In this study, a recombinant Pichia pastoris expression system was developed to express HPV16 L1 protein that was driven by a strong AOX1 promoter. HPV16L1 gene was cloned into vector pPICZ,αB. HPV16 L1 protein expre...In this study, a recombinant Pichia pastoris expression system was developed to express HPV16 L1 protein that was driven by a strong AOX1 promoter. HPV16L1 gene was cloned into vector pPICZ,αB. HPV16 L1 protein expression induced by methanol was screened by using sodium dedecyl sulfate-polyacrylamide gel electrophoresis （SDSPAGE） and Western blotting. The results indicate that the HPVl6 L1 protein is secreted by the recombinant P. pastoris, and the purified HPV16 L1 protein can self-assemble into vires-like particles（ VLPs）, which show a good immunogenicity and induces high-titer antibody in mice.展开更多
This study bioinformatically analyzed the non-VP1 capsid proteins（VP2-VP4） of Coxasckievirus A6（CVA6）, with an attempt to predict their basic physicochemical properties, structural/functional features and linear B...This study bioinformatically analyzed the non-VP1 capsid proteins（VP2-VP4） of Coxasckievirus A6（CVA6）, with an attempt to predict their basic physicochemical properties, structural/functional features and linear B cell eiptopes. The online tools Sub Loc, Target P and the others from Ex PASy Bioinformatics Resource Portal, and SWISS-MODEL（an online protein structure modeling server）, were utilized to analyze the amino acid（AA） sequences of VP2-VP4 proteins of CVA6. Our results showed that the VP proteins of CVA6 were all of hydrophilic nature, contained phosphorylation and glycosylation sites and harbored no signal peptide sequences and acetylation sites. Except VP3, the other proteins did not have transmembrane helix structure and nuclear localization signal sequences. Random coils were the major conformation of the secondary structure of the capsid proteins. Analysis of the linear B cell epitopes by employing Bepipred showed that the average antigenic indices（AI） of individual VP proteins were all greater than 0 and the average AI of VP4 was substantially higher than that of VP2 and VP3. The VP proteins all contained a number of potential B cell epitopes and some eiptopes were located at the internal side of the viral capsid or were buried. We successfully predicted the fundamental physicochemical properties, structural/functional features and the linear B cell eiptopes and found that different VP proteins share some common features and each has its unique attributes. These findings will help us understand the pathogenicity of CVA6 and develop related vaccines and immunodiagnostic reagents.展开更多
Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus（HEV）. HEV is a nonenveloped virus that bas been classified in the family of Caliciviridae. The virus appears to be a...Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus（HEV）. HEV is a nonenveloped virus that bas been classified in the family of Caliciviridae. The virus appears to be a polya-denylated, positive-stranded RNA virus with three major open reading frames（ORFs）. The capsid protein of HEV is encoded by the open reading frame 2（ORF2）. We attempted to produce a truncated capsid protein, designed p293, in Pichia pastoris. The p293 gene encoding amino acids（aa） 382-674 of HEV ORF2 was designed based on the full length of HEV ORF2, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS 115. SDS-PAGE and Western blotting demonstrated that the recombinant protein p293 could well be expressed in P pastoris. Under optimized conditions （culture medium pH, 6.0-6.5; methanol concentration added daily, 3.0%; inoculum density, OD600=60; induction time point, 72-96 h）, the yield of soluble p293 was approximately 80 mg/L. We also observed p293 secretory expressed in P. pastoris to be 30 nm viral like particles by using electron microscopy. These results show that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV, and serve as a useful antigen for both diagnostic and vaccine purposes.展开更多
Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprise...Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.展开更多
Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NL...Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % (73/264). Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-2 ORF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.展开更多
VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability...VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.展开更多
We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constr...We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.展开更多
