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。     

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。     

Efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie1 promoter, a novel shuttle promoter between insect cells and mammalian cells

Recombinant baculoviruses have emerged as a new gene delivery vehicle for mammalian cells. Thus, a shuttle promoter that mediates gene expression in both insect and mammalian cells will facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle. This study described the generation of three recombinant baculoviruses with an EGFP reporter gene under the control of the white spot syndrome virus (WSSV) ie1 promoter, or either of two control promoters, the baculovirus early-to-late (ETL) promoter and polyhedrin promoter. The resulting recombinant baculoviruses were used to infect insect Sf9 cells and transduce several mammalian cell lines to test the expression of EGFP. We found that the WSSV ie1 promoter displayed a strong promoter activity in both insect and mammalian cells, and showed a stronger promoter activity than the ETL promoter in some mammalian cell lines. The activity of the WSSV ie1 promoter, but not the ETL promoter, can be enhanced by sodium butyrate, a histone deacetylase inhibitor. A transient plasmid transfection assay indicated that the WSSV ie1 promoter activity in mammalian cells is independent of baculovirus gene expression, differing from the ETL promoter, which was shown to be baculovirus-dependent. This study demonstrates, for the first time, that the WSSV ie1 promoter can function as a baculovirus-independent shuttle promoter between insect cells and mammalian cells. This novel shuttle promoter will facilitate the application of baculovirus-based vectors in gene expression, gene therapy, and non-replicative vector vaccines.     

Baculovirus expression systems for recombinant protein production in insect and mammalian cells

The baculovirus vector systems has been extensively used for the expression of foreign gene products in insect and mammalian cells. New advances increase the possibilities and applications of the baculovirus expression system, which has the capability to express multiple genes simultaneously within a single infected insect cells and to use recombinant virus with mammalian cell-active expression cassettes to permit expression of recombinant proteins in mammalian cells in vitro and in vivo. Future investigations of the baculovirus expression system designed for specific target cells, can open wide variety of applications. This review summarizes the recent achievements in applications the baculovirus vector systems and optimization recombinant protein expression in both insect and mammalian cell lines.     

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。     

Genetic modification of baculovirus expression vectors

As a protein expression vector,the baculovirus demonstrates many advantages over other vectors.With the development of biotechnology,baculoviral vectors have been genetically modified to facilitate hig...     

Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery.

Recombinant baculoviruses have been extensively used as vectors for abundant expression of a large variety of foreign proteins in insect cell cultures. The appeal of the system lies essentially in easy cloning techniques and virus propagation combined with the eukaryotic post-translational modification machinery of the insect cell. Recently, a novel molecular biology tool was established by the development of baculovirus surface display, using different strategies for presentation of foreign peptides and proteins on the surface of budded virions. This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a versatile system in molecular biology. Surface display strategies play an important role, as they may be used to enhance the efficiency and specificity of viral binding and entry to mammalian cells. In addition, baculovirus surface display vectors have been engineered to contain mammalian promoter elements designed for gene delivery both in vitro and in vivo. Moreover, baculovirus capsid display has recently been developed; this holds promise for intracellular targeting of the viral capsid and subsequent cytosolic delivery of desired protein moieties. Finally, the viruses can accommodate large insertions of foreign DNA and replicate only in insect cells. Together, these are attributes that are very likely to make them important tools in functional genomics and proteomics.     

Baculovirus vectors for efficient gene delivery and expression in mammalian cells

Baculovirus expression vectors are extensively used for the delivery of foreign genes and expression of recombinant proteins in insect and mammalian cells. Modified baculoviruses containing mammalian promoter elements (BacMam viruses) for an efficient transient and stable transduction of diverse mammalian cells ensure a high level of heterologous protein expression both in vitro and in vivo. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus promoter, green or red fluorescent protein gene, SV40pA polyadenylation signal, and polylinker MCS were constructed for the delivery of genes encoding hepatitis C virus structural proteins into mammalian cells. In HEK293T and Huh7 cells, formation of glycoprotein complexes and HCV4ike particles was observed. A high efficiency of the baculovirus-medi-ated gene transfer and expression of the virus envelope proteins in mammalian cells was demonstrated using fluorescence, flow cytometry, and immunoblot techniques.     

Enhancement and prolongation of baculovirus-mediated expression in mammalian cells: focuses on strategic infection and feeding

