Infectious bursal disease(IBD)causes considerable economic losses in the commercial poultry industry worldwide.The principal way to control IBD virus(IBDV),the causative agent of IBD,is still through vaccination progr...Infectious bursal disease(IBD)causes considerable economic losses in the commercial poultry industry worldwide.The principal way to control IBD virus(IBDV),the causative agent of IBD,is still through vaccination programs.Virus-like particles(VLPs)are recognised as a safe and potent recombinant vaccine platform.This research work explores the characterisation and separation of infectious bursal disease virus-like particles(IBD-VLPs)from crude feedstock.Various characteristics were studied with highperformance size-exclusion chromatography(HP-SEC),sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS-PAGE)and transmission electron microscopy(TEM)analyses.Subsequently,the separation of IBD-VLPs using polyethylene glycol(PEG)/sodium citrate-based aqueous two-phase systems(ATPSs)was conducted and optimised.Moreover,a scale-up study of the best ATPS constituted of 15%PEG 6000,11%sodium citrate and 10%crude feedstock was performed to compare the separation performance of IBD-VLPs with and without centrifugation-assisted.The results indicated that the optimised ATPS with centrifugation-assisted for both 5 g and 50 g systems showed good recovery of IBDVLPs of>97%in the interphase between the PEG-rich top and salt-rich bottom phases.These optimised systems also showed high removal efficiencies of impurities of>95%.The results demonstrated that aqueous two-phase extraction could be a promising technology for efficient VLPs separation.展开更多
Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe ge...Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichiapastorisunder the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualiza-tion. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoriswith an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immuno-genicity on surface-displayed HEnAg. Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.展开更多
Many flaviviruses are emerging and reemerging pathogens,such as West Nile virus(WNV) ,dengue virus(DENV) ,yellow fever virus(YFV) ,and Japanese encephalitis virus. Serological assay is the dominant method for diagnosi...Many flaviviruses are emerging and reemerging pathogens,such as West Nile virus(WNV) ,dengue virus(DENV) ,yellow fever virus(YFV) ,and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members,plaque reduction neutralization test(PRNT) is the only available assay to determine the infecting flavivirus type. Since PRNT requires culturing raw viruses,it must be performed in biosafety level-3 or level-4 containment for many flaviviruses,and takes more than ten days to complete. To overcome these problems,we have developed flavivirus viral-like particles(VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps:(i) VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h;(ii) the neutralized VLPs are used to infect Vero cells;and(iii) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen(as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept,we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the "gold standard" of the classic PRNT;importantly,it shortens the assay time from >10 days to <1 day,and can be performed in biosafety level-2 facility.展开更多
Filoviruses are hemorrhagic fever viruses endemic to parts of Africa and the Philippines. Infection carries with it a mortality rate of up to 90% and currently there are no effective vaccines or therapeutics available...Filoviruses are hemorrhagic fever viruses endemic to parts of Africa and the Philippines. Infection carries with it a mortality rate of up to 90% and currently there are no effective vaccines or therapeutics available to combat infection. However, the filovirus virus-like particles (VLP), which are currently under development, have been shown to be a promising vaccine candidate. They provide protection from infection in the mouse, guinea pig, and nonhuman primate models of infection, eliciting high anti-glycoprotein antibody titers and T cell responses to viral proteins. In this review, we will highlight the development of the filovirus VLP and describe the current understanding of VLP immunogenicity and correlates of protection.展开更多
The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus's serological diagnostic methods....The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus's serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid (Opt-Cap) gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% (v/v) methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What's more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles (VLPs). These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.展开更多
Summary: To prepare carboxyl terminus truncated human papillomavirus type 58 L1(HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1...Summary: To prepare carboxyl terminus truncated human papillomavirus type 58 L1(HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing-HPV58L1 protein-were harvested and analyzed by SDS-PAGE and Western blot. The ProBond~TM purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.展开更多
