A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1–4 CELLS

Abstract  The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B1–4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins, however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B1–4 cells. The growth characteristics, productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B1–4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing ß -galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B1–4 cells.     

杆状病毒表达系统的改进及IL-6在昆虫细胞内的表达和纯化

使用同源重组方法,在昆虫细胞内将多角体启动子驱动的EGFP表达盒插入杆状病毒穿梭载体Bacmid的p74位相,经5轮空斑纯化获得重组穿梭载体Bacmid-egfp。然后将Bacmid-egfp转化含转座助手质粒的E.coliDH10B,获得受体菌E.coliDH10Bac-egfp,由于Bacmid-egfp保留了完整的转座结构和α互补功能,因此该菌株和原始E.coliDH10Bac一样能有效的利用各种pFastBac系列的载体进行转座并构建出能指示病毒繁殖和目的基因表达的重组病毒。使用红色荧光蛋白DsRed对系统进行了验证,结果表明重组病毒Bac-egfp-DsRed感染的细胞中绿色荧光蛋白和红色荧光蛋白均得到了高效表达。进一步使用该系统在昆虫细胞中高效表达并纯化了IL-6蛋白,为研究和应用该细胞因子提供物质基础,同时也进一步证明所改造的杆状病毒表达系统的可靠性和实用性。     

Porcine interleukin 18: cloning, characterization of the cDNA and expression with the baculovirus system

We have isolated and sequenced a cDNA that contains the coding sequence of porcine interleukin 18 (IL-18) and the recombinant protein of porcine IL-18 was expressed using the baculovirus system. The open reading frame (ORF) of the porcine IL-18 cDNA is 579 base pairs (bp) in length and encodes 192 amino acids. The predicted amino acid sequence is 76.7%, 64.7% and 61.6% homologous to the predicted human, murine and rat amino acid sequences, respectively. The porcine precursor and mature IL-18 protein were expressed respectively in Trichoplusia ni -derived (Tn5) cells using the baculovirus Autografha californica nuclear polyhedorosis virus (AcNPV) as a vector. Tn5 cells infected with recombinant virus containing a whole IL-18 protein coding region sequence secreted porcine precursor IL-18 into the culture medium. On the other hand, Tn5 cells infected with recombinant virus containing a mature IL-18 protein coding region sequence expressed several proteins in the cell lysates, but did not secrete mature protein into the culture medium efficiently. Immunoblotting analysis of recombinant protein showed cross-reactivity with anti-human IL-18 polyclonal antibody. The mature form of porcine IL-18 protein induced IFN-gamma production in suboptimal doses of anti-CD3 antibody and concanavalin A- (ConA) stimulated porcine peripheral blood mononuclear cells (PBMC), but the precursor form had little effect.     

Hepatitis C virus structural proteins and virus-like particles produced in recombinant baculovirus-infected insect cells

Three proteins, namely, the core protein C and envelope glycoproteins E1 and E2, are main structural proteins forming a hepatitis C virus (HCV) virion. The virus structure and assembly and the role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Highly efficient heterologous expression systems make it possible to obtain self-assembled, nonreplicating, genome-lacking particles that are morphologically similar to intact virions. Using recombinant baculoviruses expressing the HCV structural protein genes in insect cells, the individual HCV structural proteins were expressed to 25–35% of the total cell protein, and the CE1 and E1E2 heterodimers and HCV-like particles were obtained. It was demonstrated that the recombinant C, E1, and E2 proteins underwent posttranslational modification, the glycoproteins formed a noncovalent heterodimer, and HCV- like particles were located in endoplasmic reticulum membranes of infected cells. The formation of E1E2 dimers and HCV-like particles was used to study the effect of E1 glycosylation on the expression and processing of the coat proteins.     

Helicoverpa armigera nucleopolyhedrovirus ORF80 encodes a late, nonstructural protein

The Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ORF80 (ha80) has 765 bp encoding a protein with approximately 254 amino acids and a predicted molecular weight of 30.8 kDa. Homologues of ha80 are found in most baculovirus sequences, including those from lepidopteran NPVs, lepidopteran granuloviruses (GVs), hymenopteran baculoviruses, and one dipteran baculovirus, yet their functions remain unclear. In this study we characterized ha80, and showed that it was transcribed late in infected host cells (HzAM1). The product of ha80 was a 31 kDa protein that was not a structural protein of budded virus (BV) or occlusion-derived virus (ODV) particles. Ha80 was first detected in the cytoplasm of infected HzAM1 cells at 12 h p.i., and was observed in the nucleus at later stages of infection, suggesting that it may be involved in transporting viral proteins into the host cell nucleus or play its roles in the nucleus.     

