Baculoviridae ; genetics ; metabolism ; Phylogeny ; Viral Proteins ; chemistry ; classification ; genetics ; metabolism

Limulus Factor C, a serine protease zymogen from the amoebocytes of the limulus, has high affinity for endotoxin. When Factor C is activated by endotoxin, it hydrolyses artificial tripeptide substrate and measurable products are released, so it can be used as an alternative reagent for endotoxin analysis. Factor C gene of Tachypleus tridentatus was obtained through RT-PCR and the recombinant protein was expressed by Bac-to-Bac/BmNPV baculovirus expression system in silkworm larvae. The activity of Factor C was detected with diluted serum of silkworm larvae, and the sensitivity of endotoxin detected was 0.2 EU/mL when the serum was diluted at 1:500. The silkworm larvae expressed limulus Factor C could be used to develop a new low-cost endotoxin test reagent.
Animals ; Arthropod Proteins ; biosynthesis ; Baculoviridae ; Bombyx ; metabolism ; Enzyme Precursors ; biosynthesis ; Genetic Vectors ; Larva ; metabolism ; Recombinant Proteins ; biosynthesis ; Serine Endopeptidases ; biosynthesis

OBJECTIVETo construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells.

METHODSHuman group A rotavirus was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene (egfp) driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection. The expression baculoviruse was constructed by Tn7 and Cre-LoxP recombinant and transfected into BmN cells. The gene expression was determined by detecting 6-His tag fused into VP7 C-terminus, and the assembled VLPs were observed by transmission electron micrography.

RESULTSThree genes of rotavirus were cloned and BmMultiBac was constructed. The genes were expressed and the rotavirus-like particles assembled in BmN cells successfully as verified by ELISA and electron microscope.

CONCLUSIONWe have successfully constructed the recombinant baculovirus co-expressing the 3 structural genes of rotavirus, which provide the basis for producing protein complex containing multiple subunits and investigation of the structure of the macromolecules.

Animals ; Baculoviridae ; genetics ; metabolism ; Bombyx ; virology ; Capsid Proteins ; genetics ; Cell Line ; Gene Expression ; Genetic Vectors ; Rotavirus ; genetics ; metabolism

OBJECTIVETo establish a method for the expression of glycogen synthase kinase 3β with high purity and biological activity.

METHODSE.coli expression system and baculovirus-insect cell expression system were used to produce the kinase, followed by purification using His-tag and GST-tag and determination of its purity and activity by SDS-PAGE and kinase reaction, respectively.

RESULTSGlycogen synthase kinase 3β produced from E.coli represented 54% of the total bacterial protein, as compared with 96% of the total protein from the insect cell system .Glycogen synthase kinase 3β produced from insect cell exhibited an one-fold higher biological activity than the protein obtained from E.coli.

CONCLUSIONSCompared with the protein from E.coli system, glycogen synthase kinase 3β from the insect cell expression system is endowed with a higher purity and bioactivity.

Animals ; Baculoviridae ; metabolism ; Escherichia coli ; metabolism ; Genetic Vectors ; Glycogen Synthase Kinase 3 ; biosynthesis ; isolation & purification ; Insecta ; cytology

To produce the recombinant baculovirus transfer plasmid pFast-ORF2, the ORF2 gene of Porcine Circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector (pFastBac(TM1) ) using Bac-to-Bac baculovirus expression system. E. coli DH10Bac (Gibco BRL) containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bac. ORF2) were collected by blue/white selection. The Bac. ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene, referred to as Ac. ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay (IIFA), SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2. The results indicated the ORF2 gene was properly expressed in sf9 cell. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in purified and phosphotungstic acid (PTA) stained PCV2 ORF2 protein by electron microscope. The particles were of similar morphology to the PCV2 virion and some self-assembled virus-like particles had darkly stained centers that made them appear to be empty capsids. Both PCV2 particles and self-assembled particles were approximately 17 nm in diameter.
Animals ; Baculoviridae ; genetics ; metabolism ; Circovirus ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Insecta ; cytology ; metabolism ; Open Reading Frames ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Swine ; Viral Proteins ; genetics ; metabolism ; Virion

Baculoviruses are a family of arthropod-specific viruses that produce two morphologically distinct types of virions (budded and occlusion-derived) in their lifecycle. Baculoviruses establish infection in the midgut of their host via the oral route: occlusion-derived virions have pivotal roles in these processes. This review summarizes the basic characteristics of baculoviruses, and discusses the composition and classification of baculovirus occlusion-derived virions. The latter focuses mainly on the evolution and role of multiple occlusion-derived virions in the lifecycle of baculoviruses. These achievements should aid understanding the evolution and infection mechanisms of baculoviruses.
Animals ; Baculoviridae ; genetics ; growth & development ; physiology ; Insecta ; virology ; Viral Proteins ; genetics ; metabolism ; Virion ; genetics ; growth & development ; physiology

OBJECTIVEThe S gene of a Hanta Virus (HV) Z10 strain was cloned into a baculovirus shuttle bacmid pDual-CMV contained a CMV promoter to generated a recombinant baculovirus BAC-pDual-CMV-HVS, then the recombinant baculovirus was transfected into Vero-E6 cell. The cells with recombinant baculovirus were applied to the detection of HV antiserum.

