A Baculovirus Expression System for Insect Cells

The review considers the biology of baculoviruses, construction of transfer vectors for the baculovirus expression system (BES), selection of recombinant baculoviruses, approaches to expression of multimeric proteins, and BES potentialities and prospects.     

Expression profiling of herpesvirus and vaccinia virus proteins using a high-throughput baculovirus screening system

We have developed a high-throughput system for generating baculoviruses and testing the expression, solubility, and affinity column purification of encoded proteins. We have used this system to generate baculoviruses for and analyze the expression of 337 proteins from three different herpesviruses (HSV-1, EBV, and CMV) and vaccinia virus. Subsets of these proteins were also tested for expression and solubility in E. coli. Comparisons of the results in the two systems are presented for each virus.     

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。     

Baculoviruses as Vectors in Mammalian Cells

The Baculoviridae are a large family of enveloped DNA viruses exclusively pathogenic to arthropods. Baculoviruses have been extensively used in insect cell-based recombinant protein expression system and as biological pesticides. They have been deomostrated to be safe to mammals, birds and fish. Recently, baculoviruses has been shown to transduce different mammalian cells in spite of the fact that they cannot replicate in mammalian cells (11, 73, 76). This has resulted in the development of baculoviruses as mammalian expression systems and even as vestors for gene therapy.     

Cell-specific expression of enhanced green fluorescence protein under the control of neuropeptide gene promoters in the brain of the silkworm,Bombyx mori,using Bombyx mori nucleopolyhedrovirus-derived vectors

Using the larvae of the silkworm, Bombyx mori, we examined the baculovirus expression vector system for the expression of the enhanced green fluorescence protein (EGFP) gene under the control of several gene promoters in vivo. To investigate the gene-delivery efficiency of the baculovirus into various larval tissues, we constructed two recombinant baculoviruses carrying the EGFP gene downstream of the Drosophila melanogaster hsp70 gene promoter from B. mori nucleopolyhedrovirus (BmNPV) and Autographa californica nucleopolyhedrovirus (AcNPV). After injection of these recombinant baculoviruses into newly ecdysed 5th instar larvae, hsp70::EGFP-BmNPV, but not hsp70::EGFP-AcNPV, caused intense expression of EGFP not only in various non-neural tissues, but also in the neural organs including the brain 5 days postinfection. To investigate the cell-specific expression in the brain, we constructed recombinant C4/B3::EGFP-BmNPV and PTTH::EGFP-BmNPV which carry the EGFP gene under the control of bombyxin B3 and prothoracicotropic hormone (PTTH) gene promoters, respectively. Injection of these recombinant baculoviruses caused specific expression of EGFP with a high gene-expression efficiency in the neurosecretory cells of the brain depending on the neurohormone gene promoters. Present results indicate that this in vivo gene-expression system mediated by the baculovirus can serve as an efficient system permitting gene delivery into neural tissues in insects.     

Generation of baculovirus expression vectors

The baculovirus expression system has become an important tool for the expression of heterologous genes because it has several positives attributes. First, high quantities of protein are produced because the target genes are driven by strong viral promoters. Second, most eukaryotic posttranslational modifications are carried out in insect cells in an authentic manner. Thus, proteins expressed with the baculovirus expression system usually have the same activities as the authentic protein. Several approaches have been developed to obtain recombinant baculoviruses easily and nowadays many modified baculoviral DNAs and a huge variety of transfer plasmids are available. Here, we described the rapid generation of recombinant baculoviruses using parental viral DNA that incorporates a lethal deletion and can be selected against. This basic approach should be suitable for the majority of applications.     

杆状病毒用于哺乳动物细胞快速高效表达外源基因的研究

现已发现杆状病毒可进入某些培养的哺乳动物细胞,这提示可将杆状病毒作为一种对哺乳动物细胞的新型基因转移载体。对杆状病毒转移载体的改造及对哺乳动物细胞的基因转移方式进行了进一步的研究。以绿色荧光蛋白基因为报告基因,利用Bac-to-Bac系统构建了分别含有正向和反向CMV启动子表达盒的两种重组杆状病毒。可观察到CMV启动子在Sf9细胞中可启动报告基因的表达,但表达效率较低。用重组杆状病毒感染后Sf9细胞的培养上清直接与HepG2细胞作用,以流式细胞术检测基因转移效率及荧光表达强度,发现这两种病毒在相同的感染复数下对HepG2细胞具有相似的基因转移及表达效率。同时,利用流式细胞术进一步研究了直接使用重组杆状病毒感染4d后Sf9细胞的培养上清对哺乳动物细胞进行基因转移的方法。通过对HepG2细胞的实验结果显示,将带毒Sf9细胞培养上清(1.2×10⁷PFU/mL)用哺乳动物细胞培养基1倍稀释后,37℃下孵育靶细胞12h(moi=50),可达到较高的基因转移及表达效率,同时不会对细胞造成明显损伤。将重组杆状病毒与脂质体和逆转录病毒这两种系统对HepG2及CV1细胞的基因转移效率进行了比较,结果发现在同样未经浓缩等特殊处理的条件下重组杆状病毒对这两种细胞的基因转移效率是最高的。因此可以认为,经过适当改造后的Bac-to-Bac重组杆状病毒系统可作为一种对哺乳动物细胞简便高效的基因转移表达载体。     

