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. Th...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.展开更多
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 fu...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.展开更多
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 c...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.展开更多
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 N...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.展开更多
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 baculo...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.展开更多
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 i...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.展开更多
We trans fected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 geneand neomycin-reslstant gene (as a seIection marker). By G418 screening, we got transformed cells that ap-peared resistant ...We trans fected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 geneand neomycin-reslstant gene (as a seIection marker). By G418 screening, we got transformed cells that ap-peared resistant to G418 and picked one clone named Sf9-35. By hybridization in situ, it was found that p35gene had integrated into the chromosome of Sf9-35 cells; By using actinomycin D treatment and cellular DNAelectrophoresls, Sf9-35 cells were found to reslst apoptosis lnduced by infection of vAcAp35 deleting p35 geneand actinomycin D treatment; And it was aIso found that apoptosis induced by viral infection and actinomycinD treatment can only be deIayed, hut can not be stopped in Sf9-35-展开更多
Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene intothe Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect tothe polyhedrin pro...Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene intothe Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect tothe polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 elementfrom the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda(Sf9). cells to get the recombinant virus. Fresh insect Sf9 cells were infected with therecombinant virus containing p24 to express the target protein. The target protein expressed was analyzed ona 15% polghcrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positiveresult shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISAand other reliable diagnostic methods or HIV-1 Infection.展开更多
In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) ...In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) gene, and green florescence protein (GFP) gene. Baculovirus gp64 TM and gp64 CTD in the pBacSC vector were designed to display heterologous proteins on the baculovirus envelope. After cloning the VP2 gene of CPV-2 into pBacSC vector, the recombinant plasmid pBacSC-VP2 was transformed into E. coli DH10Bac competent cells to form recombinant bacmid DNA. One recombinant baculovirus BacSC-VP2 that expresses the VP2 protein of CPV-2 was obtained. Confocal microscopy and immunogold electron microscopy were used to verify whether VP2 expressing on baculovirus envelope or cell membrane. Immunization of BALB/c mice with recombinant baculovirus BacSC-VP2, demonstrated that serum from the BacSC-VP2 treated models had higher levels of virus neutralization titers than the control groups. The results show that the recombinant baculovirus BacSC-VP2 can induce a strong immune response in a mouse model, suggesting that the pseudotyped baculovirus BacSC-VP2 can serve as a potential vaccine against CPV infections.展开更多
The aim of this study was to investigate the molecular identification and assess the genetic relationship of baculovirus isolated from Southern Vietnam. The diseased insect samples were collected from the different fi...The aim of this study was to investigate the molecular identification and assess the genetic relationship of baculovirus isolated from Southern Vietnam. The diseased insect samples were collected from the different fields. The partial sequence of 450 base pairs of lef-8 gene was amplified and sequenced to assess the genetic variations of baculovirus isolates specific for Spodoptera litura, Helicoverpa zea, and Helicoverpa armigera. The sequences alignment demonstrated that Helicoverpa zea specific isolates exhibited six single nucleotide polymorphic sites. Whereas, twenty five single polymorphic sites were found in Spodoptera litura specific isolates. Thus, Spodoptera litura specific isolates were higher polymorphic than Helicoverpa zea specific isolates. The genetic distance analyses showed that the distance between Vietnamese baculovirus isolates and Group II Alphabaculovirus isolates was lower than other Baculovirus groups. The phylogeny of Vietnamese isolates in relation to other baculovirus isolates was also determined using partial sequences of lef-8 gene. The phylogenetic tree placed all Vietnamese isolates in Group II Alphabaculovirus, where seven Vietnamese Helicoverpa zea specific isolates were most closely related to Helicoverpa zea SNPV, fourteen Vietnamese Spodoptera litura specific isolates were located with Spodoptera litura NPV-G2 in one clade and a Vietnamese Helicoverpa armigera isolate was appeared to be closely related to Helicoverpa armigera SNPV-NNg1, Helicoverpa armigera NPV-C1, Helicoverpa armigera NPV-G4.展开更多
Baculoviruses infect insects in nature by targeting their larvae.They can also pass on genes into mammalian cells(including human cells)without producing progeny viruses via a process termed transduction.For that,they...Baculoviruses infect insects in nature by targeting their larvae.They can also pass on genes into mammalian cells(including human cells)without producing progeny viruses via a process termed transduction.For that,they are promising vectors for human gene therapy.There is,however,an unsolved obstacle that the mammalian transduction is very inefficient.To address that,scientists from the CAS Wuhan Institute of Virology(WHIOV)sought to figure out the major roadblock to mammalian cell entry of baculovirus and find out a solution to overcome it.展开更多
In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper a...In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome,and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive(ts) plasmid, p SIM6. Using the resulting ts helper plasmid p MON7124 ts and the ts donor plasmid p FB1ts-PH-GFP, a recombinant bacmid,b Ac WT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For b Ac WT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.展开更多
基金supported financially by Iran National Science Foundation(INSF)grant number 91004026
文摘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.
