Transformation efficiency, virus multiplication and foreign gene expression were characterized in the insect cells transformed with Autographa calfornica nuclear polyhedrosis virus (AcNPV) immediate early 1 gene (IE1). Transformation efficiency of insect cells by AcNPV IE1 gene vector horboring foreign gene was approximately 8-fold higher in the Sf9 cells transformed previously with AcNPV IE1 gene than in the normal Sf9 cells. Virus multiplication and foreign gene expression of recombinant baculovirus in the Sf9 cells transformed with AcNPV IE1 gene were similar to those of the normal Sf9 cells. These results suggest that transformed cells displaying foreign gene product by using AcNPV IE1 gene promoter will be useful for the diverse applications of insect cells.
Baculoviridae ; Gene Expression ; Insects* ; Nucleopolyhedrovirus* ; Sf9 Cells

Transformation efficiency, virus multiplication and foreign gene expression were characterized in the insect cells transformed with Autographa calfornica nuclear polyhedrosis virus (AcNPV) immediate early 1 gene (IE1). Transformation efficiency of insect cells by AcNPV IE1 gene vector horboring foreign gene was approximately 8-fold higher in the Sf9 cells transformed previously with AcNPV IE1 gene than in the normal Sf9 cells. Virus multiplication and foreign gene expression of recombinant baculovirus in the Sf9 cells transformed with AcNPV IE1 gene were similar to those of the normal Sf9 cells. These results suggest that transformed cells displaying foreign gene product by using AcNPV IE1 gene promoter will be useful for the diverse applications of insect cells.
Baculoviridae ; Gene Expression ; Insects* ; Nucleopolyhedrovirus* ; Sf9 Cells

PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.
Animals ; Baculoviridae ; Genetic Vectors ; Humans ; Recombinant Proteins ; Sf9 Cells ; Spodoptera

Chinese scorpion Buthus martensii Karsch (BmK) venom is a rich source of neurotoxins which bind to various ion channels with high affinity and specificity and thus widely used as compounds to modulate channel gating or channel currents. To promote the insecticidal effects of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the gene encoding an excitatory insect toxin, BmK IT, was inserted into the genome of AcMNPV to construct a recombinant baculovirus, AcMNPV-BmK IT. Spodopter frugiperda 9 (Sf9) cells were infected with AcMNPV and AcMNPV-BmK IT respectively for 24 h. Results from the MTT assay, TUNEL assay, analysis of the expression level of apoptosis-related proteins (c-Myc, cleaved-Caspase3, Bcl-2 and Bax) of Sf9 cells, the transcription level of key genes (38K, C42, P78, F) of AcMNPV, and viral propagation assay demonstrated that AcMNPV-mediated expression of BmK IT promoted the apoptosis of Sf9 cells and replication of AcMNPV. The results laid a foundation for further structural and functional analysis of BmK IT.
Animals ; Apoptosis ; Cell Line ; Nucleopolyhedrovirus ; metabolism ; physiology ; Scorpion Venoms ; biosynthesis ; Sf9 Cells ; drug effects ; Virus Replication

Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.
Asparagine ; Baculoviridae* ; Blotting, Western ; Chromatin ; Cytoplasm ; Humans* ; Immunoprecipitation ; Insects ; Open Reading Frames ; Phosphoproteins ; Serine ; Sf9 Cells

AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2 x 10(7) cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.
Animals ; Baculoviridae ; DNA, Single-Stranded ; Dependovirus ; Gene Expression ; Genetic Vectors ; HEK293 Cells ; Humans ; Sf9 Cells ; Terminal Repeat Sequences ; Transfection

A baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. Dual Ig domain containing cell adhesion molecule (DICAM) belongs to the type I class of transmembrane proteins. It consists of a signal peptide, two V-type Ig domains in the extracellular region, and a short cytoplasmic tail of 442 amino acids. To purify the recombinant DICAM protein from cells overexpressing the mouse full-length DICAM gene, recombinant baculovirus is infected and recovered in the Sf9 cells. As a result, mouse DICAM protein was efficiently expressed and extracted from the insect cells using the BEVS. This recombinant protein can be used in further studies for functional test of DICAM protein in the cells.
Amino Acids ; Animals ; Baculoviridae ; Cell Adhesion ; Cytoplasm ; Insects ; Mice ; Protein Sorting Signals ; Proteins ; Recombinant Proteins ; Sf9 Cells

