排序方式: 共有75条查询结果,搜索用时 31 毫秒
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本实验利用AcMNPV(Autographa californica multiple nuclear polyhedrosis virus,AcMNPV)的bac-to-bac系统构建了两种重组病毒,即含GFP-actin融合基因的重组病毒AcMNPV-GFP-actin和含GFP基因的重组病毒AcMNPV-GFP。用这两种重组病毒分别感染Sf9细胞,以AcMNPV-GFP感染的Sf9细胞为对照,用共聚焦激光扫描显微镜观察了绿色荧光在病毒感染过程中的分布情况。由于肌动蛋白和绿色荧光蛋白是共定位的,所以绿色荧光的分布情况就是肌动蛋白的分布情况。实验中观察发现,AcMNPV-GFP感染的Sf9细胞中的绿色荧光,在整个感染过程中是弥散分布的,而AcMNPV-GFP-actin感染Sf9细胞后24172h这段时间内,肌动蛋白最初聚集在细胞核内,随后逐渐由细胞核向细胞质转移,最后完全聚集于细胞膜。根据实验结果,推测肌动蛋白可能参与了AcMNPV出芽型病毒粒子(BV)由细胞核向细胞质运输以及从细胞膜排出的过程。 相似文献
43.
A transposon-based random mutation library of AcMNPV, the type species of baculovirus, was constructed using a Tn5 transposon.
The green fluorescence protein gene under the control of the Drosophila hsp 70 promoter was inserted into the transposon for easy tracking in insect cells. In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon
into the virus genome. The transposed genome was then used to transfect Sf21 insect cells, and a library of mutant viruses
capable of expressing green fluorescence protein was obtained. Two mutant viruses, B9F and Li6A were isolated, and the sites
of transposon insertion were determined to be within the coding regions of the 94k and p10 genes, respectively. Both genes
were determined to be nonessential in viral replication and infection. This technique will be very useful in the functional
study of baculovirus genes.
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Translated from Journal of Fudan University(Natural Science), 2005,44(4) [译自: 复旦学报(自然科学版),2005,44(4)] 相似文献
44.
Improving Baculovirus Transduction of Mammalian Cells by Incorporation of Thogotovirus Glycoproteins
Baculovirus can transduce a wide range of mammalian cells and is considered a promising gene therapy vector. However,the low transduction efficiency of baculovirus into many mammalian cells limits its practical application. Co-expressing heterologous viral glycoproteins(GPs), such as vesicular stomatitis virus G protein(VSV G), with baculovirus native envelope protein GP64 is one of the feasible strategies for improving virus transduction. Tick-borne thogotoviruses infect mammals and their GPs share sequence/structure homology and common evolutionary origins with baculovirus GP64.Herein, we tested whether thogotovirus GPs could facilitate the entry of the prototype baculovirus Autographa californica multiple multiple nucleopolyhedrovirus(AcMNPV) into mammalian cells. The gp genes of two thogotoviruses, Thogoto virus and Dhori virus, were inserted into the AcMNPV genome. Both GPs were properly expressed and incorporated into the envelope of the recombinant AcMNPVs. The transduction rates of recombinant AcMNPVs expressing the two thogotovirus GPs increased for approximately 4–12 fold compared to the wild type AcMNPV in six of the 12 tested mammalian cell lines. It seemed that thogotovirus GPs provide the recombinant AcMNPVs with different cell tropisms and showed better performance in several mammalian cells compared to VSV G incorporated AcMNPV. Further studies showed that the improved transduction was a result of augmented virus-endosome fusion and endosome escaping, rather than increased cell binding or internalization. We found the AcMNPV envelope protein GP64-mediated fusion was enhanced by the thogotovirus GPs at relatively higher p H conditions. Therefore, the thogotovirus GPs represent novel candidates to improve baculovirus-based gene delivery vectors. 相似文献
45.
Xiaoqian Zhang Ming Chen Xinlei Ma Xiaofan Zhao Jinxing Wang Honglian Shao Qisheng Song David Stanley 《Archives of insect biochemistry and physiology》2013,82(3):158-171
Host cytoskeletons facilitate the entry, replication, and egress of viruses because cytoskeletons are essential for viral survival. One mechanism of resisting viral infections involves regulating cytoskeletal polymerization/depolymerization. However, the molecular mechanisms of regulating these changes in cytoskeleton to suppress viral replication remain unclear. We established a cell line (named Ha‐shl‐t) from the pupal testis of Helicoverpa armigera (Lepidoptera: Noctuidae). The new testis cell line suppresses Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) replication via disassembly of cytoskeleton. Up‐regulation of thymosin (actin disassembling factor) and adf (actin depolymerizing factor) reduces F‐actin. Silencing thymosin or adf or treating cells with the F‐actin stabilizer phalloidin led to increased AcMNPV replication, while treating cells with an F‐actin assembly inhibitor cytochalasin B decreased viral replication. We infer that Ha‐shl‐t cells utilize F‐actin depolymerization to suppress AcMNPV replication by up‐regulating thymosin and adf. We propose Ha‐shl‐t as a model system for investigating cytoskeletal regulation in antiviral action and testicular biology generally. 相似文献
46.
