核衣壳蛋白VP39融合TAT转导肽对杆状病毒转导哺乳动物细胞的影响

为了提高昆虫杆状病毒在哺乳动物细胞中转导基因的效率,构建了重组杆状病毒AcRed-tat和AcRed。两者都能在哺乳动物细胞内表达红色荧光蛋白作为报告基因。同时,AcRed-tat带有HIV-1Tat转导肽、病毒主要衣壳蛋白基因vp39及增强型绿色荧光蛋白(egfp)三者的融合基因,并由杆状病毒多角体启动子表达,能够在昆虫细胞中表达该Tat融合蛋白,并掺入子代病毒粒子。而AcRed作为相应的对照病毒,带有多角体启动子表达vp39和egfp的融合基因。2株病毒分别转导哺乳动物细胞后,利用流式细胞仪检测报告基因的表达水平,发现在CHO和Vero细胞中AcRed-Tat介导的报告基因表达水平明显高于AcRed,而在HEK293细胞中2株病毒介导的报告基因表达水平差异不显著。结果表明Tat转导肽可以提高杆状病毒对一部分哺乳动物细胞的转导效率,为改进杆状病毒-哺乳动物细胞转导载体提供了新的思路。     

Baculovirus GP64-mediated entry into mammalian cells

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.     

Investigation of optimal transduction conditions for baculovirus-mediated gene delivery into mammalian cells

Although baculovirus-mediated gene delivery into mammalian cells has been documented in a wealth of the literature, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus is proposed for simple and efficient gene delivery into HeLa cells. We found that approximately 75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25 degrees C using Dulbecco's phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasts with previous protocols in which transduction occurs for 1 h at 37 degrees C using growth medium (e.g., DMEM) as the surrounding solution. Investigation of the physical parameters led to the findings that: 1) baculovirus uptake by HeLa cells continued for at least 4 h in the event of high virus dosage, which led to higher gene expression; 2) the half-life of baculovirus dramatically decreased at 37 degrees C; 3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; and 4) lower transduction efficiency and gene expression were obtained when DMEM was used (in comparison with D-PBS and TNM-FH), suggesting that DMEM contains certain inhibitory factors for baculovirus transduction. Our data uncovered several aspects that were not investigated before and the optimized transduction conditions allowed for gene delivery as efficient as that by the protocols commonly employed by others, but eliminated the need for virus ultracentrifugation. The protocol not only represented a simpler approach, but also considerably reduced possible virus inactivation during ultracentrifugation, thus making it easier to convert the baculovirus/mammalian cell system to a tool for eukaryotic protein production on a larger scale.     

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.     

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.     

Baculovirus-mediated gene transfer is attenuated by sodium bicarbonate

BACKGROUND: Baculovirus transduction of cultured mammalian cells is typically performed by incubating the cells with virus using culture medium (e.g. Dulbecco's modified Eagle's medium (DMEM)) as the surrounding solution. However, we previously uncovered that DMEM hinders the baculovirus-mediated gene transfer. METHODS: In this study, we systematically explored the influences of promoter and medium constituents on the transduction efficiency by using different recombinant viruses and surrounding solutions for transduction, followed by flow cytometric analyses. Whether the key medium component impeded baculovirus binding to the cells and subsequent virus entry was investigated by immunofluorescence/confocal microscopy and quantitative real-time polymerase chain reaction (Q-PCR). RESULTS: We demonstrated that the poorer transduction by using DMEM as the surrounding solution is independent of the promoter. Examination of the medium constituents group by group revealed that the balanced salt solution suppresses the baculovirus transduction. By omitting individual salt species in the balanced salt solution, we surprisingly uncovered that NaHCO(3), a common buffering agent, exerts the inhibitory effects in a concentration-dependent manner. Intriguingly, NaHCO(3) did not debilitate the baculovirus, nor did it inhibit virus binding to the cells. Instead, NaHCO(3) inhibited baculovirus transduction by reducing the intracellular virus number. CONCLUSIONS: To our best knowledge, this is the first report unraveling the significance of NaHCO(3) in gene transfer. Our finding suggests that baculovirus-mediated gene transfer can be readily enhanced by omitting NaHCO(3) from the medium during the transduction period.     

