Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

Objective: To express human Vascular endothelial growth factor121(VEGF121) in insect cells. Methods: A gene construct containing VEGF was cloned in the p Fast Bac-HTA vector, followed by transformation in DH10 BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by Western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. Results: Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. Conclusions: Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

High-Level Production of a Functional Recombinant Hepatitis B Virus Polymerase in Insect Cells with a Baculovirus Expression System

HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain ac- tive polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombi- nant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Re- combinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were in- fected with the recombinant virus containing HBV polymerse gene to express the target protein. HBV polymerse expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system

Objective：CYP1A2 and NADPH-CYP450 oxidoreductase（POR）were expressed in the baculovirus/ Spodoptera frugiperda（sf9）system.The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR.Methods：The heme precursors[δ-Aminolaevulinic Acid（5-ALA）,Fe^3＋and hemin]were introduced into the system to evaluate their effects on the expression of CYP1A2,POR and their co-expression.All the proteins were identified using immunoblotting,CO-difference spectroscopy,or cytochrome c assay.Results：In the present study,functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system,and both of them showed high activities.Co-addition of 5-ALA and Fe^3＋significantly improved expression of CYP1A2 by about 50%compared with the addition of 5-ALA,Fe^3＋or hemin alone.Either co-addition of 5-ALA and Fe^3＋or addition of 5-ALA or Fe^3＋alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control（no addition）.However,unlike CYP1A2,there was no difference between the co-addition and addition of these heme precursors alone.Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR,with a 3：1 ratio of BvCYP1A2/BvPOR significantly increasing their co-expression.Surprisingly,the addition of 0.1 mM 5-ALA or Fe^3＋alone,but not their co- addition,could significantly improve the CYP1A2 and POR co-expression（P〈0.05）.Conclusion：5-ALA and Fe^3＋increased the expression of CYP1A2 and POR in a baculovirus/sf9 system,but the pattern of their expression was different between their expression alone and co-expression.

Expression of p30, the major surface antigen of Toxoplasma gondii, in baculovirus-insect cell system and the evaluation of immune response induced by p30

Objective: To produce the major surface antigen (p30) of Toxoplasma gondii from the Baculovirus Expression System. Methods: The p30 coding sequence was cloned into a transfer vector, then the recombinant baculovirus containing p30 gene was cloned and purified by the co-transfection and plaque assay. The expression and immunoactivity of the recombinant p30 were analyzed by SDS-PAGE and Western blot. The immune responses in mice for being immunized with recombinant p30 were tested. Results: About 750μg of purified (95% purity) p30 was obtained from a culture of 108 in- sect Sf21 cells. Mice in injected with the recombinant protein produced specific humoral and cellular immune responses. Immunization with p30 also prolonged the period of mice survival infected by Toxoplasma gondii. Conclusion: It is indicated that the recombinant p30 from baculovirus expression system can stimulate mice to produce effective protection from Toxo- plasma gondii infection.

Sequence and Molecular Evolution Analysis of Ubiquitin Proteins Encoded by Baculoviruses

[Objective] The aim of this study was to analyze the sequence characteristics and molecular evolution of ubiquitins encoded by baculoviruses.[Methods]Clustal W software was used for multiple sequence alignment analysis,and neighbor-joining method（NJ）and maximum parsimony method（MP）were used for the construction of phylogenetic tree.[Results]The baculoviral ubiquitins showed 73%-86% sequence identity to eukaryotic ubiquitin.Two heterogeneous regions of baculoviral ubiquitins were observed：one was the residues from 15-32,the other was located from residues 53 to 60.The else parts were conserved,where many functional amino acids were also observed.Phylogenetic analysis indicated that baculoviral ubiquitins could be divided into three sub-families,including sub-family GV,sub-family I and sub-family II.The molecular evolution of baculoviral ubiquitins might be under negative selection to maintain their functional and structural stability.[Conclusion]The analysis had provided reference for the researches on functional characterization of baculoviral ubiquitins.

Detection of prawn white spot baculovirusby polymerase chain reaction

INTRODUCTIONAprawnbaculovirushasbeenresponsibleformostoftheseriousshrimpdiseaseinChinasince1992.Studiesonthepathology,Pathogenesisandmorphologyofthevirusshowedthatitwasanon-occlUSiontheybaculoviruswhichcouldinfectPenaeusjaponicus,P.nzonham,P.chinests...