基金a grant from the National Natu-ral Sciences Foundation of China (No. 30330680)
文摘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.
基金National Natural Science Foundation of China No39400115 and Natural Science Foundation of Guang Dong No940292
文摘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.
基金Supported by Grants from the National Natural Science Foundation of China,No.30570525the Shanghai Leading Academic Discipline Project,No.S30203
文摘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.
基金supported by the National Key Research and Development Program of China (2016YFD0500801)
文摘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.
文摘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.
文摘We trans fected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 geneand neomycin-reslstant gene (as a seIection marker). By G418 screening, we got transformed cells that ap-peared resistant to G418 and picked one clone named Sf9-35. By hybridization in situ, it was found that p35gene had integrated into the chromosome of Sf9-35 cells; By using actinomycin D treatment and cellular DNAelectrophoresls, Sf9-35 cells were found to reslst apoptosis lnduced by infection of vAcAp35 deleting p35 geneand actinomycin D treatment; And it was aIso found that apoptosis induced by viral infection and actinomycinD treatment can only be deIayed, hut can not be stopped in Sf9-35-
文摘Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene intothe Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect tothe polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 elementfrom the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda(Sf9). cells to get the recombinant virus. Fresh insect Sf9 cells were infected with therecombinant virus containing p24 to express the target protein. The target protein expressed was analyzed ona 15% polghcrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positiveresult shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISAand other reliable diagnostic methods or HIV-1 Infection.
文摘In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) gene, and green florescence protein (GFP) gene. Baculovirus gp64 TM and gp64 CTD in the pBacSC vector were designed to display heterologous proteins on the baculovirus envelope. After cloning the VP2 gene of CPV-2 into pBacSC vector, the recombinant plasmid pBacSC-VP2 was transformed into E. coli DH10Bac competent cells to form recombinant bacmid DNA. One recombinant baculovirus BacSC-VP2 that expresses the VP2 protein of CPV-2 was obtained. Confocal microscopy and immunogold electron microscopy were used to verify whether VP2 expressing on baculovirus envelope or cell membrane. Immunization of BALB/c mice with recombinant baculovirus BacSC-VP2, demonstrated that serum from the BacSC-VP2 treated models had higher levels of virus neutralization titers than the control groups. The results show that the recombinant baculovirus BacSC-VP2 can induce a strong immune response in a mouse model, suggesting that the pseudotyped baculovirus BacSC-VP2 can serve as a potential vaccine against CPV infections.
文摘The aim of this study was to investigate the molecular identification and assess the genetic relationship of baculovirus isolated from Southern Vietnam. The diseased insect samples were collected from the different fields. The partial sequence of 450 base pairs of lef-8 gene was amplified and sequenced to assess the genetic variations of baculovirus isolates specific for Spodoptera litura, Helicoverpa zea, and Helicoverpa armigera. The sequences alignment demonstrated that Helicoverpa zea specific isolates exhibited six single nucleotide polymorphic sites. Whereas, twenty five single polymorphic sites were found in Spodoptera litura specific isolates. Thus, Spodoptera litura specific isolates were higher polymorphic than Helicoverpa zea specific isolates. The genetic distance analyses showed that the distance between Vietnamese baculovirus isolates and Group II Alphabaculovirus isolates was lower than other Baculovirus groups. The phylogeny of Vietnamese isolates in relation to other baculovirus isolates was also determined using partial sequences of lef-8 gene. The phylogenetic tree placed all Vietnamese isolates in Group II Alphabaculovirus, where seven Vietnamese Helicoverpa zea specific isolates were most closely related to Helicoverpa zea SNPV, fourteen Vietnamese Spodoptera litura specific isolates were located with Spodoptera litura NPV-G2 in one clade and a Vietnamese Helicoverpa armigera isolate was appeared to be closely related to Helicoverpa armigera SNPV-NNg1, Helicoverpa armigera NPV-C1, Helicoverpa armigera NPV-G4.
文摘Baculoviruses infect insects in nature by targeting their larvae.They can also pass on genes into mammalian cells(including human cells)without producing progeny viruses via a process termed transduction.For that,they are promising vectors for human gene therapy.There is,however,an unsolved obstacle that the mammalian transduction is very inefficient.To address that,scientists from the CAS Wuhan Institute of Virology(WHIOV)sought to figure out the major roadblock to mammalian cell entry of baculovirus and find out a solution to overcome it.
基金supported by the National Nature Science Foundation of China (31370188)Shenzhen Municipal Development and Reform Commission (Shenzhen Research and Development Center,Code:[2012]318)
文摘In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome,and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive(ts) plasmid, p SIM6. Using the resulting ts helper plasmid p MON7124 ts and the ts donor plasmid p FB1ts-PH-GFP, a recombinant bacmid,b Ac WT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For b Ac WT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.