Circoviridae represent a growing family of small animal viruses. Some of these viruses have veterinary and medical importance, although, a vast amount of these newly discovered viruses have unknown effects on their ho...Circoviridae represent a growing family of small animal viruses. Some of these viruses have veterinary and medical importance, although, a vast amount of these newly discovered viruses have unknown effects on their hosts. The capsid-associated protein (Cap) of circoviruses is of interest because of its role in viral structure, immune evasion, host cell entry, and nuclear shuttling of viral components. The structure of the porcine circovirus 2 (PCV2) Cap has been solved and offered insight to these functions. Based on the crystallographic PCV2 Cap structure, models from circoviruses isolated from avian, fish, and mammalian hosts have been constructed and analyzed to better understand the roles of these proteins in the virus family. A high degree of conservation is observed in the models, however, the surface antigens differ among viruses. This is likely a reflection of the small genome harbored by circoviruses, and therefore the requirement of their few proteins to carry out specific vital functions, while maintaining enough variation to successfully infect their hosts. Here we describe the putative structures of a range of Cap proteins from circoviruses based on the crystallographic determination of porcine Cap, identifying key regions for function and inhibition of crystal formation.展开更多
Capsid protein enterovirus 71 （EV71） is one of the major viruses that cause the severe encephalitis and thus result in a high mortality in children less than 5 years of age.In an effort to discover new potent inhibi...Capsid protein enterovirus 71 （EV71） is one of the major viruses that cause the severe encephalitis and thus result in a high mortality in children less than 5 years of age.In an effort to discover new potent inhibitors against EV71,a novel three-dimensional pharmacophore model was developed on 24 inhibitors with different molecular structures and bioactivities.The best hypothesis （Hypo1） has a high predictive power and consists of four features,namely,one hydrophobic point （HY） and three hydrogen-bond acceptors （HA）.Two key features of the best Hypo1,HY1 and HA3 match well with an important narrow hydrophobic canyon and with the surface of LYS274 in the target EV71 active site,respectively.The more versatile feature,HA1,is firstly found to be very influential on these compounds’ bioactivities,which may interact with the other side of the active site in the EV71 receptor.The application of the model is successful in predicting the activities of 30 known EV71 inhibitors with a correlation coefficient of 0.831.Furthermore,Hypo1 demonstrates a superior screening capability for retrieving inhibitors from the database with a high enrichment factor of 70.This study provides some important clues in search for more potent inhibitors against EV71 infection.展开更多
An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the U...An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.展开更多
Cervical cancer is one of the most common malignant gynecological tumors and has the second highest incidence of all malignancies in females.Chronic and persistent infection with High Risk Human Papillomavirus(HR-HPV)...Cervical cancer is one of the most common malignant gynecological tumors and has the second highest incidence of all malignancies in females.Chronic and persistent infection with High Risk Human Papillomavirus(HR-HPV)is the main cause of cervical cancer.There is a distinct lack of methodology by which to determine whether cervical epithelial dysplasia is cancerous following HPV infection.HPV L1 capsid protein is a major structural protein of human papillomavirus(HPV),and it is the main target of the local cellular immune response aiming to combat human papillomavirus after HPV infection within cervical cells.Greater understanding of HPV L1 capsid protein and its association with cervical cytology,histopathology,patient age and human papillomavirus viral load has the potential to contribute toward improved the diagnosis and management of cervical cancer,providing useful information for gynecological clinicians in the hope of improving patient treatment and quality of life.This article reviews the predictive utility of HPV L1 capsid protein for cervical lesions.展开更多