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to invest...The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to investigate papillomavirus life cycle which hampered by the unavailability of conventional cell culture system, we constructed a chimeric bovine papillomavirus (BPV) type 1 virus like particles (VLPs) containing GFP. It was found that fluorescent VLPs could be assembled from L2 protein in which GFP is inserted into the N terminal region of L2 (aa 88). The fluorescent VLPs could also be assembled from a GFP/L2 fusion protein in which part of the L2 sequence had been deleted. In vitro , fluorescent VLPs could bind to CV 1 cells, and this VLP/cell interaction could be analyzed by FACS assay. These results demonstrated that GFP could incorporate into BPV1 VLPs without disruption of the VLP structure. Fluorescent VLPs might be a useful tool for study of papillomavirus virus/cell interaction.展开更多
Dengue virus type 2 ThNH7/93 retained infectious activity after purification by ceramic hydroxyapatite chromatogra-phy. Dengue virus type 2 culture fluid was loaded onto the ceramic hydroxyapatite column and eluted wi...Dengue virus type 2 ThNH7/93 retained infectious activity after purification by ceramic hydroxyapatite chromatogra-phy. Dengue virus type 2 culture fluid was loaded onto the ceramic hydroxyapatite column and eluted with a linear gradient of sodium phosphate buffer. Culture fluid and protein contaminants derived from host cells were eluted initially, followed by elutions of dsDNA, and then dengue virus type 2. The recoveries of dengue virus type 2 were 64 ± 14% (n = 11) in the hemagglutination (HA) test and 60% (n = 2) determined by focus assay for viral infectivity. This protocol was highly reproducible, simple, rapid, and appears applicable to other virus species such as influenza virus, Japanese encephalitis virus and adenovirus.展开更多
Objective.To test whether intramuscular,intranasal,intrarectal and intravagina l administration of HPV6b L 1 virus-like particlescould induce immune response in mice and to as sess whether intra-muscular and mucosal v...Objective.To test whether intramuscular,intranasal,intrarectal and intravagina l administration of HPV6b L 1 virus-like particlescould induce immune response in mice and to as sess whether intra-muscular and mucosal vaccination against HPV is feasible.Me thods.HPV6b L1proteins self-assembled into VLPs in Sf-9cell in vitro.Mic e were immunized on day0and21with50ìg HPV6b L1VLPs intramuscularly,int ranasally,intrarectally and intravagi-nally respectively.Sera were collected for testing IgG titer after a further7days and3months respec-tively.Results .After immunizations,all mice developed significant anti-HPV6b L1antibody titers in serum by7days after the second immunization.The titer of the serum I gG antibody against HPV6b L1VLPs in the intramuscularly immunized group was h igher than that in the intranasally,intrarectally and intravaginally immunized groups respectively,indicating that both muscular and mucosal administration of HPV6b L1VLPs can stimulate a systemic HPV-specific antibody response.Sera of the mice in the in-tramuscularly immunized group still maintained a high tit er of the serum IgG antibody against HPV6b L1VLPs 3months after the immunizat ion.Conclusion.The results demonstrated that the HPV6b L1VLPs maintain stro ng antigenicity.Immu-nization with HPV6b L1VLPs via intramuscular and mucos al routes,without adjuvant ,can elicit spe-cific antibody in sera.These fin dings suggest that the VLPs are able to induce protective antibodies.展开更多
Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family,which is widespread and causes high fatality.The nucleocapsid of CCHFV is comprised of N prote...Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family,which is widespread and causes high fatality.The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment.In this research,the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus.Under an electron microscope,Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells.Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation,which was confirmed by Western blot analysis and immuno-electron microscopy (IEM).展开更多
Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects,offering a promising insecticidal component for insect pest control.H...Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects,offering a promising insecticidal component for insect pest control.However,effective delivery systems are required to help neurotoxic peptides pass through the gut barrier into the hemolymph,where they can act.Here,we investigated the potential of a novel nanocarrier,Drosophila X virus-like particle(DXV-VLP),for delivering a neurotoxin from the scorpion Androctonus australis Hector(AaIT)against the invasive pest fruit fly,Drosophila suzuki.Our results show that the fusion proteins of DXV polyproteins with AalT peptide at their Ctermini could be sufficiently produced in Lepidoptera Hi5 cells in a soluble form using the recombinant baculovirus expression system,and could self-assemble into VLPs with similar particle morphology and size to authentic DXV virions.In addition,the AalT peptides displayed on DXV-VLPs retained their toxicity,as demonstrated in injection bioassays that resulted in severe mortality(72%)in adults after 72 h.When fed to adults,mild mortality was observed in the group treated with DXV-AalT(38%),while no mortality occurred in the group treated with AaIT peptide,thus indicating the significant role of DXV-VLPs in delivering AalT peptides.Overall,this proof-of-concept study demonstrates for the first time that VLPs can be exploited to enhance oral delivery of insect-specific neurotoxic peptides in the context of pest control.Moreover,it provides insights for further improvements and potentially the development of neurotoxin-based bioinsecticides and/or transgenic crops for insect pest control.展开更多
