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

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

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

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

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

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

OBJECTIVETo obtain recombinant human CYP2E1 and to determine its activity by using the specific probe substrate.

METHODSCYP2E1 cDNA was obtained by RT-PCR using human liver RNA as template. The cloned CYP2E1 cDNA was ligated with pFastBac vector to generate recombinant pFastBac-CYP2E1, which was then transformed into E. coli DH 10 Bac. Recombinant Bacmid-CYP2E1 was generated by transposition. Then Spodoptera frugiperda (Sf9) insect cells was infected with Bacmid-CYP2E1 to generate recombinant baculoviruses carrying human CYP2E1 cDNA. Finally, Sf9 insect cells were triinfected with recombinant baculoviruses carrying human CYP2E1, CYPOR and CYPb5. The activity of the recombinant enzymes was determined using chlorzoxazone as the substrate.

RESULTThe Kmand Vmaxof recombinant CYP2E1 to chlorzoxazone was (72.4 +/-8.7) micromol. L(-1) and (2.41 +/-0.10) micromol.min(-1)?g(-1)protein, respectively.

CONCLUSIONActive recombinant CYP2E1 has been obtained by bac-to-bac expression system and its activity is similar to previous reports.

Animals ; Baculoviridae ; genetics ; metabolism ; Cytochrome P-450 CYP2E1 ; biosynthesis ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Spodoptera ; genetics ; metabolism ; Transfection

A recombinant transfer vector, pBacIL-11, containing hIL-11 cDNA of 546 nucleotides lacking leader sequence was constructed and co-transfected into BmN cells with linearized BmBacPAK(modified BmNPV) DNA for construction of a recombinant baculovirus carrying the hIL-11 gene. Southern hybridization analysis suggested that the recombinant baculovirus DNA contained hIL-11 cDNA fragment. RNA dot blotting demonstrated that the hIL-11 gene was transcribed. The recombinant baculovirus has a strong infectivity to BmN cell line and to silkworm larvae and pupae. Specific hIL-11 bands were detected from all the samples of cell extract, culture supernatant, haemolymph of larvae and pupae by SDS-PAGE analysis. Biological activity of the expressed product was determined with IL-11 dependent B9-11 cell line and by MTT colorimetric assay, which indicated that biologically active rhIL-11 protein was overexpressed in BmN cell line and in silkworm larvae and pupae.
Animals ; Baculoviridae ; genetics ; Bombyx ; cytology ; growth & development ; metabolism ; Cell Culture Techniques ; Gene Expression ; Genetic Vectors ; Humans ; Interleukin-11 ; biosynthesis ; genetics ; Larva ; genetics ; metabolism ; Pupa ; genetics ; metabolism

IL-18, as a polyphonic cytokine, is important in immune response and physiologic function. We designed one pair of primers, amplified the porcine IL-18 gene fused with a C-terminal 6xHistidine tag, and then subcloned into the pFastBacDual of Baculovirus transfer vector and transformed into DH10Bac containing a shuttle vector of Bacmid. After co-transfecting the recombinant plasmid into insect cells, the 18 kDa expressed protein of porcine IL-18 was detected by SDS-PAGE; the specificity of expressed protein was confirmed by Western blotting. The purified porcine IL-18 protein induced obvious proliferation of porcine T lymphocytes in vitro, which indicated that the expression of IL-18 had high biological activity.
Animals ; Baculoviridae ; genetics ; metabolism ; Cells, Cultured ; Genetic Vectors ; genetics ; Insecta ; genetics ; metabolism ; Interleukin-18 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transfection