Porcine circovirus type 3(PCV3)is a novel porcine circovirus associated with porcine dermatitis and nephritis syndrome(PDNS),reproductive failure,and multisystemic inflammation.Capsid protein(Cap)encoded by PCV3 ORF2 ...Porcine circovirus type 3(PCV3)is a novel porcine circovirus associated with porcine dermatitis and nephritis syndrome(PDNS),reproductive failure,and multisystemic inflammation.Capsid protein(Cap)encoded by PCV3 ORF2 gene has been identified as an immunogenic protein.Currently,there is no immunofluorescence assay(IFA)available for serological diagnosis.Here,the N-terminal 33 amino acids of Cap protein were predicted to serve as a PCV3 nuclear localization signal(NLS).Two types of recombinant plasmids were constructed for recombinant protein expression in 5f9 cells by using a baculovirus expression system:plasmid rvBac-Pc for full-length Cap protein expression and rvBac-Sc for Cap protein expression with a honeybee melittin signal peptide in place of the predicted NLS sequence.Expression of the nuclear localization sequences was further analyzed by IFA.Strong and specific fluorescence signals were observed in the nucleus of rvBac-Pc-transfected cells and in the cytoplasm of rvBac-Sc-transfected cells.No cross-reactivity was observed with porcine circovirus type 2,porcine pseudorabies virus,classical swine fever virus,or porcine reproductive and respiratory syndrome virus.In summary,we developed two fluorescence detection modes for Cap protein that can be used to detect PCV3 antibodies.This method is suitable for the diagnosis and epidemiological investigation of PCV3.This study provides a reliable detection method for monitoring PCV3 antibody level in pigs in the future.展开更多
目的观察人参皂甙Rg1对糖尿病肾病大鼠尿蛋白以及炎症因子肿瘤坏死因子-α(TNF-α)、单核细胞趋化因子蛋白-1(MCP-1)表达的影响。方法将40只雄性SD大鼠随机分为4组:正常对照组、糖尿病模型组、人参皂甙Rg1治疗组、厄贝沙坦(ARB)治疗组...目的观察人参皂甙Rg1对糖尿病肾病大鼠尿蛋白以及炎症因子肿瘤坏死因子-α(TNF-α)、单核细胞趋化因子蛋白-1(MCP-1)表达的影响。方法将40只雄性SD大鼠随机分为4组:正常对照组、糖尿病模型组、人参皂甙Rg1治疗组、厄贝沙坦(ARB)治疗组。采用链脲佐菌素(65mg/kg)腹腔注射建立糖尿病大鼠模型。于动物处死前1d测血糖,用代谢笼收集24h尿液,记录尿量行24h尿蛋白定量和肾功检查。光镜及电镜观察肾组织的病理变化。采用酶联免疫吸附法(ELISA)检测血MCP-1、血及肾TNF-α水平;免疫组织化学检测肾脏组织MCP-1蛋白的表达;实时荧光定量PCR(real-ti me PCR)检测肾脏组织中MCP-1、TNF-α基因水平的表达。结果模型组大鼠镜下见肾小球体积增大,基底膜增厚及系膜物质增多,足细胞较正常对照组减少(P<0.01)。两治疗组与模型组分别比较,肾小球基底膜未见明显增宽,足细胞数增加(P<0.05)。两治疗组之间无明显差异。模型组大鼠血糖、24h尿蛋白、血肌酐水平升高,与对照组相比差异有统计学意义(P<0.05)。两治疗组与模型组相比,血糖水平无明显变化(P>0.05),但24h尿蛋白、血肌酐水平均有所改善(P<0.05)。免疫组织化学、ELISA和real-ti me PCR结果表明,与对照组相比,模型组MCP-1、TNF-α的表达增加(P<0.05),两治疗组MCP-1、TNF-α表达较模型组减少(P<0.05),但两治疗组无明显差异。相关性分析提示MCP-1、TNF-α水平与24h尿蛋白水平(r=0.7802,0.6963)、肾小球硬化指数(r=0.8296,0.7413)、足细胞基底膜厚度(r=0.7678,0.6701)成正相关(P<0.05)。结论人参皂甙Rg1能降低TNF-α、MCP-1的水平,改善糖尿病肾病大鼠足细胞及肾脏的病理损害,能显著减少糖尿病大鼠24h尿蛋白,具有一定的肾保护作用。展开更多
基金the National Natural Science Foundation of China(No20674029) the Science and Technology Department ofJilin Province(No20050402-4)
文摘In this study, a recombinant Pichia pastoris expression system was developed to express HPV16 L1 protein that was driven by a strong AOX1 promoter. HPV16L1 gene was cloned into vector pPICZ,αB. HPV16 L1 protein expression induced by methanol was screened by using sodium dedecyl sulfate-polyacrylamide gel electrophoresis （SDSPAGE） and Western blotting. The results indicate that the HPVl6 L1 protein is secreted by the recombinant P. pastoris, and the purified HPV16 L1 protein can self-assemble into vires-like particles（ VLPs）, which show a good immunogenicity and induces high-titer antibody in mice.
基金supported by the National Natural Science Foundation of China(No.81460304)Guangxi Natural Science Foundation(No.2015GXNSFDA139020)a research program sponsored by the Health Bureau of Guangxi Zhuang Autonomous Region,China(No.Z2014298)
文摘This study bioinformatically analyzed the non-VP1 capsid proteins（VP2-VP4） of Coxasckievirus A6（CVA6）, with an attempt to predict their basic physicochemical properties, structural/functional features and linear B cell eiptopes. The online tools Sub Loc, Target P and the others from Ex PASy Bioinformatics Resource Portal, and SWISS-MODEL（an online protein structure modeling server）, were utilized to analyze the amino acid（AA） sequences of VP2-VP4 proteins of CVA6. Our results showed that the VP proteins of CVA6 were all of hydrophilic nature, contained phosphorylation and glycosylation sites and harbored no signal peptide sequences and acetylation sites. Except VP3, the other proteins did not have transmembrane helix structure and nuclear localization signal sequences. Random coils were the major conformation of the secondary structure of the capsid proteins. Analysis of the linear B cell epitopes by employing Bepipred showed that the average antigenic indices（AI） of individual VP proteins were all greater than 0 and the average AI of VP4 was substantially higher than that of VP2 and VP3. The VP proteins all contained a number of potential B cell epitopes and some eiptopes were located at the internal side of the viral capsid or were buried. We successfully predicted the fundamental physicochemical properties, structural/functional features and the linear B cell eiptopes and found that different VP proteins share some common features and each has its unique attributes. These findings will help us understand the pathogenicity of CVA6 and develop related vaccines and immunodiagnostic reagents.