Objective To eliminate the side effects of aluminum adjuvant and His-tag,we constructed chimeric VLPs displaying the epitope of EV71(SP70) without His-tagged.Then evaluating whether the VLPs could efficiently evoke ...Objective To eliminate the side effects of aluminum adjuvant and His-tag,we constructed chimeric VLPs displaying the epitope of EV71(SP70) without His-tagged.Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.Methods The fusion protein was constructed by inserting SP70 into the MIR of truncated HBc Ag sequence,expressed in E.Coli,and purified through ion exchange chromatography and density gradient centrifugation.Mice were immunized with the VLPs and sera were collected afterwards.The specific antibody titers,Ig G subtypes and neutralizing efficacy were detected by ELISA,neutralization assay,and EV71 lethal challenge.IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.Results HBc-SP70 proteins can self-assemble into empty VLPs.After immunization with HBc-SP70 VLPs,the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge.There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not.The specific Ig G subtypes were mainly IgG1 and IgG2 b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.Conclusion The fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation.In the absence of adjuvant,they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant.Furthermore,the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.展开更多
The virus-like particle(VLPs) vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have bee...The virus-like particle(VLPs) vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have been used, one can express the HIV-1 main structure proteins, Gagpol and Env, and the other contains an antibiotic gene. The two kinds of plasmids have been cotransfected into 293 cells. A stable cell line that can express Gagpol and Env proteins efficiently and lastingly has been screened. It has been confirmed that Gagpol and Env proteins in the cell culture supernatant can be self-assembled into virus-like particles. The authors have detected the secretion of VLPs in the cell medium, defined the peak of the secretion, and followed and monitored the stability of expression.展开更多
基金Zhejiang University and TalentIntroduction Program of China for Postdoctoral Researcher for the financial supportfinancially supported by the National Key Research&Development Program of China (2021YFE0113300)the National Natural Science Foundation of China (22078286)。
文摘Infectious bursal disease(IBD)causes considerable economic losses in the commercial poultry industry worldwide.The principal way to control IBD virus(IBDV),the causative agent of IBD,is still through vaccination programs.Virus-like particles(VLPs)are recognised as a safe and potent recombinant vaccine platform.This research work explores the characterisation and separation of infectious bursal disease virus-like particles(IBD-VLPs)from crude feedstock.Various characteristics were studied with highperformance size-exclusion chromatography(HP-SEC),sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS-PAGE)and transmission electron microscopy(TEM)analyses.Subsequently,the separation of IBD-VLPs using polyethylene glycol(PEG)/sodium citrate-based aqueous two-phase systems(ATPSs)was conducted and optimised.Moreover,a scale-up study of the best ATPS constituted of 15%PEG 6000,11%sodium citrate and 10%crude feedstock was performed to compare the separation performance of IBD-VLPs with and without centrifugation-assisted.The results indicated that the optimised ATPS with centrifugation-assisted for both 5 g and 50 g systems showed good recovery of IBDVLPs of>97%in the interphase between the PEG-rich top and salt-rich bottom phases.These optimised systems also showed high removal efficiencies of impurities of>95%.The results demonstrated that aqueous two-phase extraction could be a promising technology for efficient VLPs separation.
文摘Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichiapastorisunder the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualiza-tion. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoriswith an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immuno-genicity on surface-displayed HEnAg. Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.
基金supported by National Institute of Health grants U01 AI061193 and U54-AI057158 (Northeast Biodefense Center).
文摘Many flaviviruses are emerging and reemerging pathogens,such as West Nile virus(WNV) ,dengue virus(DENV) ,yellow fever virus(YFV) ,and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members,plaque reduction neutralization test(PRNT) is the only available assay to determine the infecting flavivirus type. Since PRNT requires culturing raw viruses,it must be performed in biosafety level-3 or level-4 containment for many flaviviruses,and takes more than ten days to complete. To overcome these problems,we have developed flavivirus viral-like particles(VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps:(i) VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h;(ii) the neutralized VLPs are used to infect Vero cells;and(iii) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen(as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept,we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the "gold standard" of the classic PRNT;importantly,it shortens the assay time from >10 days to <1 day,and can be performed in biosafety level-2 facility.