Suppression of gastric cancer growth by baculovirus vector-mediated transfer of normal epithelial cell specific-1 gene

AIM: To study the inhibitory effect of baculovirus- mediated normal epithelial cell specific-1 (NES1) gene therapy on gastric cancer (GC) in vitro and in vivo. METHODS: We first constructed recombinant baculovirus vectors and then transfected them into gastric cancer cells (SGC-7901). Efficiency of the baculovirus for gene transfer into SGC-7901 cells and cell growth curves were detected by fluorescence microscopy, Western blot and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro, respectively. The therapeutic effect of this gene therapy on GC was confirmed in xenografted nude mice. Tumor growth was determined by tumor volume, and expression of NES1 in tumor was analyzed by immunohistochemistry. RESULTS: Baculovirus vectors were successfully transfected into SGC-7901 cells. SGC-7901 cells transfected with the NES1 gene inhibited cell growth. In the Bac-NES1 treated group, tumor growth was significantly reduced with a high level of NES1 expression CONCLUSION: Baculovirus-mediated NES1 gene can be used in gene therapy for GC.

Efficient expression of histidine-tagged large hepatitis delta antigen in baculovirus-transduced baby hamster kidney cells

AIM： To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen （L-HDAg）. METHODS： A recombinant baculovirus expressing histidine-tagged L-HDAg （L-HDAgH） was constructed to transduce baby hamster kidney （BHK） cells by a simplified transduction protocol. RESULTS： The recombinant baculovirus transduced BHK cells with efficiencies higher than 90% as determined by flow cytometry. The expression level was significantly higher than that obtained by plasmid transfection and was further enhanced 3-fold to around 19 pg/cell by the addition of 10 mmol/L sodium butyrate. Importantly, the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated as determined by immunofluorescence labeling and confocal microscopy. Moreover, L-HDAgH interacted with hepatitis B surface antigen to form virus-like particles. CONCLUSION： The fusion with histidine tags as well as overexpression of L-HDAgH in the baculovirus-transduced BHK cells does not impair the biological functions. Taken together, the baculovirus/mammalian cell system offers an attractive alternative for high level expression of L-HDAgH or other proteins that require extensive posttranslational modifications.

Baculovirus vector-mediated transfer of NIS gene into colon tumor cells for radionuclide therapy

AIM:To investigate the feasibility of radionuclide therapy of colon tumor cells by baculovirus vector-mediated transfer of the sodium/iodide symporter(NIS) gene.METHODS:A recombinant baculovirus plasmid carrying the NIS gene was constructed,and the viruses(BacNIS) were prepared using the Bac-to-Bac system.The infection efficiency in the colon cancer cell line SW1116 of a green fluorescent protein(GFP) expressing baculovirus(Bac-GFP) at different multiplicities of infection(MOI) with various concentrations of sodium butyrate was determined by flow cytometry.An in vitro cytotoxicity assay was also conducted after infection of SW1116 cells with Bac-NIS.Iodine uptake of Bac-NIS infected SW1116 cells and inhibition of this uptake by sodium perchlorate was examined,and the effect of Bac-NISmediated 131 I in killing tumor cells was evaluated by cell colony formation tests.RESULTS:Infection and transgene expression in SW1116with Bac-GFP were significantly enhanced by sodium butyrate,as up to 72% of SW1116 cells were infected with the virus at MOI of 400 and sodium butyrate at 0.5 mmol/L.No obvious cytotoxicity was observed under these conditions.Infection of SW1116 with Bac-NIS allowed uptake of 131 I in these tumor cells,which could be inhibited by sodium perchlorate.The viability of SW1116 cells infected with Bac-NIS was significantly lower than with Bac-GFP,suggesting that NIS gene-mediated 131 I uptake could specifically kill tumor cells.CONCLUSION:Baculovirus vector-mediated NIS gene therapy is a potential approach for treatment of colon cancer.

Genetic Modification of Baculovirus Expression Vectors

As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells. These modifications include utilization of different promoters and signal peptides, deletion or replacement of viral genes for increasing protein secretion, integration of polycistronic expression cassette for producing protein complexes, and baculovirus pseudotyping, promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery. This review summarizes the development and the current state of art of the baculovirus expression system. Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications.

Study on purification and ultrastructure of a baculovirus in Penaeus chinensis

A kind of baculovirus was isolated from the cephalothorax homogenate of sick or morbid Penaeus chinensis by differential centrifugation and density gradient centrifugation. Electron microscopic examination of ultrathin section of the gills, stomach and mid-gut tissues also revealed the presence of rod-shaped baculoviral particles with the same size in the affected cell nuclei, where most of the virions arranging in cluster assembled and caused a series of cytopathic changes. The virion covered with bilaminal envelope was 320 ~ 400 nm × 100 ~ 130 nm in size, whereas the nucleocapsid ranged in size of 250~ 300 nm in length and 70 ~ 100 nm in breadth respectively. No nuclear polyhedron or granulin occlusion theies have been found in cells. According to the principle of viral classification, this newly found virus could probably belong to the non-occluded subgroup of insect baculoviridae, i. e., C subgroup baculovirus.

