经常用到外源表达系统和CRISPR/Cas9系统。外源表达系统结合记录系统可用于目标OR表达和配体筛选,CRISPR/Cas9系统可从昆虫染色体上敲除目标OR基因,进而利用电生理技术和行为学实验研究其功能。本文系统总结了鳞翅目、直翅目、半翅目、双翅目、膜翅目和鞘翅目6个目30个种的农业昆虫OR的气味分子反应谱及功能,尤其是鳞翅目农业昆虫。农业昆虫的性信息素往往是由雌虫产生的两种或两种以上成分按一定比例组成的混合物,包括在种间生殖隔离中起作用的行为拮抗剂。每个种因此具有多个性信息素的受体,共同感受这些信息素信息,调控种内和种间性行为。有些OR可调谐至主要针对植物挥发物,包括花香气味,在寄主植物选择和雌虫选择产卵地等行为中起作用。聚集信息素受体可特异地被聚集信息素激活,引起聚集行为;报警信息素受体可特异地被报警信息素激活,引起排斥行为。研究农业昆虫OR的气味分子反应谱和功能可为开发在害虫防控中应用的性诱剂、食诱剂、拒食剂、聚集信息素等奠定理论基础。最后我们对未来的主要研究方向提出了以下建议：(1)开发新的外源OR表达系统;(2)研究在雌虫中特异调谐至针对雄虫产生的性信息素的性信息素受体的功能;(3)探讨OR与气味分子之间特异性相互作用的分子机制。     

昆虫嗅觉相关蛋白的结构和功能

昆虫在长期进化的过程中形成了复杂的嗅觉系统,气味剂结合蛋白(odorant binding proteins,OBPs)、嗅觉受体(olfactory receptors,ORs)是其最主要的组分.其主要作用是结合外围挥发性的气味分子并将信号传递给细胞内的第二信使.OBPs和ORs的结构、功能、表达、进化是昆虫行为与进化关系的重要研究领域和研究热点.本文主要总结了近年来昆虫OBPs和ORs的结构特点、生理功能、表达特点、遗传进化等方面研究的最新进展,对OBPs和ORs的研究趋势进行了展望,为昆虫嗅觉系统进化研究及寻找害虫防治新途径提供参考信息.     

昆虫病毒表达关系的模型建立

昆虫杆状病毒作为生物杀虫剂以及在基因治疗方面的应用有着独特的优越性,而昆虫杆状病毒表达系统(BEVS)也是当今基因工程领域四大表达系统之一,得到广泛的应用.在我们对部分昆虫杆状病毒分子生物学已有较深入认知的情况下,研究其病毒表达系统及生物过程,将有助于我们有效地利用其优越的性能,开拓新的实用价值.类病毒颗粒(Virus...     

基于VOSviewer和CiteSpace的昆虫肠道微生物领域可视化分析

【背景】近年来，昆虫肠道微生物领域受到科研工作者的广泛关注，相应发表了大量的科研论文和著作。然而目前尚缺乏对昆虫肠道微生物全面、系统的文献计量分析。【目的】了解国内外昆虫肠道微生物领域的历史、研究热点和新兴趋势。【方法】以昆虫肠道微生物为搜索主题对中国知网(China National Knowledge Infrastructure, CNKI)数据库和Web of Science (WOS)数据库进行检索，利用文献计量工具VOSviewer和Cite Space对关键词进行分析。【结果】1991-2022年间该领域全球研究发文量整体呈现上升趋势，国内外对昆虫肠道微生物领域的关注点和研究方向逐渐扩大。关键词聚类锚定了3个新兴研究领域：昆虫饮食(insect meal)、次生代谢物(secondary metabolites)和生物降解(biodegradation)。【结论】基于文献计量学研究的发现提供了昆虫肠道微生物领域的现状和趋势，可能有助于确定该领域的热点话题和探索新的方向。     

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.     