The Bombyx mori decapentaplegic gene is one of the conserved genes in vertebrate and invertebrates. The TGF-beta superfamily contains conserved polypeptide growth factors that play important roles in different cellular processes such as proliferation, apoptosis, differentiation and cell-fate determination. The B. mori dpp gene shares genetic homology with hBMPs and Drosophila dpp. Until now, only few studies have been conducted to examine the functions of B. mori dpp; and hence, its function is not yet well understood. In this study, the baculovirus expression vector system (BEVS) was used for expression of the recombinant B. mori dpp protein and in which the recombinant baculovirus is recovered in the host Sf9 cells. The selected pure recombinant baculovirus containing B. mori dpp gene (rBV-egfp-Bm dpp) was used to increase the effective protein purification by using His-tag extraction strategy. After selection of recombinant baculovirus, recombinant B. mori dpp proteins were extracted from the re-infected cells with pure rBV-egfp-Bm dpp. Herein, we summarize the efficient expression and purification of B. mori dpp proteins from the insect cells using the BEVS. This recombinant protein could be suitable for functional test and various application studies.
Apoptosis ; Baculoviridae* ; Bombyx* ; Drosophila ; Insects ; Intercellular Signaling Peptides and Proteins ; Invertebrates ; Sf9 Cells ; Transforming Growth Factor beta ; Vertebrates

OBJECTIVETo investigate the optimal conditions of tri-expression of CYP3A4, POR and cyt b5 in Sf 9 cells.

METHODSThe Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined.

RESULTSWhen the temperature and rotation speed of the shaker were 27 degree and 90 r/min, the cell density and culture volume were 5X105 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km, Vmax, and CLint for testosterone metabolism were 119.6 μmol/L,0.52 μmol/(min*g protein) and 4.34 ml/(min*g protein), respectively.

CONCLUSIONThe conditions of tri-expressing of CYP3A4, POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.

Animals ; Cytochrome P-450 CYP3A ; biosynthesis ; Cytochromes b5 ; biosynthesis ; Humans ; Insecta ; NADPH-Ferrihemoprotein Reductase ; biosynthesis ; Sf9 Cells

BACKGROUND: Reactive Oxygen species have been known to be a key factor to promote atherosclerosis. DDR2(Discoidin Domain Receptor 2) is a cell surface receptor tyrosine kinase which is activated by fiber collagen. Recently, DDR2 was suggested to be involved in activation of smooth muscle cell in blood vessel of atherosclerosis. METHODS: The effect of antioxidant, N-acetyl cysteine and H2O2(Hydrogen peroxide) in the activation of DDR2 by collagen was studied using HEK293 cells expressing DDR2. The direct activation of DDR2 tyrosine kinase domain by tyrosine phosphorylation upon the treatment of H2O2 was analysed after the kinase domain was expressed in sf9 cells. RESULTS: H2O2 enhanced DDR2 auto-phosphorylation and its cellular signaling to induce MMP-1 expression. However N-acetyl cysteine suppressed the DDR2 activation. The reactive oxygen induced tyrosine phosphorylation in DDR2 tyrosine kinase domain to activate its tyrosine kinase activity. CONCLUSIONS: DDR2 activity can be up-regulated by oxidative stress and this provides a mechanism that DDR2 plays a critical role when reactive oxygen species promote atherosclerosis. Therefore inhibition of the activated DDR2 could be a new therapeutic strategy for atherosclerosis.
Atherosclerosis* ; Blood Vessels ; Collagen ; Cysteine ; HEK293 Cells ; Myocytes, Smooth Muscle ; Oxidative Stress* ; Oxygen ; Phosphorylation ; Phosphotransferases ; Protein-Tyrosine Kinases ; Reactive Oxygen Species ; Sf9 Cells ; Tyrosine