Baculoviruses as vectors in mammalian cells

The Baculoviridae are a large family of enveloped DNA viruses exclusively pathogenic to arthropods. Baculoviruses have been extensively used in insect cell-based recombinant protein expression system and as biological pesticides. They have been deomostrated to be safe to mammals, birds and fish. Recently, baculoviruses has been shown to transduce different mammalian cells in spite of the fact that they cannot replicate in mammalian cells (11, 73, 76). This has resulted in the development of baculoviruses as mammalian expression systems and even as vestors for gene therapy. Foundation item: National Nature Science Foundations of China (30325002, 30470075).     

A baculovirus superinfection system: efficient vehicle for gene transfer into Drosophila S2 cells

The baculovirus expression vector system is considered to be a safe, powerful, but cell-lytic heterologous protein expression system in insect cells. We show here that there is a new baculovirus system for efficient gene transfer and expression using the popular and genetically well-understood Drosophila S2 cells. The recombinant baculovirus was constructed to carry an enhanced green fluorescent protein under the control of polyhedrin promoter as a fluorescent selection marker in the Sf21 cell line. Recombinant baculoviruses were then used to transduce S2 cells with target gene expression cassettes containing a Drosophila heat shock protein 70, an actin 5C, or a metallothionein promoter. Nearly 100% of the S2 cells showed evidence of gene expression after infection. The time course for the optimal protein expression peaked at 24 to 36 h postinfection, which is significantly earlier than a polyhedrin-driven protein expression in Sf21 cells. Importantly, S2 cells did not appear to be lysed after infection, and the protein expression levels are comparable to those of proteins under the control of polyhedrin promoter in several lepidopteran cell lines. Most surprisingly, S2 cells permit repetitive infections of multiple baculoviruses over time. These findings clearly suggest that this baculovirus-S2 system may effect the efficient gene transfer and expression system of the well-characterized Drosophila S2 cells.     

A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms.     

Alzheimer's amyloid precursor protein produced by recombinant baculovirus expression. Proteolytic processing and protease inhibitory properties

The baculovirus expression system was used to generate recombinant Alzheimer's amyloid precursor (AAP) proteins. Recombinant baculoviruses were constructed, designed to express full-length 695-, 751-, and 770-amino acid forms. Recombinant baculoviruses designed for constitutive secretion were engineered by placing a termination codon between the beta-protein domain and cytoplasmic anchor of the full-length forms. Insect cells infected with each of these baculoviruses produced both secreted and cell-associated AAPs. Full-length constructs produced secreted derivatives which were COOH-terminally cleaved within the beta-protein domain at Gln15 or Lys16, essentially identical to previous reports utilizing mammalian cell systems. Rare secreted forms (less than 5%) appeared to extend to Lys28. Secretion constructs produced these same forms, but in different ratios. Most (approximately 60%) terminated at Gln15 or Lys16, while the remainder apparently extended to Lys28. AAPs containing the Kunitz-type serine protease inhibitory domain (AAP-751 and -770) were shown to be active inhibitors. No differences were observed in the inhibitors activities of these two forms. The similarities in AAP processing by insect and mammalian systems, together with the large amounts of recombinant protein produced by baculovirus expression, make this an attractive system for studies of AAP processing and biochemical properties.     