The baculovirus/insect cell system has been widely used for recombinant protein production. Since the finding that baculovirus was able to infect hepatocytes in 1995, various attempts to utilize baculovirus as a gene delivery vehicle into mammalian cells have been reported. In this study, we intended to explore the possibility of utilizing a baculovirus/mammalian cell system as a nonlytic, continuous protein production system. A recombinant baculovirus vector carrying enhanced green fluorescent protein (EGFP) under the control of cytomegalovirus immediate-early (CMV-IE) promoter was constructed. This virus was used to infect four common mammalian cell lines, and HeLa was found to yield the highest expression level. Additions of butyrate and valproic acid both enhanced the expression level, but butyrate exhibited a more profound effect. More importantly, HeLa cells were found to be superinfected by baculovirus, a result not observed in the conventional baculovirus/insect cell system. The effects of multiplicity of infection (MOI) and infection timing were also compared. High MOI up to 800 increased the expression in the short term (4 days), but the relatively higher cell death and lower cell density compromised the overall protein yield thereafter. The highest overall expression for a long term was obtained at MOI = 200 when the cells were initially infected at the mid-exponential phase and superinfected with additional baculovirus (MOI = 200) together with a one-time supplement of butyrate. In summary, the strategic infection and feeding enhanced the expression level 9-fold (compared with unsuperinfected culture) and prolonged the duration of expression to 16 days. This study reveals that this baculovirus/mammalian cell system has great potential to become a novel continuous, nonlytic expression system.     

Recombinant baculoviruses as expression vectors for insect and mammalian cells.

Baculovirus expression vectors are widely used for expressing heterologous proteins in cultured insect cells. Recent advances include further development of the system for production of multi-subunit protein complexes, co-expression of protein-modifying enzymes to improve heterologous protein production, and additional applications of baculovirus display technology. The application of modified baculovirus vectors for gene expression in mammalian cells continues to expand.     

Baculovirus as versatile vectors for protein expression in insect and mammalian cells

Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.     

Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters

It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation.     

Baculovirus expression systems for production of recombinant proteins in insect and mammalian cells

Baculovirus vector systems are extensively used for the expression of foreign gene products in insect and mammalian cells. New advances increase the possibilities and applications of the baculovirus expression system, which makes it possible to express multiple genes simultaneously within a single infected insect cell and to obtain multimeric proteins functionally similar to their natural analogs. Recombinant viruses with expression cassettes active in mammalian cells are used to deliver and express genes in mammalian cells in vitro and in vivo. Further improvement of the baculovirus expression system and its adaptation to specific target cells can open up a wide variety of applications. The review considers recent achievements in the use of modified baculoviruses to express recombinant proteins in eukaryotic cells, advantages and drawbacks of the baculovirus expression system, and ways to optimize the expression of recombinant proteins in both insect and mammalian cell lines.     

An OriP/EBNA-1-based baculovirus vector with prolonged and enhanced transgene expression

BACKGROUND: The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been explored as a gene delivery vehicle for a variety of mammalian cell lines. However, the transient expression nature due to its incapability to replicate in mammalian cells and insufficient transduction efficiency limit its application. METHODS: Recombinant baculovirus vectors containing genetic elements from Epstein-Barr virus (EBV), OriP and EBNA-1, which are essential for the episomal maintenance of the EBV genome in latently infected cells, were constructed and tested for their ability to sustain and express transgene (enhanced green fluorescence protein (egfp)) in mammalian cells. RESULTS: The recombinant baculovirus containing OriP and EBNA-1 genes driven by the cytomegalovirus (CMV) promoter was capable of persisting in a significant proportion of infected mammalian cells, HEK293, Vero, Cos-7, and Hone-1, without any selective pressure. In HEK293, the expression of EGFP lasted for 60 days with markedly enhanced expression level. The persistence of baculovirus genome correlated with the expression of EBNA-1. CONCLUSIONS: The improved baculovirus vector could mediate prolonged and enhanced foreign gene expression in some mammalian cells. Furthermore, an adequate level of the EBNA-1 protein was essential for the maintenance of the OriP-containing baculovirus genome. The new vector has potential for use in gene therapy.     

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。     

A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells

Baculoviruses are widely used for protein production in insect cells, and their potential for gene transfer to mammalian cells is increasingly being recognized. Here we describe a baculovirus vector with a bicistronic mammalian expression cassette and demonstrate its suitability for efficient transient and stable protein expression in human glioblastoma cells. Bicistronic baculovirus vectors are safe, cost efficient, and easy to produce; thus, they represent an excellent gene transfer system for mammalian cells.     

Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-β) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-β promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIM_S, a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses.     

Transient transfection coupled to baculovirus infection for rapid protein expression screening in insect cells

Baculovirus infected insect cells are widely used for heterologous protein expression. Despite the power of this system, the use of baculovirus techniques for protein expression screening is hampered by the time and resources needed to generate each recombinant baculovirus. Here, we show that a transfection/infection based expression system is suitable for screening of expression constructs in insect cells and represents a valid alternative to other traditional screening methodologies using recombinant baculovirus. The described method is based on gene delivery by transfection coupled to the induction of protein expression by non-recombinant baculovirus infection. Vectors that control expression by a combination of the baculovirus promoters ie1 and p10 and the enhancer element hr5 are among the ones suitable for this method. Infection with non-recombinant baculovirus drastically increases the basal activity of these elements, leading to protein over-expression. Multiple vectors can be simultaneously co-transfected/infected, making transfection/infection amenable for screening of multiple co-expressed proteins and protein complexes. Taken together, our results prove that the transfection/infection protocol is a valid and innovative approach for increasing speed and reducing costs of protein expression screening for structural and functional studies.