The VP2 protein of grass carp reovirus(GCRV) expressed in a baculovirus exhibits RNA polymerase activity

The double-shelled grass carp reovirus （GCRV） is capable of endogenous RNA transcription and processing.Genome sequence analysis has revealed that the protein VP2,encoded by gene segment 2 （S2）,is the putative RNA-dependent RNA polymerase （RdRp）.In previous work,we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity （denoted as rVP2390-900） in E.coil and have prepared a polyclonal antibody against VP2.To characterize the GCRV RNA polymerase,a recombinant full-length VP2 （rVP2） was first constructed and expressed in a baculovirus system,as a fusion protein with an attached His-tag.Immunofluorescence （IF） assays,together with immunoblot （IB） analyses from both expressed cell extracts and purified Histagged rVP2,showed that rVP2 was successfully expressed in Sf9 cells.Further characterization of the replicase activity showed that purified rVP2 and GCRV particles exhibited poly（C）-dependent poly（G） polymerase activity.The RNA enzymatic activity required the divalent cation Mg2＋,and was optimal at 28 ℃.The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.

Baculovirus-expressed FAdV-4 penton base protein protects chicken against hepatitis-hydropericardium syndrome

Hepatitis-hydropericardium syndrome(HHS)is an infectious disease caused by fowl adenovirus serotype 4(FAdV-4).Several structural and non-structural proteins of FAdV-4 have been expressed in Escherichia coli and baculovirus expression system to develop candidate subunit vaccines.However,the protective efficiency of baculovirus-expressed penton base protein has not been assessed.In this study,two recombinant capsid proteins,penton base and fiber-2,were constructed.And then,penton base and fiber-2 were administrated alone or together to specific pathogen-free(SPF)chickens at 14 days of life and boosted at 28 days of life.At 42 days of life,the immunized groups and the control group were challenged with FAdV-4 virulent strain.Results show that inoculating penton base or penton base+fiber-2 provided 100%protection to the chickens.All groups vaccinated with the recombinant protein produced detectable antibodies and showed no apparent lesions.Thus,baculovirus-expressed penton base protein is a promising candidate subunit vaccine.

Interstitial tissue-specific gene expression in mouse testis by intra-tunica albuguineal injection of recombinant baculovirus

The purpose of this study is to establish a gene delivery system for interstitial tissue-specific protein expression in mice testes using modified recombinant baculovirus. Green fluorescent protein （GFP）-expressing recombinant baculovirus （GFP-baculovirus）, in which the insect cell-specific polyhedron promoter was replaced by the cytomegalovirus （CMV）-IE promoter, was used to transfect testicular cells in vitro, and for intra-tunica albuguineal injection of the interstitial tissue of the testis. GFP expression was monitored in frozen testes sections by fluorescence microscopy. Expression of GFP in testicular tissues was also assessed by reverse transcription polymerase chain reaction （RT-PCR）, and protein expression was assessed by Western blot. Testicular cells in vitro were infected efficiently by modified recombinant GFP-baculovirus. lntra-tunica albuguineal injection of GFP- baculovirus into the mouse testis resulted in a high level of GFP expression in the interstitial tissues. RT-PCR analysis clearly showed GFP gene expression in the testis, particularly interstitial tissues. Intra-tunica albuguineal injection of a modified baculovirus that encoded recombinant rat insulin-like growth factor binding protein （IGFBP）-5 resulted in an increase in IGFBP-5 in testis and semen. In conclusion, we have developed an efficient delivery system for gene expression in vivo in testicular cells, particularly cells of the interstitial tissue using intratunica albuguineal injection of a modified recombinant baculovirus. This method will be particularly relevant for application that requires gene delivery and protein expression in the testicular cells of the outer seminiferous tubule of the testis.

Transgene expression and differentiation of baculovirus-transduced adipose-derived stem cells from dystrophin-utrophin double knock-out mouse

In this study, recombinant baculovirus carrying the microdystrophin and β-catenin genes was used to infect adipose-derived stem cells from a dystrophin-utrophin double knock-out mouse. Results showed that, after baculovirus transgene infection, microdystrophin and β-catenin genes were effectively expressed in adipose-derived stem cells from the dystrophin-utrophin double knock-out mouse. Furthermore, this transgenic expression promoted adipose-derived stem cell differentiation into muscle cells, but inhibited adipogenic differentiation. In addition, protein expression related to the microdystrophin and Wnt/β-catenin signaling pathway was upregulated. Our experimental findings indicate that baculovirus can successfully deliver the microdystrophin and β-catenin genes into adipose-derived stem cells, and the microdystrophin and Wnt/β-catenin signaling pathway plays an important role in myogenesis of adipose-derived stem cells in the dystrophin-utrophin double knock-out mouse.