基金funded by the Chemical-Biological Medical System-Joint Vaccine Acquisition Program (CBMS-JVAP) as well as the Defense Threat Reduction Agency (DTRA) (CBM.VAXV.03.11.RD.009 and A151 A.41)
文摘Filoviruses are hemorrhagic fever viruses endemic to parts of Africa and the Philippines. Infection carries with it a mortality rate of up to 90% and currently there are no effective vaccines or therapeutics available to combat infection. However, the filovirus virus-like particles (VLP), which are currently under development, have been shown to be a promising vaccine candidate. They provide protection from infection in the mouse, guinea pig, and nonhuman primate models of infection, eliciting high anti-glycoprotein antibody titers and T cell responses to viral proteins. In this review, we will highlight the development of the filovirus VLP and describe the current understanding of VLP immunogenicity and correlates of protection.
基金supported by the National Science and Technology Support Program(2015BAD12B05)the China Agricultural Research System(CARS-43-8)+2 种基金the Integration and Demonstration of Key Technologies for Duck Industrial in Sichuan Province,China(2014NZ0030)the Ministry of Education Program of China(20125103110013)the Sichuan Province Research Programs,China(2013HH0042/2013 TD0015/2014-002)
文摘The capsid (Cap) protein, which is the only structural protein of duck circovirus (DuCV), is the most important antigen for the development of vaccines against DuCV and the virus's serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid (Opt-Cap) gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% (v/v) methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What's more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles (VLPs). These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.
文摘Summary: To prepare carboxyl terminus truncated human papillomavirus type 58 L1(HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing-HPV58L1 protein-were harvested and analyzed by SDS-PAGE and Western blot. The ProBond~TM purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.
文摘The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to investigate papillomavirus life cycle which hampered by the unavailability of conventional cell culture system, we constructed a chimeric bovine papillomavirus (BPV) type 1 virus like particles (VLPs) containing GFP. It was found that fluorescent VLPs could be assembled from L2 protein in which GFP is inserted into the N terminal region of L2 (aa 88). The fluorescent VLPs could also be assembled from a GFP/L2 fusion protein in which part of the L2 sequence had been deleted. In vitro , fluorescent VLPs could bind to CV 1 cells, and this VLP/cell interaction could be analyzed by FACS assay. These results demonstrated that GFP could incorporate into BPV1 VLPs without disruption of the VLP structure. Fluorescent VLPs might be a useful tool for study of papillomavirus virus/cell interaction.
文摘Dengue virus type 2 ThNH7/93 retained infectious activity after purification by ceramic hydroxyapatite chromatogra-phy. Dengue virus type 2 culture fluid was loaded onto the ceramic hydroxyapatite column and eluted with a linear gradient of sodium phosphate buffer. Culture fluid and protein contaminants derived from host cells were eluted initially, followed by elutions of dsDNA, and then dengue virus type 2. The recoveries of dengue virus type 2 were 64 ± 14% (n = 11) in the hemagglutination (HA) test and 60% (n = 2) determined by focus assay for viral infectivity. This protocol was highly reproducible, simple, rapid, and appears applicable to other virus species such as influenza virus, Japanese encephalitis virus and adenovirus.
文摘Objective.To test whether intramuscular,intranasal,intrarectal and intravagina l administration of HPV6b L 1 virus-like particlescould induce immune response in mice and to as sess whether intra-muscular and mucosal vaccination against HPV is feasible.Me thods.HPV6b L1proteins self-assembled into VLPs in Sf-9cell in vitro.Mic e were immunized on day0and21with50ìg HPV6b L1VLPs intramuscularly,int ranasally,intrarectally and intravagi-nally respectively.Sera were collected for testing IgG titer after a further7days and3months respec-tively.Results .After immunizations,all mice developed significant anti-HPV6b L1antibody titers in serum by7days after the second immunization.The titer of the serum I gG antibody against HPV6b L1VLPs in the intramuscularly immunized group was h igher than that in the intranasally,intrarectally and intravaginally immunized groups respectively,indicating that both muscular and mucosal administration of HPV6b L1VLPs can stimulate a systemic HPV-specific antibody response.Sera of the mice in the in-tramuscularly immunized group still maintained a high tit er of the serum IgG antibody against HPV6b L1VLPs 3months after the immunizat ion.Conclusion.The results demonstrated that the HPV6b L1VLPs maintain stro ng antigenicity.Immu-nization with HPV6b L1VLPs via intramuscular and mucos al routes,without adjuvant ,can elicit spe-cific antibody in sera.These fin dings suggest that the VLPs are able to induce protective antibodies.