Expression of rotavirus VP2 produces empty corelike particles.

The complete VP2 gene of bovine rotavirus strain RF has been inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. Cotransfection of Spodoptera frugiperda 9 cells with wild-type baculovirus DNA and transfer vector DNA led to the formation of recombinant baculoviruses which contain bovine rotavirus gene 2. Infection of S. frugiperda cells with this recombinant virus resulted in the production of a protein similar in size and antigenic properties to the authentic rotavirus VP2. The protein binds double-stranded RNA and DNA in an overlay protein blot assay. Expressed VP2 assembles in the cytoplasm of infected cells in corelike particles 45 nm in diameter. These corelike particles were purified by sucrose gradient centrifugation and found to be devoid of nucleic acid. Coexpression of VP2 and VP6 from heterologous rotavirus strains (bovine and simian) resulted in the formation of single-shelled particles. These results definitively show the existence of an innermost protein shell in rotavirus which is formed independently of other rotavirus proteins. These results have implications for schemes of rotavirus morphogenesis.     

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.     

Cell-based analysis of Chikungunya virus E1 protein in membrane fusion

Background

Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored.

Methods

A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity.

Results

Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178A mutation exhibited a slight decrease in cholesterol-dependence and a higher-pH threshold for fusion.

Conclusions

Cells expressing amino acid substitutions of conserved protein E1 residues of E1-G91 and E1-H230 lost most of the CHIKV E1-mediated membrane fusion activity. Cells expressing mutations of less-conserved amino acids, E1-V178A and E1-A226V, retained membrane fusion activity to levels similar to those expressing wild type E1, but their fusion properties of pH threshold and cholesterol dependence were slightly altered.     

Expression and self-assembly of empty virus-like particles of hepatitis E virus.

Hepatitis E virus (HEV) is a pathogenic agent that causes fecally-orally transmitted acute hepatitis. The genome, a single-stranded positive-sense RNA, encodes three forward open reading frames (ORFs), in which an approximately 2-kb structural protein is located in the 3' end. To produce HEV-like particles the structural protein, with its N terminus truncated (amino acid residues 112 to 660 of ORF2), was expressed in insect Tn5 cells by a recombinant baculovirus. In addition to the primary translation product with a molecular mass of 58 kDa, a large amount of a further-processed molecule with a molecular mass of 50 kDa was generated and efficiently released into the culture medium. Electron microscopic observation of the culture medium revealed that the 50-kDa protein self-assembled to form empty virus-like particles (VLPs). The buoyant density of the VLPs in CsCl was 1.285 g/cm3 and their diameter was 23.7 nm, a little smaller than the 27 nm of native HEV particles secreted into the bile or stools of experimentally infected monkeys. The yield of the VLPs was 1 mg per 10(7) cells as a purified form. The particles possess antigenicity similar to that of authentic HEV particles and, consequently, they appear to be a good antigen for the sensitive detection of HEV-specific immunoglobulin G (IgG) and IgM antibodies. Furthermore, the VLP may be the most promising candidate yet for an HEV vaccine, owing to its potent immunogenicity.     

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.     

Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.     

Effect of MOI ratio on the composition and yield of chimeric infectious bursal disease virus-like particles by baculovirus co-infection: deterministic predictions and experimental results

Virus-like particles (VLPs) are empty particles consisting of virus capsid proteins that closely resemble native virus but are devoid of the native viral nucleic acids and therefore have attracted significant attention as noninfectious vaccines. A recombinant baculovirus, vIBD-7, which encodes the structural proteins (VP2, VP3, and VP4) of infectious bursal disease virus (IBDV), produces native IBD VLPs in infected Spodoptera frugiperda insect cells. Another baculovirus, vEDLH-22, encodes VP2 that is fused with a histidine affinity-tag (VP2H) at the C-terminus. By co-infection with these two baculoviruses, hybrid VLPs with histidine tags were formed and purified by immobilized metal affinity chromatography (Hu et al., 1999). Also, we demonstrated that varying the MOI ratio of these infecting viruses altered the extent of VP2H incorporated into the particles. A dynamic mathematical model that described baculovirus infection and VLP synthesis (Hu and Bentley, 2000) was adapted here for co-infection and validated by immunofluorescence labeling. It was shown to predict the VLP composition as a dynamic function of MOI. A constraint in the VP2H content incorporated into the particles was predicted and shown by experiments. Also, the MOI ratio of both infecting viruses was shown to be the major factor influencing the composition of the hybrid particles and an important factor in determining the overall yield. ELISA results confirmed that VP2H was exhibited to a varied extent on the outer surface of the particles. This model provides insight on the use of virus co-infection in virus-mediated recombinant protein expression systems and aids in the optimization of chimeric VLP synthesis.     

