棉铃虫核型多角体病毒凋亡抑制基因对p35基因失活病毒的功能拯救

野生型苜蓿银纹夜蛾核型多角体病毒（ＡｃＮＰＶ）能感染棉铃虫细胞,不引起细胞凋亡,用ＬａｃＺ基因使ＡｃＮＰＶ凋亡抑制基因ｐ３５插入失活,得到缺陷病毒Ａｃｐ３５Ｚ能迅速引起棉铃虫细胞凋亡,但缺陷病毒本身不能复制。用ＡｃＮＰＶ即早期基因ＩＥ１启动子带动棉铃虫杆状病毒（ＨａＮＰＶ）ｐ３５基因在棉铃虫细胞中瞬时表达,能拯救ｐ３５基因失活病毒Ａｃｐ３５Ｚ复制。通过Ｘ－ｇａｌ显色反应和ｄｏｔＥＬＩＳＡ分别检测到了半乳糖苷酶的活性和ｐ３５基因的表达,证明了所克隆的ＨａＮＰＶｐ３５基因不仅是一个凋亡抑制基因,也是一个与杆状病毒复制相关的基因,它的瞬时表达能支持Ａｃｐ３５Ｚ在棉铃虫细胞中复制。     

Construction of Stably Transformed Bm5 and Sf9 Cells Displaying Green Fluorescence by Using Autographa californica Nuclear Polyhedrosis Virus IE1 Gene

Transformed Bm5 or Sf9 cells displaying green fluorescence were constructed by using Autographa californica nuclear polyhedrosis virus (AcNPV) immediate early gene (ie 1). Green fluorescent protein (gfp) gene was introduced under the control of the AcNPV ie 1 promoter to yield expression plasmid pAcIE1-GFP. It was transfected into Sf9 or Bm5 cells and cell clones expressing GFP were selected by fluorescence microscopy. Genomic DNA from transformed cells was isolated and integration of AcNPV ie 1 gene harboring gfp gene was confirmed by PCR using AcNPV ie 1 gene-specific primers. The GFP was successfully expressed in the cytoplasm of insect cells transformed with pAcIEI-GFP and the expressed GFP was maintained during cell division. Furthermore, GFP expression by AcNPV ie 1 promoter in transformed cells was not interfered with viral replication. This suggests that transformed cells displaying foreign gene product by using AcNPV ie 1 promoter will be useful for the diverse applications of the insect cells.     

Functional analysis of the inhibitor of apoptosis genes in <Emphasis Type="Italic">Antheraea pernyi</Emphasis> nucleopolyhedrovirus

The inhibitor of apoptosis proteins (IAP) plays an important role in cell apoptosis. We cloned two novel IAP family members, Ap-iap1 and Ap-iap2, from Antheraea pernyi nucleopolyhedrovirus (ApNPV) genome. Ap-IAP1 contains two baculoviral IAP repeat (BIR) domains followed by a RING domain, but Ap-IAP2 has only one BIR domain and RING. The result of transient expression in Spodoptera frugiperda (Sf21) showed that Ap-iap1 blocked cell apoptosis induced by actinomycin D treatment and also rescued the p35 deficient Autographa californica nucleopolyhedrovirus (AcNPV) to replicate in Sf9 cells, while Ap-iap2 does not have this function. Several Ap-IAP1 truncations were constructed to test the activity of BIRs or RING motif to inhibit cell apoptosis. The results indicated that BIRs or RING of Ap-IAP1 had equally function to inhibit cell apoptosis. Therefore deletion of above both of the above domains could not block apoptosis induced by actinomycin D or rescue the replication of AcMNPVΔp35. We also screened two phage-display peptides that might interact with Ap-IAP1.     

苜宿银纹夜蛾核型多角体病毒vp39基因的克隆及其对S_f9细胞形态的影响

最近的研究发现：ＡｃＮＰＶ的ｖｐ３９基因与侵染密切相关［１］．在侵染过程中,ＶＰ３９蛋白与宿主的肌动蛋白结合,使其重排形成缆索（ｃａｂｌｅ）．导致细胞骨架发生变化有利于病毒编码的蛋白酶的水解．最后,子代病毒颗粒大量形成,宿主昆虫体全部液化成为脓水．可...     