XianDong Zeng Fang Nan ChangYong Liang JianHua Song Qian Wang Just M. Vlak XinWen Chen 《中国科学:生命科学英文版》2009,52(8):761-770
The Autographa californica nucleopolyhedrovirus (AcMNPV) contains three apoptosis suppressor genes: p35, iap1 and iap2. AcMNPV P35 functions as a pancaspase inhibitor, but the function of IAP1 and IAP2 has not been entirely resolved. In this paper, we analyze the function of IAP1 and IAP2 in de-tail. AcMNPV with p35-deletion inhibited the apoptosis of BTI-Tn-5B1-4 (Tn-Hi5) cells induced by a Helicoverpa armigera single nucleocapsid NPV (HearNPV) infection and rescued the replication of HearNPV and BV production in these cells. Transient-expression experiments indicated that both IAP1 and IAP2 suppress apoptosis of Tn-Hi5 cells during HearNPV infection. Recombinant HearNPVs ex-pressing AcMNPV iap1, iap2 and p35, respectively, not only prevented apoptosis but also allowed HearNPV to replicate in Tn-Hi5 cells. However, the iap1, iap2 and p35 genes when expressed in HearNPV were unable to rescue BV production. These results indicate that both AcMNPV iap1 and iap2 function independently as apoptosis inhibitors of and are potential host range factors. 相似文献
47.
Ascorbic acid, known to be a free radical scavenger and vital to insect development, is important in larval resistance to baculovirus infection. We sequentially elevated the ascorbic acid content in an ascorbic acid-depleted diet and evaluated the effect on larval Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) development and immunocompetence. Dietary ascorbic acid levels lower than 0.7 g l−1 slowed the growth rate of larvae, reduced pupal weights significantly, and severely inhibited adult emergence. Larvae developing on ascorbic acid-free diet experienced far higher levels of mortality following per os infection with virus. Additionally, viral infection in larvae fed an ascorbic acid-free diet, as monitored by epifluorescence microscopy, showed signs of infection much earlier than larvae fed control levels of ascorbic acid. These results demonstrate an indirect correlation between the level of ascorbic acid in the food stream of larval H. virescens and the susceptibility of the insect to baculoviral infection. 相似文献
48.
Gatehouse LN Markwick NP Poulton J Young VL Ward VK Christeller JT 《Bioprocess and biosystems engineering》2008,31(5):469-475
The yield of two proteins, avidin and green fluorescent protein (GFP), expressed from a modified Autographa californica nucleopolyhedrovirus (AcMNPV), was compared in Sf9 cell culture monolayer, Sf21 cell suspension culture and intact Spodoptera litura larvae. GFP expressed from the p10 promoter yielded up to 1.5% of total soluble protein in larvae, 20-fold higher than that in monolayer suspension culture. Avidin, expressed from the polh promoter, yielded up to 2.3% of total soluble protein in larvae, 10-fold higher than that in suspension culture and 40-fold higher than that in monolayers. Avidin expression did not affect amounts of GFP in dual-expressing baculovirus compared with those detected from a GFP-only expressing AcMNPV. A biotin-binding assay showed that all avidin expressed in larvae was fully active. Glycosylation patterns of chicken-avidin and Spodoptera-avidin were very similar, though the latter showed a proportion of partially glycosylated material. 相似文献
49.
A recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) strain showing higher virulence against Trichoplusia ni larvae than the wild-type virus was developed. The 'enhancin' (VEF) gene of T. ni granulovirus (TnGV) and the AcMNPV polyhedrin gene were cloned into the baculovirus transfer vector pAcUW31. This plasmid and AcMNPV BacPAK6 DNA were co-transfected into the BTI-Tn5B1-4 cell line. A recombinant AcMNPV strain (BacVEFPol) was purified, amplified, and bioassayed against T. ni first instar larvae. Its estimated LC50 (0.184 OB/mm2) was 2.18 times lower than the LC50 estimated for the wild-type AcMNPV (0.402 OB/mm2). Likewise, an LT50 of 67.7 h was estimated for the recombinant AcMNPV strain while the LT50 of wild-type AcMNPV was estimated at 81.9 h. This indicates a 17.4% reduction of the time required to kill the larvae. The higher virulence of the recombinant strain, evidenced by its LC50 and LT50 values being lower than those of the wild-type strain, indicates that the VEF protein is expressed properly and may be occluded in the OBs. 相似文献
50.
Goodman CL McIntosh AH El Sayed GN Grasela JJ Stiles B 《In vitro cellular & developmental biology. Animal》2001,37(6):374-379
Summary One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high
levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10
lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected
baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus
(AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers
were determined using TCID50 assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines
were tested, with eight producing AcMNPV ECV titers of 1.1–47.3×106 TCID50/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5–62.3×106 TCID50/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4–5.0×106TCID50/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7–232.6×106TCID50/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for
AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs
and generate moderate to high levels of ECVs of one or more baculoviruses.
All programs and services of the USDA Department of Agriculture are offered on a nondiscriminatory basis without regard to
race, color, national origin, religion, sex, age, marital status or handicap. 相似文献