6-O- and N-Sulfated Syndecan-1 Promotes Baculovirus Binding and Entry into Mammalian Cells

Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show that baculovirus requires HSPG sulfation, particularly N- and 6-O-sulfation, to bind to and transduce mammalian cells. According to our results, baculovirus binds specifically to syndecan-1 (SDC-1) but does not interact with SDC-2 to SDC-4 or with glypicans. Competition experiments performed with SDC-1 antibody or recombinant SDC-1 protein inhibited baculovirus binding, and SDC-1 overexpression enhanced baculovirus-mediated transduction. In conclusion, we show that SDC-1, a commonly found cell surface HSPG molecule, has a role in the binding and entry of baculovirus in vertebrate cells. The results presented here reveal important aspects of baculovirus entry and can serve as a basis for next-generation baculovirus vector development for gene delivery.     

Enhanced baculovirus-mediated transduction of human cancer cells by tumor-homing peptides

Tumor cells and vasculature offer specific targets for the selective delivery of therapeutic genes. To achieve tumor-specific gene transfer, baculovirus tropism was manipulated by viral envelope modification using baculovirus display technology. LyP-1, F3, and CGKRK tumor-homing peptides, originally identified by in vivo screening of phage display libraries, were fused to the transmembrane anchor of vesicular stomatitis virus G protein and displayed on the baculoviral surface. The fusion proteins were successfully incorporated into budded virions, which showed two- to fivefold-improved binding to human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells. The LyP-1 peptide inhibited viral binding to MDA-MB-435 cells with a greater magnitude and specificity than the CGKRK and F3 peptides. Maximal 7- and 24-fold increases in transduction, determined by transgene expression level, were achieved for the MDA-MB-435 and HepG2 cells, respectively. The internalization of each virus was inhibited by ammonium chloride treatment, suggesting the use of a similar endocytic entry route. The LyP-1 and F3 peptides showed an apparent inhibitory effect in transduction of HepG2 cells with the corresponding display viruses. Together, these results imply that the efficiency of baculovirus-mediated gene delivery can be significantly enhanced in vitro when tumor-targeting ligands are used and therefore highlight the potential of baculovirus vectors in cancer gene therapy.     

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.     

Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells

BACKGROUND: Mesenchymal stem cells (MSCs) have drawn considerable attention as vehicles for cell- or gene-based therapies, yet various problems still exist for current gene delivery vectors. On the other hand, baculovirus has emerged as a novel gene therapy vector, but its transduction of stem cells has not been reported. METHODS: A recombinant baculovirus expressing the enhanced green fluorescent protein (EGFP) was constructed to transduce human MSCs derived from umbilical cord blood (uMSCs) or bone marrow (bMSCs). RESULTS: In this study, we demonstrated for the first time that human uMSCs or bMSCs could be transduced by baculovirus with high efficiencies (up to approximately 72.8% and 41.1%, respectively) and significantly elevated transgene (enhanced green fluorescent protein, EGFP) expression upon incubation with unconcentrated virus and phosphate-buffered saline for 4 h at 25 degrees C. The transduction efficiency into bMSCs could be further increased to approximately 72.2% by lowering the cell density. The improved transgene expression was partly attributed to the enhanced virus uptake upon transduction, as determined by quantitative real-time polymerase chain reaction (Q-PCR). MSC growth was not obstructed by baculovirus transduction itself, but was somewhat hampered by EGFP expression. Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic lineage. The adipogenic progenitors appeared more permissive to baculovirus transduction than the undifferentiated bMSCs, thus allowing for the maintenance and enhancement of transgene expression by repeated transduction after subculture. CONCLUSIONS: These findings implicate the potential applications of baculovirus as an alternative vector to genetically modify MSCs for ex vivo gene therapy.     

应用离心方法提高杆状病毒对哺乳动物细胞转导实验效率

重组杆状病毒作为一种对哺乳动物细胞的新型基因转移载体已获得了日益广泛的应用。为进一步提高转导实验的效率,本研究利用已构建的带有CMV启动子-增强型绿色荧光蛋白(eGFP)表达盒的重组杆状病毒BacV-CMV-EGFPA,在CV-1细胞中探索了应用离心方法提高转导实验效率的可行性。结果显示离心方法可显著提高单位时间内重组杆状病毒对哺乳动物细胞的转导效率,同时不会对靶细胞造成损伤。通过对离心时间、离心后孵育时间、病毒上清的稀释缓冲液的进一步摸索优化,结果显示病毒上清以PBS为稀释缓冲环境室温600g水平离心1h即可获得高水平的转导效率,优于在PBS环境中27℃孵育8h的效果。该方法可在获得高转导效率的同时显著缩短实验时间,具有快捷、高效、低损伤的特点,可作为一种常规操作方法用于日常实验。本研究进一步应用该方法对多种不同来源和类型的哺乳动物细胞株进行基因转导,结果显示该方法可适用于多数不同种属和组织来源的哺乳动物细胞,其中对贴壁细胞的效果最为显著。     

Ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can infect a variety of mammalian cells, as well as insect cells, facilitating its use as a viral vector for gene delivery into mammalian cells. Glycoprotein gp64, a major component of the budded AcMNPV envelope, is involved in viral entry into cells by receptor-mediated endocytosis and subsequent membrane fusion. We examined the potential production of pseudotype baculovirus particles transiently carrying ligands of interest in place of gp64 as a method of ligand-directed gene delivery into target cells. During amplification of a gp64-null pseudotype baculovirus carrying a green fluorescent protein gene in gp64-expressing insect cells, however, we observed the high-frequency appearance of a replication-competent virus incorporating the gp64 gene into the viral genome. To avoid generation of replication-competent revertants, we prepared pseudotype baculoviruses by transfection with recombinant bacmids without further amplification in the gp64-expressing cells. We constructed gp64-null recombinant bacmids carrying cDNAs encoding either vesicular stomatitis virus G protein (VSVG) or measles virus receptors (CD46 or SLAM). The VSVG pseudotype baculovirus efficiently transduced a reporter gene into a variety of mammalian cell lines, while CD46 and SLAM pseudotype baculoviruses allowed ligand-receptor-directed reporter gene transduction into target cells expressing measles virus envelope glycoproteins. Gene transduction mediated by the pseudotype baculoviruses could be inhibited by pretreatment with specific antibodies. These results indicate the possible application of pseudotype baculoviruses in ligand-directed gene delivery into target cells.     

RGD motifs on the surface of baculovirus enhance transduction of human lung carcinoma cells

Baculovirus vectors have been shown to enter a variety of mammalian cell lines and gene transfer with wild-type baculovirus (WT) has been demonstrated both in vitro and in vivo. Different protein motifs have been displayed on the viral surface to serve as ligands for cell-specific receptor molecules. We have generated recombinant baculovirus vectors displaying an RGD-motif, recognized by alphaV integrin, on the viral surface. The RGD motifs within the C-terminus of coxsackie virus A9 and human parechovirus 1 VP1 proteins were fused to the N-terminus of the major envelope glycoprotein, gp64, of Autographa californica multiple nucleopolyhedrovirus. The recombinant RGD-presenting viruses bound more efficiently to the surface of human lung carcinoma cells (A549), known to contain alphaV integrins, as compared to WT baculovirus. In addition, the binding pattern of the RGD-displaying baculovirus showed extensive clustering. This most likely represents clustering of the integrin molecules on the cell surface, induced by binding of the RGD-displaying baculovirus. Finally, the transduction efficiency of an RGD-representing virus increased by almost three-fold as monitored by light emission measurements. In conclusion, these results suggest that the RGD-motif is functional on the surface of baculovirus and thereby these tropism-modified viruses bind more efficiently as well as enhance the transduction efficiency of human cancer cells expressing alphaV integrins.     

杆状病毒转导不同哺乳动物骨髓来源间充质干细胞

杆状病毒作为一种新型基因载体,若能有效转导不同哺乳动物骨髓来源间充质干细胞(bone marrow-derived mesenchymal stem cells, BMSCs),将会成为干细胞基因修饰研究领域中更理想的一种基因载体.本文探讨了重组杆状病毒(BacV-CMV-EGFP)对不同哺乳动物BMSCs的转导效率.体外原代培养小鼠、大鼠、猪、恒河猴及人的BMSCs.用培养3代以上的哺乳动物BMSCs进行病毒转导实验,转导2d后用倒置荧光显微镜观察绿色荧光蛋白在不同哺乳动物BMSCs中的表达,并用流式细胞仪检测重组杆状病毒对不同哺乳动物BMSCs的转导效率.结果显示:原代培养的小鼠、大鼠、猪、恒河猴及人的BMSCs于体外传代3次以上后,细胞呈现较均一的梭形,漩涡状生长;倒置荧光显微镜观察显示,与小鼠、大鼠、猪的BMSCs相比,恒河猴及人有更多BMSCs表达绿色荧光蛋白,且荧光强度较强;杆状病毒对小鼠、大鼠、猪、恒河猴及人的BMSCs的转导效率分别为(21.21±3.02)%、(22.51±4.48)%、(39.13±5.79)%、(71.16±5.36)%及(70.67±3.74)%.上述结果表明,重组杆状病毒对不同哺乳动物BMSCs的转导效率不同,对恒河猴及人的BMSCs转导效率较高,说明重组杆状病毒可作为人或灵长类动物BMSCs基因修饰研究领域中更理想的基因载体.     