Stable cell lines expressing baculovirus P35: resistance to apoptosis and nutrient stress, and increased glycoprotein secretion

Summary The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells byAutographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9^P35AcV5-1 and Sf9^P35AcV5-3) showed increased resistance to actinomycin D-induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9^P35AcV5-1 and Sf9^P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.     

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order + 35 > + 1 > − 3 > − 8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60–72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in ‘glowing silkworms’. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.     

Persistent Baculovirus Infection Results from Deletion of the Apoptotic Suppressor Gene p35

Infection with the wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) results in complete death of Spodoptera frugiperda (Sf) cells. However, infection of Sf cells with AcMNPV carrying a mutation or deletion of the apoptotic suppressor gene p35 allowed the cloning of surviving Sf cells that harbored persistent viral genomes. Persistent infection established with the virus with p35 mutated or deleted was blocked by stable transfection of p35 in the host genome or by insertion of the inhibitor of apoptosis (iap) gene into the viral genome. These artificially established persistently virus-infected cells became resistant to subsequent viral challenge, and some of the cell lines carried large quantities of viral DNA capable of early gene expression. Continuous release of viral progenies was evident in some of the persistently virus-infected cells, and transfection of p35 further stimulated viral activation of the persistent cells, including the reactivation of viruses in those cell lines without original continuous virus release. These results have demonstrated the successful establishment of persistent baculovirus infections under laboratory conditions and that their establishment may provide a novel continuous, nonlytic baculovirus expression system in the future.     

Synthesis of the herpes simplex virus type 1 trans-activator Vmw65 in insect cells using a baculovirus vector

Vmw65, the Herpes Simplex Virus trans-activator of immediate-early genes, was expressed in insect cells using a recombinant baculovirus expression vector and partially purified. Insect cell-derived Vmw65 was shown to be indistinguishable from authentic Vmw65 present in purified HSV-1 virions based on electrophoretic mobility, immunoreactivity with a monoclonal antibody, and ability to interact with cellular factors to form a protein/DNA complex with oligonucleotides containing a TAATGARAT element.Abbreviations AcNPV Autographica californica nuclear polyhedrosis virus - HSV Herpes Simplex Virus - IE Immediate Early - moi multiplicity of infection - Sf9 Spodoptera frugiperda cells     

Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 × 10⁶ cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) cross-reacted with re-PNA. The subunit molecular weight (30kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.     

Stress-induced apoptosis in Spodoptera frugiperda (Sf9) cells: baculovirus p35 mitigates eIF2 alpha phosphorylation

Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.     

用杆状病毒为载体在昆虫细胞中表达马立克病病毒pp38基因

鸡马立克病病毒(MDV)38kd磷蛋白(pp38)基因中包括起始密码子和终止密码子的完整编码序列被整合进杆状病毒AcNPV的转移载体质粒pVL1392,用所得的含pp38基因的重组转移载体质粒pVLpp38I与野生型杆状病毒AcNPV的DNA共转染昆虫传代细胞系Sf9细胞后,用荧光抗体法以抗MDV单克隆抗体H_(19)筛选到能表达MDVpp38的重组杆状病毒克隆BP38 I。免疫印迹试验表明,在重组病毒BP38 I感染的Sf9细胞溶解物中,可表现一条分子量约为35—36kd的为单克隆抗体H_(19)识别的MDV特异性蛋白带。     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV,and knockout of cysteinase gene for more efficient expression

AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insectbaculovirus expression systems, each having different characteristics. AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression, but the small size of the host insect,A. californica, makes AcNPV less suitable for large scale protein synthesis. In contrast, BmNPV can only infect the silkworm,Bombyx mori, which is wellknown for its easy rearing and large size. These characteristics make the BmNPV system especially suitable for largescale industrial expression. To utilize the advantages of both AcNPV and BmNPV, we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV, designated as HyNPV The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV. Taking the human basic fibroblast growth factor (bFGF) gene as an application example, we constructed a recombinant, HyNPV-bFGF. This construct is able to express the bFGF protein both in silkworm larvae and in commonuse cell lines, sf21, sf9 and High-five. Moreover, to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus, we knocked out the cysteinase gene coding for one of the most important baculovirus proteases. This knockout mutation improves the production efficiency of the bFGF recombinant protein.     