构建基于杆状病毒的BV-T7杂合表达体系及利用其在哺乳动物和禽类细胞中表达eGFP基因

【目的】研究重组杆状病毒BV-T7杂合表达体系能否有效转导禽类细胞并在禽类细胞中表达外源基因(eGFP),从而构建能在禽类细胞中高效稳定表达外源基因的重组杆状病毒表达系统。【方法】本研究利用Bac-to-Bac杆状病毒表达系统,结合T7表达系统,通过对eGFP表达水平的调控来把握噬菌体T7 RNA聚合酶(T7 RNAP)的功能。利用两支重组杆状病毒,pFastBac-CMV-T7 RNAP重组杆状病毒为哺乳动物细胞启动子CMV调控的噬菌体T7 RNA聚合酶的cDNA;pFB-T7pro-IRES-GFP-T7ter重组杆状病毒为T7启动子控制的eGFP报告基因。将两支重组杆状病毒共同侵染哺乳动物OL(oligodendrocyte)细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞。【结果】两支重组杆状病毒利用T7启动子和T7 RNAP,在OL细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞中成功表达eGFP报告基因,而且未引起细胞病变,但在鸡胚原代细胞中eGFP的表达相对弱于在OL细胞中的表达。在OL细胞中重组杆状病毒对细胞的转导效率为59.5%,在鸡胚成纤维细胞和鸡胚骨骼肌细胞中转导效率分别为23.2%和33.1%。【结论】本研究构建的基于杆状病毒、T7RNA聚合酶、T7启动子(BV-T7)杂合表达体系能够在哺乳类细胞及禽类细胞中表达T7 RNAP,并利用T7RNAP继续高效而稳定地表达外源基因。这为难于体外操作的RNA病毒提供了有效的研究方法,并对新型基因工程疫苗的研制提供了一个高效而稳定的表达载体系统。     

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).     

Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system

Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.     

杆状病毒作为基因治疗载体的研究进展

杆状病毒是昆虫专一性的病原病毒.近来的研究表明杆状病毒能进入哺乳动物细胞, 但病毒自身不能在哺乳动物细胞中复制, 感染也不引起细胞病变.另外,已经证明杆状病毒能在体外或体内高效地转导许多类型哺乳动物细胞,并且能得到固定表达细胞系,显示了杆状病毒作为基因治疗载体有着良好的应用前景.综述了该领域的最新研究进展并探讨了其发展趋势.     

Baculoviruses: diversity,evolution and manipulation of insects

Baculoviruses, members of the family Baculoviridae, are large, enveloped viruses that contain a double‐stranded circular DNA genome of 80–180 kbp, encoding 90–180 putative proteins. These viruses are exclusively pathogenic for arthropods, particularly insects, and have been developed, or are being developed, as environmentally sound pesticides and eukaryotic vectors for foreign protein expression, surface display, gene delivery for gene therapy, vaccine production and drug screening. The baculoviruses contain a set of approximately 30 core genes that are conserved among all baculovirus genomes sequenced to date. Individual baculoviruses also contain a number of lineage‐ or species‐specific genes that have greatly impacted the diversification and evolution of baculoviruses. In this review, we first describe the general properties and biology of baculoviruses and then focus on the baculovirus genes and mechanisms involved in the replication, spread and survival of baculoviruses within the context of their diversity, evolution and insect manipulation.     

Production of recombinant human calcitonin from silkworm (B. mori) larvae infected by baculovirus

A synthetic modified gene encoding the human calcitonin analog (hmCT) was expressed by use of the baculovirus expression system. After injection with recombinant baculoviruses, the hmCT-GST fusion protein was produced within the silkworm larvae. The fusion protein was purified by affinity chromatography. Biological activity of hmCT for hypercalcemic effect was determined in normal rats.     

Recombinant baculovirus-based multiple protein expression platform for Drosophila S2 cell culture

A platform for selective and controllable expression of multiple foreign protein types was developed in insect cell culture. Based on the fact that baculovirus cannot replicate in nonpermissive Drosophila melanogaster Schneider line 2 (S2) cells, S2 cells that stably express human erythropoietin (hEPO) under the control of the S2-derived inducible metallothionein (MT) promoter were infected with three types of recombinant baculoviruses, each of which expressed a different fluorescent protein gene under the control of MT promoter. Addition of copper sulfate as an inducer to infected, stably transfected S2 cells resulted in simultaneous expression of hEPO and three fluorescent proteins. Expression profiles and levels of the three induced fluorescent proteins were similar in all single infected cells. Importantly, expression profiles and levels of hEPO were similar in both non-infected and infected cells, indicating that baculovirus expressed recombinant proteins do not adversely affect expression of host cell recombinant proteins. Expressions of the three fluorescent proteins were able to be selectively regulated by altering combination ratios of the three types of recombinant baculoviruses. Collectively, these data indicate that the baculovirus/stably transfected S2 cell system can be successfully used to express multiple foreign proteins in a controlled and selective manner without the burden of additional selection markers. Such a system would be expected to be attractive as a multiple protein expression platform for engineering metabolic or glycosylation pathways.