基金The Knowledge Innovation Program of the Chinese Academy of Sciences,Grant No.KSCX2-YW-N-065the Knowledge Innovation Program of the Chinese Academy of Sciences,Grant No.KSCX2-EW-G-8+1 种基金National Key Basic Research Program(973Program),Grant No.2010CB530103National Basic Research Priorities Program of China,Grant No.2007FY210700
文摘Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family,which is widespread and causes high fatality.The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment.In this research,the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus.Under an electron microscope,Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells.Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation,which was confirmed by Western blot analysis and immuno-electron microscopy (IEM).
基金supported by the Special Research Fund of Ghent University and Research Foundation Flanders(FWO).CNTT is recipient of a senior postdoctoral fellowship from FWO(grant number 12V5722N).
文摘Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects,offering a promising insecticidal component for insect pest control.However,effective delivery systems are required to help neurotoxic peptides pass through the gut barrier into the hemolymph,where they can act.Here,we investigated the potential of a novel nanocarrier,Drosophila X virus-like particle(DXV-VLP),for delivering a neurotoxin from the scorpion Androctonus australis Hector(AaIT)against the invasive pest fruit fly,Drosophila suzuki.Our results show that the fusion proteins of DXV polyproteins with AalT peptide at their Ctermini could be sufficiently produced in Lepidoptera Hi5 cells in a soluble form using the recombinant baculovirus expression system,and could self-assemble into VLPs with similar particle morphology and size to authentic DXV virions.In addition,the AalT peptides displayed on DXV-VLPs retained their toxicity,as demonstrated in injection bioassays that resulted in severe mortality(72%)in adults after 72 h.When fed to adults,mild mortality was observed in the group treated with DXV-AalT(38%),while no mortality occurred in the group treated with AaIT peptide,thus indicating the significant role of DXV-VLPs in delivering AalT peptides.Overall,this proof-of-concept study demonstrates for the first time that VLPs can be exploited to enhance oral delivery of insect-specific neurotoxic peptides in the context of pest control.Moreover,it provides insights for further improvements and potentially the development of neurotoxin-based bioinsecticides and/or transgenic crops for insect pest control.
基金supported by the National Science-technology Support Plan Projects 'The development of EV71 genetic engineering vaccine'[2008BAI69B02]
文摘Objective To eliminate the side effects of aluminum adjuvant and His-tag,we constructed chimeric VLPs displaying the epitope of EV71(SP70) without His-tagged.Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.Methods The fusion protein was constructed by inserting SP70 into the MIR of truncated HBc Ag sequence,expressed in E.Coli,and purified through ion exchange chromatography and density gradient centrifugation.Mice were immunized with the VLPs and sera were collected afterwards.The specific antibody titers,Ig G subtypes and neutralizing efficacy were detected by ELISA,neutralization assay,and EV71 lethal challenge.IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.Results HBc-SP70 proteins can self-assemble into empty VLPs.After immunization with HBc-SP70 VLPs,the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge.There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not.The specific Ig G subtypes were mainly IgG1 and IgG2 b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.Conclusion The fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation.In the absence of adjuvant,they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant.Furthermore,the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.
基金the National Natural Science Foundation of China(No.30371317).
文摘The virus-like particle(VLPs) vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have been used, one can express the HIV-1 main structure proteins, Gagpol and Env, and the other contains an antibiotic gene. The two kinds of plasmids have been cotransfected into 293 cells. A stable cell line that can express Gagpol and Env proteins efficiently and lastingly has been screened. It has been confirmed that Gagpol and Env proteins in the cell culture supernatant can be self-assembled into virus-like particles. The authors have detected the secretion of VLPs in the cell medium, defined the peak of the secretion, and followed and monitored the stability of expression.