Efficient membrane targeting of the chick nicotinic acetylcholine receptor α-subunit in stable insect cell lines

We have synthesised the -subunit of the chick nicotinic acetylcholine receptor (nAChR) in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovius immediate early gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.nAChR, encoding the -subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising the chick nAChR -subunit. Using fluorescence microscopy and ligand binding studies we were able to demonstrate efficient membrane targeting of the receptor subunit in the insect cell plasma membrane. Stable insect cell lines may thus have significant advantages over transient baculovirus vectors for the synthesis and characterisation of heterologous receptor proteins.Abbreviations AcNPV Autographa californica nuclear polyhedrosis virus - BTX -bungarotoxin - BSA bovine serum albumin - FITC Fluoroscein isothiocyanate - G418 geneticin-418 - hpi hours post-infection - ie-1 immediate early 1 gene - nAChR nicotinic acetylcholine receptor alpha subunit - Sf Spodoptera frugiperda - tPA tissue plasminogen activator     

p13 from group II baculoviruses is a killing-associated gene

p13 gene was first described in Leucania separata multinuclear polyhedrosis virus (Ls-p13) several years ago, but the function of P13 protein has not been experimentally investigated to date. In this article, we indicated that the expression of p13 from Heliothis armigera single nucleocapsid nucleopolyhedrovirus (Ha-p13) was regulated by both early and late promoter. Luciferase assay demonstrated that the activity of Ha-p13 promoter with hr4 enhancer was more than 100 times in heterologous Sf9 cells than that in nature host Hz-AM1 cells. Both Ls-P13 and Ha-P13 are transmembrane proteins. Confocal microscopic analysis showed that both mainly located in the cytoplasm membrane at 48 h. Results of RNA interference indicated that Ha-p13 was a killing-associated gene for host insects H. armigera. The AcMNPV acquired the mentioned killing activity and markedly accelerate the killing rate when expressing Ls-p13. In conclusion, p13 is a killing associated gene in both homologous and heterologous nucleopolyhedrovirus. [BMB Reports 2012; 45(12): 730-735]     

Transfection of insect cell lines using polyethylenimine

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85–90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

Baculovirus-mediated expression of the Na+/glucose cotransporter in Sf9 cells.

We have used baculovirus (AcNPV) to express the Na+/glucose cotransporter protein in cultured Sf9 cells. We constructed a baculovirus transfer vector containing the cDNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) under the control of the polyhedrin gene promoter. Recombinant baculovirus was obtained by cotransfection of SF9 cells with wild-type AcNPV DNA and the transfer vector. Recombinant virus was identified by Southern blotting and then purified. Recombinant infected Sf9 cells expressed a protein which was recognized by anti-peptide antibodies raised to sequences of the cloned Na+/glucose cotransporter. This protein migrated with a molecular mass of 55 kD by SDS-PAGE, similar to the in vitro translation product of SGLT1. An identical protein was metabolically labeled with [35S]methionine. Cells which synthesized the transport protein showed Na(+)-dependent alpha MeGlc transport. Micromolar phlorizin inhibited transport. Uninfected and wild-type virus infected Sf9 cells did not have Na(+)-dependent glucose transport. All transport protein migrated at 45% sucrose (w/w) by density gradient sedimentation, suggesting that the expressed transporter is membrane associated. We conclude that we have functionally expressed the rabbit intestinal Na+/glucose cotransporter in Sf9 cells. The transporter is not heavily glycosylated, and this is consistent with previous work showing that glycosylation is not necessary for function. We are poised to purify and characterize this protein from a structure-function perspective.     