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro

Baculoviruses produce two viral phenotypes, the budded virus （BV） and the occlusion-derived virus （ODV）. ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-l, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.

The Parasitoid Factor in the Virulence and Spread of Lepidopteran Baculoviruses

Insect parasitoids and baculoviruses play important roles in the natural and strategic biological control of insects. The two parasites are frequent competitors within common hosts and much research has focused on the negative impact that baculoviral host infections have on parasitoids. This review summarizes the impacts that parasitoids may have on the virulence and spread of lepidopteran baculoviruses. By changing host behavior and development, parasitoids have been shown to decrease baculovirus virulence and productivity within parasitized baculovirus-susceptible hosts; however, studies of the tools used by hymenopteran parasitoids to overcome their hosts'immune systems, suggest that parasitoids may, in some cases, facilitate baculoviral infections in less susceptible hosts. Laboratory and field research have demonstrated that parasitoids can mechanically transmit baculoviruses between insects, and in this way, increase the efficacy of the viruses. Instances of new, more virulent isolates of baculoviruses have been recorded from specifically parasitoid-targeted hosts suggesting other possible benefits from the transmission or activation of baculoviruses by parasitoids.

Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy in Lung Adenocarcinoma

Objective: To investigate the feasibility of temporally and spatially restricted Kringle5 expression induced by radiation, as well as the dual effect of radiotherapy and antiangiogenic therapy in lung adenocarcinoma in vitro. Methods: We first constructed recombinant baculovirus vectors containing Egr1 promoter and human plasminogen Kringle5 gene (rhK5), then transfected them into lung adenocarcinoma cells (A549). Transfect efficiency of the baculovirus for gene transfer in A549 cells and the activity of Egr1 promoter induced by X-radiation were detected by fluorescence microscopy. The rhK5 mRNA transcription and rhK5 protein expression were detected by Real-time PCR and Western blot assay, respectively. The apoptosis asssay of human umbilical veins endothelial cells (HUVEC) was analyzed by flow cytometry. Results: The recombinant baculovirus were successfully transfected into A549 and HUVEC cells. As for the temporal regulation, the rhK5 mRNA transcription and rhK5 protein expression were elevated with the irradiation time significantly. And the HUVEC apoptotic percentage increased in relation to the irradiation time as well. As for the spatial regulation, rhK5 mRNA transcription level of A549 cell lines transfected with recombinant baculovirus Egr1-K5 was significantly higher than that of control groups after the same dose of X-radiation. When we analyzed the dose and frequency of X-radiation, no difference was observed among each dose after continuously three-times of irradiation. Conclusion: Baculovirus-mediated Egr1-K5 can be used in gene radiotherapy for its temporary and spatial controllable rhK5 expression by X-radiation and the consequent HUVEC apoptosis in vitro study. And low dose and more times of irradiation might be more effective. It would provide a promising way for the tumor treatment.

Promising Additives to Protect the Activity of Baculovirus Biocontrol Agent under Field-Sunlight Conditions in Egypt

Baculoviruses are effective biocontrol agents except of their short persistence under sunlight conditions. Four promising additives containing different groups of antioxidants were tested on cotton plant foliage. Spodoptera littoralis test insect and its nuclepolyhedrovirus （SpliNPV） are the standard material used in the investigation. Results are based on leaf-bioassays, to test Original Activity Remaining （OAR） and Lethal Infectivity Time to 50% （LIT50）of tested population of the virus after exposure to natural sunlight. The results showed that cacao additive at 10%, sustained 50% of virus activity for five days post application （113.11 hours） and three days and a half （83.33 hours） at 5% concentration. The virus alone treatment sustained 50% of its activity for only 24.07 hours. The obtained results suggested the possibility of prolonging the virus activity on plant foliage under field applications.

AcMNPV As A Model for Baculovirus DNA Replication

Baculoviruses were first identified as insect-specific pathogens, and it was this specificity that lead to their use as safe, target specific biological pesticides. For the past 30 years, AcMNPV has served as the subject of intense basic molecular research into the baculovirus infectious cycle including the interaction of the virus with a continuous insect cell line derived from Spodoptera frugiperda. The studies on baculoviruese have led to an in-depth understanding of the physical organization of the viral genomes including many complete genomic sequences, the time course of gene expression, and the application of this basic research to the use of baculoviruses not only as insecticides, but also as a universal eukaryotic protein expression system, and a potential vector in gene therapy. A great deal has also been discovered about the viral genes required for the replication of the baculovirus genome, while much remains to be learned about the mechanism of viral DNA replication. This report outlines the current knowledge of the factors involved in baculovirus DNA replication, using data on AcMNPV as a model for most members of the Baculoviridae.