The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 μM, and appeared to plateau above a concentration of 1 μM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.     

In vitro and in vivo gene delivery by recombinant baculoviruses

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo 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。     

杆状病毒对不同哺乳动物细胞基因转移及表达效率的研究

研究已证实杆状病毒可进入某些哺乳动物细胞,这提示了可将重组杆状病毒作为一种对哺乳动物细胞的新型基因转移载体。利用已构建的重组杆状病毒BacV-CMV-EGFPA,以含病毒的Sf9细胞培养上清同时孵育多种哺乳动物细胞,利用流式细胞术检测报告基因在不同细胞株中的转移效率及表达效率。共使用了20种哺乳动物细胞株,其中有12种人类组织细胞,7种小鼠组织细胞,1种猴组织细胞。实验结果显示携带CMV启动子的重组杆状病毒可有效进入多数哺乳动物细胞,其中对人、猴来源细胞的基因转移效率显著高于对鼠源细胞,对贴壁细胞的基因转移效率显著高于对悬浮细胞。同时,通过脂质体LipofectAMINE将携带有CMV启动子和EGFP基因的哺乳动物细胞表达质粒pCDNA3-1-EGFP分别转染部分特别是被认为杆状病毒进入能力较低的细胞株,结果显示CMV启动子在这些细胞中均可有效引导EGFP基因的表达,因此认为携带了CMV启动子的重组杆状病毒对不同哺乳动物细胞基因转移效率能基本反映出杆状病毒对不同种哺乳动物细胞的进入能力。通过综合比较携带CMV启动子的杆状病毒对不同种属及组织来源细胞的基因转移及表达效率,可看出杆状病毒对灵长类动物贴壁细胞的基因转移及表达效果是较好的,而对小鼠来源的细胞及悬浮培养细胞却并不十分理想,这表明将重组杆状病毒作为一种对哺乳动物细胞的基因转移工具,仍有其局限性,不一定对所有的细胞都合适,在应用时应予以充分考虑。     

杆状病毒AcMNPV p48基因在昆虫细胞中的表达

【目的】p48(ac103)基因在昆虫杆状病毒中高度保守,暗示其具有重要的生物学功能。为了研究该基因的功能,我们首先对该基因的表达特征进行描述。【方法】以杆状病毒代表种——苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)的p48基因为研究对象,利用Bac-to-Bac杆状病毒表达载体系统分别构建了在P48蛋白N-端和C-端融合HA-标签,并且携带绿色荧光蛋白基因和多角体蛋白基因的重组Bacmid。将重组Bacmid转染Sf9细胞,收集含病毒的上清去感染Sf9细胞,在感染后不同时间点收集细胞进行SDS-PAGE电泳,利用商业化的HA抗体进行Western blot分析以检测融合蛋白在昆虫细胞中的表达情况。【结果】用C-端融合HA-标签的重组病毒感染细胞后12h即可检测到一条43kDa左右、能与HA抗体发生特异性结合的蛋白条带,该特异性蛋白的表达一直持续到病毒感染后96h。从感染后48h起一直到96h,均能检测到另外一条约26kDa的蛋白条带也能与HA抗体发生特异性结合。在N-端融合HA-标签的重组病毒感染的细胞中没有检测到与HA抗体特异结合的蛋白。【结论】结果表明,p48基因是个晚期基因,在病毒感染的晚期表达,并且该蛋白在昆虫细胞中表达时N-端可能被剪切。     

Improving promiscuous mammalian cell entry by the baculovirus Autographa californica multiple nuclear polyhedrosis virus

The insect baculovirus AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells, we show that entry is favoured by low pH and by increasing the available cell-surface area through a transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268–281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell-to-cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies, suggesting that additional factors also govern entry.