Baculovirus as a highly efficient gene delivery vector for the expression of hepatitis delta virus antigens in mammalian cells

Baculovirus has been employed for a wide variety of applications. In this study, we further expanded the application to the high-level expression of hepatitis delta virus (HDV) antigens and the formation of virus-like particles (VLP) in transduced mammalian cells. To this end, two recombinant baculoviruses were constructed to express large hepatitis delta antigen (L-HDAg) and hepatitis B surface antigen (HBsAg) under mammalian promoters. With a simplified transduction protocol using unconcentrated virus, high transduction efficiencies were achieved in hepatoma cells, in which L-HDAg and HBsAg were expressed abundantly, allowing for easy colorimetric detection in Western blots. L-HDAg alone was nucleus-bound and HBsAg alone was secreted; formation and secretion of HDV-like particles were readily detected upon coexpression, indicating that the baculovirus-expressed proteins were processed correctly as the authentic proteins. Quantitative real-time PCR (Q-PCR) analyses quantitatively revealed that baculovirus transduction was more efficient than plasmid transfection with respect to DNA uptake and DNA transport to the nucleus. Furthermore, superinfection introduced more baculovirus DNA into cells in the long-term culture as revealed by Q-PCR, thereby enhancing and prolonging the expression. In summary, baculovirus transduction can be an attractive method as an alternative to the plasmid transfection commonly employed for HDV research thanks to the significantly higher gene delivery efficiencies as well as the abundant expression and proper processing. Baculovirus can also be envisaged as a useful tool for investigating protein-cell interactions and virus assembly.     

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。     

Baculovirus Superinfection: A Probable Restriction Factor on the Surface Display of Proteins for Library Screening

In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells co-expressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the re-infection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides.     

尼帕病毒融合蛋白、受体结合蛋白的表达及特异性高免血清制备

尼帕病毒膜融合蛋白F和受体结合蛋白G在病毒感染和诱导机体产生保护性免疫中起重要的作用。通过PCR扩增获得尼帕病毒F1和G基因片段(均去掉信号肽和跨膜区),克隆至原核表达载体,IPTG诱导大肠杆菌表达目的蛋白,Western blot表明重组F1、G蛋白与兔抗尼帕病毒血清具有良好的反应原性;同时将F1和G基因克隆至经改造过的杆状病毒表达载体,获得了含有目的基因的重组杆状病毒,接种sf9单层细胞,间接免疫荧光检测表明F1、G蛋白在杆状病毒中正确表达,并与抗尼帕病毒血清具有良好的反应原性。以纯化原核表达的F1、G蛋白免疫兔获得了抗F1和抗G重组蛋白的特异血清,Western blot和间接免疫荧光检测表明所制备的血清具有特异性。试验所表达的抗原和制备的特异血清可用于尼帕病的诊断。     

猪瘟病毒E2(gp55)基因的克隆表达及其DNA疫苗的初步研究

用RTPCR方法从中国标准强毒株石门毒的细胞培养物中 扩增获得了其结构蛋白E2基因cDNA,将之克隆到pGEM5Z T载体,用双脱氧链终止法测定其核苷酸序列,并推导出其对应氨基酸序列,与几个代表毒株Alfort株、Brescia株和C株相应序列进行比较,所测核苷酸序列与各株的同源性分别为84.7%、92.6%和95.2%,氨基酸序列的同源性分别为89.4%,92.6%和94.6%;将此E2片段亚克隆至真核表达载体pcDNA3.1,构建表达CSFV E2蛋白的重组质粒pcE2,用脂质体转染法将pcE2导入cos7细胞进行瞬时表达,用针对E2蛋白的特异性单抗以间接免疫荧光法检测,结果E2蛋白在cos7细胞中获得了正确表达,随之将pcE2质粒DNA进行小鼠肌内接种免疫,ELISA法检测证实在免疫后2周和4 周的小鼠体内可诱导出较为明显的阳性血清,并高于E2单抗的阳性对照,病毒中和试验也表明DNA免疫后小鼠体内可诱导产生CSFV中和抗体;同时构建了能在昆虫细胞Sf9中表达GSTE2和GSTGFPE2融合蛋白的重组杆状病毒;上述研究结果为研制针对CSFV的DNA疫苗,亚单位疫苗及其诊断试剂打下了基础。