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。     

Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry

The GM2-activator protein (GM2AP) is a small non-enzymatic cofactor assisting the enzyme beta-hexosaminidase A in the lysosomal degradation of ganglioside GM2. Mutations in the gene encoding this glycoprotein lead to a fatal neurological disorder, the AB variant of GM2-gangliosidoses. In this paper, we describe the overexpression of GM2AP in Sf21 cells using both the baculovirus expression vector system (BEVS) and a non-lytic, plasmid-based insect cell expression system (InsectSelect). For the BEVS, the cDNA encoding human GM2AP-preproprotein was cloned in the expression vector pAcMP3. The recombinant virus generated by cotransfection with linearized baculovirus DNA was used to infect Sf21 cells. For the non-lytic expression system, the cDNA of GM2AP was inserted into the vector pIZ/V5-His, which was used for the constitutive expression in stably transformed Sf21 cells. As it was shown by immunoblot analysis of the cell culture supernatant, in both expression systems the GM2AP precursor protein was efficiently secreted into the medium. Following expression in the BEVS, the GM2AP was purified by sequential chromatography on Ni-NTA-agarose and Con A-Sepharose, resulting in a yield of up to 9 mg purified protein from 1L of cell culture supernatant. Following expression in stably transformed insect cells, the secreted protein was first concentrated by cation-exchange and purified by metal-ion affinity chromatography, with a yield of 0.1 mg/L cell culture supernatant. The biological activity of the recombinant protein was demonstrated by its ability to stimulate the hexosaminidase A-catalyzed degradation of ganglioside GM2, and the homogeneity and glycosylation were assessed by ESI-TOF mass spectrometry. While the protein expression in the BEVS led to partly glycosylated and partly non-glycosylated protein, the stably transformed cells produced only glycosylated protein. In both expression systems, the glycosylation was found to be identical and corresponded to the structure (GlcNAc)(2)Fuc(Man)(3).     

Avidin is a promising tag for fusion proteins produced in baculovirus-infected insect cells.

The baculovirus expression vector system (BEVS) has become one of the most versatile and powerful eukaryotic systems for recombinant protein expression. We have constructed a novel baculovirus transfer vector (pbacAVs+C) which allows for the efficient production, detection, and single-step purification of the desired molecule as a secretion-compatible avidin fusion protein in insect cells. It also enables fast construction of the baculoviruses by site-specific transposition in Escherichia coli. To demonstrate the power of this vector, we report here on the production of immunologically intact hevein, a major cysteine-rich latex allergen, as avidin fusion protein. Our results indicate that avidin is a stable and versatile tag in the BEVS. It retains its extraordinarily high biotin-binding activity and also enables independent folding of the fusion partner. The versatility with which avidin fusion proteins can be detected, purified, and immobilized is the basis for the use of our system as a useful alternative in eukaryotic fusion protein production.     

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.     

Galatosylation and sialylation of mammalian glycoproteins produced by baculovirus-madiated gene expression in insect cells

The baculovirus expression vector system (BEVS) is used extensively for the production of proteins from exogenous cDNAs. However, BEVS is not ideal for pharmaceutical production of glycoproteins owing to the properties of the N-glycans in the expressed products and that insect cells lack several of the enzymes required for mammalian-type N-glycan synthesis. This study describes the effective mammalian-like production of glycoproteins, such as β-1,4-galactosyltransferase and α-2,6-sialyltransferase, in the insect cell line Sf9.Revisions requested 13 April 2005; Revisions received 17 May 2005     

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.     

Co-expression vs. co-infection using baculovirus expression vectors in insect cell culture: Benefits and drawbacks

The baculovirus expression vector system (BEVS) is a versatile and powerful platform for protein expression in insect cells. With the ability to approach similar post-translational modifications as in mammalian cells, the BEVS offers a number of advantages including high levels of expression as well as an inherent safety during manufacture and of the final product. Many BEVS products include proteins and protein complexes that require expression from more than one gene. This review examines the expression strategies that have been used to this end and focuses on the distinguishing features between those that make use of single polycistronic baculovirus (co-expression) and those that use multiple monocistronic baculoviruses (co-infection). Three major areas in which researchers have been able to take advantage of co-expression/co-infection are addressed, including compound structure-function studies, insect cell functionality augmentation, and VLP production. The core of the review discusses the parameters of interest for co-infection and co-expression with time of infection (TOI) and multiplicity of infection (MOI) highlighted for the former and the choice of promoter for the latter. In addition, an overview of modeling approaches is presented, with a suggested trajectory for future exploration. The review concludes with an examination of the gaps that still remain in co-expression/co-infection knowledge and practice.     

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.