基金Supported by the National Natural Science Foundation of China(No.30771609)the National High-tech Research and Development Program of China(No.2007AA021004)
文摘Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus（HEV）. HEV is a nonenveloped virus that bas been classified in the family of Caliciviridae. The virus appears to be a polya-denylated, positive-stranded RNA virus with three major open reading frames（ORFs）. The capsid protein of HEV is encoded by the open reading frame 2（ORF2）. We attempted to produce a truncated capsid protein, designed p293, in Pichia pastoris. The p293 gene encoding amino acids（aa） 382-674 of HEV ORF2 was designed based on the full length of HEV ORF2, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS 115. SDS-PAGE and Western blotting demonstrated that the recombinant protein p293 could well be expressed in P pastoris. Under optimized conditions （culture medium pH, 6.0-6.5; methanol concentration added daily, 3.0%; inoculum density, OD600=60; induction time point, 72-96 h）, the yield of soluble p293 was approximately 80 mg/L. We also observed p293 secretory expressed in P. pastoris to be 30 nm viral like particles by using electron microscopy. These results show that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV, and serve as a useful antigen for both diagnostic and vaccine purposes.
基金National Basic Research Program ofChina (973 Program) (2009CB118701)National NaturalScientific Foundation of China (30671615, 30871940)+1 种基金Innovation Project of the Chinese Academy of Sciences(KSCX2-YW-N-021)Science and Technology Foundation of Zhejiang Province (2007C22052)
文摘Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.
基金supported by the special studies for social welfare researches in institutes (2005DIB4J041)
文摘Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % (73/264). Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-2 ORF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.
文摘VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.
基金supported by the National Programs for High Technology Research and Development of China (2006AA10A204)the Gansu Key Technologies R&D Program(ZGS-052-A41-0006-03)the Programs for Director Fund of Lanzhou Veterinary Research Institute
文摘We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.
文摘Circoviridae represent a growing family of small animal viruses. Some of these viruses have veterinary and medical importance, although, a vast amount of these newly discovered viruses have unknown effects on their hosts. The capsid-associated protein (Cap) of circoviruses is of interest because of its role in viral structure, immune evasion, host cell entry, and nuclear shuttling of viral components. The structure of the porcine circovirus 2 (PCV2) Cap has been solved and offered insight to these functions. Based on the crystallographic PCV2 Cap structure, models from circoviruses isolated from avian, fish, and mammalian hosts have been constructed and analyzed to better understand the roles of these proteins in the virus family. A high degree of conservation is observed in the models, however, the surface antigens differ among viruses. This is likely a reflection of the small genome harbored by circoviruses, and therefore the requirement of their few proteins to carry out specific vital functions, while maintaining enough variation to successfully infect their hosts. Here we describe the putative structures of a range of Cap proteins from circoviruses based on the crystallographic determination of porcine Cap, identifying key regions for function and inhibition of crystal formation.
基金Sponsored by the National Natural Science Foundation of China (No. 30800719)
文摘Capsid protein enterovirus 71 （EV71） is one of the major viruses that cause the severe encephalitis and thus result in a high mortality in children less than 5 years of age.In an effort to discover new potent inhibitors against EV71,a novel three-dimensional pharmacophore model was developed on 24 inhibitors with different molecular structures and bioactivities.The best hypothesis （Hypo1） has a high predictive power and consists of four features,namely,one hydrophobic point （HY） and three hydrogen-bond acceptors （HA）.Two key features of the best Hypo1,HY1 and HA3 match well with an important narrow hydrophobic canyon and with the surface of LYS274 in the target EV71 active site,respectively.The more versatile feature,HA1,is firstly found to be very influential on these compounds’ bioactivities,which may interact with the other side of the active site in the EV71 receptor.The application of the model is successful in predicting the activities of 30 known EV71 inhibitors with a correlation coefficient of 0.831.Furthermore,Hypo1 demonstrates a superior screening capability for retrieving inhibitors from the database with a high enrichment factor of 70.This study provides some important clues in search for more potent inhibitors against EV71 infection.
基金National Natural Science Funds(30570081, 30670094 and 30700028)
文摘An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.