Bruton酪氨酸激酶在昆虫细胞中表达的初步研究

用杆状病毒表达载体系统表达小鼠Ｂｒｕｔｏｎ酪氨酸激酶（Ｂｒｕｔｏｎｔｙｒｏｓｉｎｅｋｉｎａｓｅ,Ｂｔｋ）．构建重组转染载体时,于Ｂｔｋ起始码的上游插入了一段Ｈ９０２序列．用重组转染载体、苜蓿夜蛾核型多角体病毒线性ＤＮＡ和质脂体共转染Ｓｆ９昆虫细胞,经过对重组杆状病毒三轮扩增后,用Ｈ９０２抗体检测表明,昆虫细胞中Ｂｔｋ的表达已达最高水平．对表达后Ｂｔｋ自身磷酸化检测表明,该激酶具有自身磷酸化活性．从而证实Ｂｔｋ在昆虫细胞中的表达获得了成功     

Expression of lacZ reporter gene under the control of the polyhedrin promoter of Spodoptera litura nuclear polyhedrosis virus

Promoter function of the putative polyhedrin-encoding gene (polh) of Spodoptera litura nuclear polyhedrosis virus (S1MNPV) was determined by transferring it to the Autographa californica nuclear polyhedrosis virus (AcMNPV) through the AcNPV polh based vector, pVL1393. Three transfer vectors pCBT2, pCBT3 and pCBT4 were constructed by substituting the promoter and the neighbouring sequences of AcNPV in pVL1393 by that of SINPV. The Escherichia coli lacZ gene was placed downstream from the S1NPV polh promoter in the hybrid transfer vector (pCBT) constructs. Co-transfection of Spodoptera frugiperda cells (Sf9) with each of the pCBTlacZ vector and wild-type AcNPV DNAs led to synthesis of β-galactosidase (βGal). The plaque-purified recombinant viruses (S1AcNPV.lacZ) expressing lacZ under the polh promoter of S1NPV are stable. The highest βGal activity was obtained with S1AcNPV4.lacZ. Production of βGal with recombinant virus, S1AcNPV3.lacZ in which S1NPV polh promoter is in the reverse orientation in the AcNPV genome, is 83% of that produced by S1AcNPV4.lacZ. These results indicate that the S1NPV polh promoter is active in the genetic environment of AcNPV; the polh of S1NPV is phylogenetically related to AcNPV like other baculoviruses.     

Identification and characterization of the p35 gene of Bombyx mori nuclear polyhedrosis virus that prevents virus-induced apoptosis.

Nucleotide sequence analysis of the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome revealed the existence of a gene homologous to the p35 gene of Autographa californica NPV (AcNPV), which has been shown to prevent virus-induced apoptosis. The BmNPV p35 gene showed 96.1% nucleotide and 89.6% predicted amino acid sequence identity to the AcNPV p35 gene. A mutant BmNPV (BmP35Z) lacking a functional p35 gene induced apoptosis-like cell degradation in infected BmN cells. However, unlike the p35-deleted AcNPV mutant (vAcAnh), BmP35Z replicated normally and produced polyhedral inclusion bodies. The patterns of protein synthesis and the percentages of viable BmN cells remaining following infection with either wild-type BmNPV or BmP35Z were nearly identical. BmP35Z also replicated in silkworm larvae without showing any apparent apoptotic response in infected hemocytes, fat body, or other tissues. Time to death of larvae infected with BmP35Z was similar to that for wild-type-infected larvae, and significant numbers of polyhedral inclusion bodies were produced. These results indicate that viral factors (or genes) other than p35 or host cell factors play a role in inducing, accelerating, or interfering with apoptotic processes. The evolution of baculovirus genomes is also discussed with reference to comparative analysis of the p35 and p94 gene sequences. The p94 gene is found immediately upstream of p35 in AcNPV; in BmNPV, however, the p94 gene was nearly completely missing, presumably because of large deletions in a BmNPV ancestor virus having a gene similar to the AcNPV p94 gene.