METHODSTo generate the recombinant baculovirus BAC-pDual-CMV-HVS, the sequence of CMV promoter was obtained from the plasmid pEGFP-N1 by PCR, and subsequently cloned to the baculovirus shuttle bacmid pFastBacDUAL resulting the recombinant plasmid pDual-CMV. Then the sequence of HV-S gene was inserted to the plasmid pDual-CMV, to generate the plasmid pDual-CMV-HVS. Plasmid pDual-CMV-HVS was transformed into the DH10BAC competent cells to get the recombinant baculovirus BAC-pDual-CMV-HVS. The antigen substrate slides were made by transfecting the recombinant virus into Vero-E6 cells.

RESULTSThe plasmid pDual-CMV-HVS was verified by sequencing. The recombinant virus BAC-pDual-CMV-HVS was generated according to the protocol of the baculovirus and transfected into Vero-E6 cells. The expression of the HV-S gene was verified by positive HV antiserum.

CONCLUSION[corrected] The recombinant virus were successfully generated and applied to prepare the antigen substrate slides. The antigen substrate slides was conveniently prepared without special equipments, and can be used to detect the antiserum of HV virus.

Animals ; Baculoviridae ; genetics ; metabolism ; Cercopithecus aethiops ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Hantavirus ; genetics ; metabolism ; Vero Cells ; Viral Envelope Proteins ; analysis ; genetics ; metabolism

Recombinant baculovirus containing sigmaC gene of Avian reovirus was constructed using Bac-To-Bac Baculovirus expression system, and recombinant sigmaC protein was expressed by infecting the sf9 cell with recombinant baculovirus. Firstly, sigmaC gene of Avian reovirus was cloned and inserted into donor plasmid pFastBacHTA to obtain recombinant donor plasmid pFsigmaC. Plasmid pFsigmaC was transformed into E. coli DH10Bac for integration into bacmid vector and the recombinant bacmid plasmid BacmidsigmaC was obtained. Recombinant baculovirus rBacsigmaC was obtained by transfection of the sf9 cells with BacmidsigmaC. Western blot and indirect immunofluorescence assay (IFA) were carried and the results showed that the recombinant sigmaC protein with 37 kDa molecular weight was expressed successfully.
Animals ; Baculoviridae ; genetics ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Orthoreovirus, Avian ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Spodoptera ; Transfection

As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus (MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary (GCO) cell line and displayed virion particles with atypical bullet shape, 300-500 nm in length and 100-200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein (1-458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid pFastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10Bac through screening and identification. Based on polymerase chain reaction (PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.
Animals ; Baculoviridae/metabolism* ; Bass/metabolism* ; Carps/virology* ; Cell Line ; Female ; Genetic Techniques ; Genome, Viral ; Glycoproteins/biosynthesis* ; Insecta ; Ovary/virology* ; Phylogeny ; Plasmids/metabolism* ; Recombinant Proteins/biosynthesis* ; Rhabdoviridae/metabolism*

Apolipoprotein A- I is the major apolipoprotein in high-density lipoprotein known to have a wide range of physiological functions, the best-studied one of which is in regulating cholesterol metabolism and preventing arteriosclerosis. Human blood has been the only source of this protein. To facilitate further research and application, it is essential to produce it through genetic engineering. In the current research, the baculovirus-insect cell system was used to overexpress human apolipoprotein A- I . Two recombinant baculoviruses were constructed. The first one expressed a pro form of apoA- I lacking native signal peptide. The recombinant protein was found to remain mainly inside cells in the early phase of infection, while being largely excreted to the medium late in infection. The second one used a heterologous signal peptide, snake phospholipase A2 inhibitor alpha subunit signal peptide, to lead the secretion of mature apoA- I. In contrast to the first virus, recombinant apoA- I was found in the culture medium at the early phase of virus infection. The mature apoA- I was purified from culture medium using Phenyl Sepharose hydrophobic interaction chromatography (HIC) and eluted with water and Propylene. This work shows that snake phospholipase A2 inhibitor a subunit signal peptide can be used to secret human apoA- I in insect cells, but the efficiency of its secretion is limited when the expression level is high.
Animals ; Apolipoprotein A-I ; genetics ; metabolism ; Baculoviridae ; genetics ; Blood Proteins ; genetics ; metabolism ; Blotting, Western ; Cell Line ; Chromatography ; Genetic Vectors ; genetics ; Humans ; Recombinant Proteins ; genetics ; metabolism ; Snakes ; Spodoptera ; cytology