文摘Cervical cancer is one of the most common malignant gynecological tumors and has the second highest incidence of all malignancies in females.Chronic and persistent infection with High Risk Human Papillomavirus(HR-HPV)is the main cause of cervical cancer.There is a distinct lack of methodology by which to determine whether cervical epithelial dysplasia is cancerous following HPV infection.HPV L1 capsid protein is a major structural protein of human papillomavirus(HPV),and it is the main target of the local cellular immune response aiming to combat human papillomavirus after HPV infection within cervical cells.Greater understanding of HPV L1 capsid protein and its association with cervical cytology,histopathology,patient age and human papillomavirus viral load has the potential to contribute toward improved the diagnosis and management of cervical cancer,providing useful information for gynecological clinicians in the hope of improving patient treatment and quality of life.This article reviews the predictive utility of HPV L1 capsid protein for cervical lesions.
基金This work was supported by a grant from the China Agriculture Research System(CARS-35).
文摘Porcine circovirus type 3(PCV3)is a novel porcine circovirus associated with porcine dermatitis and nephritis syndrome(PDNS),reproductive failure,and multisystemic inflammation.Capsid protein(Cap)encoded by PCV3 ORF2 gene has been identified as an immunogenic protein.Currently,there is no immunofluorescence assay(IFA)available for serological diagnosis.Here,the N-terminal 33 amino acids of Cap protein were predicted to serve as a PCV3 nuclear localization signal(NLS).Two types of recombinant plasmids were constructed for recombinant protein expression in 5f9 cells by using a baculovirus expression system:plasmid rvBac-Pc for full-length Cap protein expression and rvBac-Sc for Cap protein expression with a honeybee melittin signal peptide in place of the predicted NLS sequence.Expression of the nuclear localization sequences was further analyzed by IFA.Strong and specific fluorescence signals were observed in the nucleus of rvBac-Pc-transfected cells and in the cytoplasm of rvBac-Sc-transfected cells.No cross-reactivity was observed with porcine circovirus type 2,porcine pseudorabies virus,classical swine fever virus,or porcine reproductive and respiratory syndrome virus.In summary,we developed two fluorescence detection modes for Cap protein that can be used to detect PCV3 antibodies.This method is suitable for the diagnosis and epidemiological investigation of PCV3.This study provides a reliable detection method for monitoring PCV3 antibody level in pigs in the future.
文摘目的观察人参皂甙Rg1对糖尿病肾病大鼠尿蛋白以及炎症因子肿瘤坏死因子-α(TNF-α)、单核细胞趋化因子蛋白-1(MCP-1)表达的影响。方法将40只雄性SD大鼠随机分为4组:正常对照组、糖尿病模型组、人参皂甙Rg1治疗组、厄贝沙坦(ARB)治疗组。采用链脲佐菌素(65mg/kg)腹腔注射建立糖尿病大鼠模型。于动物处死前1d测血糖,用代谢笼收集24h尿液,记录尿量行24h尿蛋白定量和肾功检查。光镜及电镜观察肾组织的病理变化。采用酶联免疫吸附法(ELISA)检测血MCP-1、血及肾TNF-α水平;免疫组织化学检测肾脏组织MCP-1蛋白的表达;实时荧光定量PCR(real-ti me PCR)检测肾脏组织中MCP-1、TNF-α基因水平的表达。结果模型组大鼠镜下见肾小球体积增大,基底膜增厚及系膜物质增多,足细胞较正常对照组减少(P<0.01)。两治疗组与模型组分别比较,肾小球基底膜未见明显增宽,足细胞数增加(P<0.05)。两治疗组之间无明显差异。模型组大鼠血糖、24h尿蛋白、血肌酐水平升高,与对照组相比差异有统计学意义(P<0.05)。两治疗组与模型组相比,血糖水平无明显变化(P>0.05),但24h尿蛋白、血肌酐水平均有所改善(P<0.05)。免疫组织化学、ELISA和real-ti me PCR结果表明,与对照组相比,模型组MCP-1、TNF-α的表达增加(P<0.05),两治疗组MCP-1、TNF-α表达较模型组减少(P<0.05),但两治疗组无明显差异。相关性分析提示MCP-1、TNF-α水平与24h尿蛋白水平(r=0.7802,0.6963)、肾小球硬化指数(r=0.8296,0.7413)、足细胞基底膜厚度(r=0.7678,0.6701)成正相关(P<0.05)。结论人参皂甙Rg1能降低TNF-α、MCP-1的水平,改善糖尿病肾病大鼠足细胞及肾脏的病理损害,能显著减少糖尿病大鼠24h尿蛋白,具有一定的肾保护作用。
