[Objective] This study aimed to develop an indirect ELISA to detect the antibodies against Actinobacil us pleuropneumoniae （APP） using the recombinant ApxⅡA1 protein expressed in prokaryotic cells as the antigen. [...[Objective] This study aimed to develop an indirect ELISA to detect the antibodies against Actinobacil us pleuropneumoniae （APP） using the recombinant ApxⅡA1 protein expressed in prokaryotic cells as the antigen. [Method] The major epi-tope domain of ApxⅡ was cloned into the prokaryotic expression vector pET-28a （＋） to obtain the recombinant plasmid pET-ApxⅡA1, which was then transformed into E. coli BL21 （DE3） for expression. The immunogenicity of the recombinant pro-tein was analyzed by western-blotting. After that, the purified recombinant protein was used as the coating antigen in the indirect ELISA for detecting the antibodies against APP. Final y, the concentration of coated antigen and the dilution of serum were optimized. [Result] Proved by enzyme digestion and sequencing, the recombi-nant plasmid pET-ApxⅡA1 was constructed successful y. The recombinant protein was highly expressed in prokaryotic cells, and Western-blotting analysis showed that it was recognized specifical y by positive serum of APP. The indirect ELISA could detect the antibody against APP with the purified recombinant protein as the coating antigen. The optimal concentration of coated antigen was 1.23 μg/ml and the opti-mal dilution of serum was 1：100. Compared with a commercial ELISA kit detecting antibody against ApxⅣ, the coincidence rate of the indirect ELISA was 90.4%. [Conclusion] Our results indicated that the indirect ELISA is sensitive and specific, and suitable for evaluating the effect of APP vaccine and epidemiological surveys.展开更多
The nueleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (Nl and N2) of the N protein of SARS-CoV were predicted by bio...The nueleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (Nl and N2) of the N protein of SARS-CoV were predicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodies were isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-induced polyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SAR,S-CoV. Furthermore, it was confirmed that Nl peptide-specific IgG antibodies were detectable in the sera of severe acute respiratory syndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified and N protein specific Abs were produced by peptide immunization, which will be useful for the study of SARS-CoV.展开更多
Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods:...Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods: The recombinant baculovirus bac-S0.7 with the 700 bp fragment of S gene 5' terminal of Hantaan virus was constructed, and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay, micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0.7 infecting insect cells, specific antibody with the highest titer of 1∶1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0.7 gene fragment in insect cells is immunogenic.展开更多
AIM:To evaluate the antitumoral effect of combined inhibitors of angiogenesis and histone deacetylases in an experimental rat hepatoma model.METHODS:MH7777A hepatoma cells were injected into the liver of male Buffalo ...AIM:To evaluate the antitumoral effect of combined inhibitors of angiogenesis and histone deacetylases in an experimental rat hepatoma model.METHODS:MH7777A hepatoma cells were injected into the liver of male Buffalo rats.After 7 d treatment with the vascular endothelial growth factor receptor antagonist PTK787/ZK222584(PTK/ZK),the histone deacetylase inhibitor MS-275,tamoxifen(TAM) and/or retinoic acid was initiated(n ≥ 8 animals/group).Natural tumor development was shown in untreated control groups(control 1 with n = 12,control 2 with n = 8).The control groups were initiated at different time points to demonstrate the stability of the hepatoma model.For documentation of possible side effects,we documented any change in body weight,loss of fur and diarrhea.After 21 d treatment,the rats were euthanized.Main target parameters were tumor size and metastasis rate.Additionally,immunohistochemistry for the proliferating cell nuclear antigen(PCNA) and TdT-mediated dUTP-biotin nick end labeling(TUNEL) assay were performed.RESULTS:The control groups developed large tumor nodules with extrahepatic tumor burden in the lung and abdominal organs(control 1:6.18 cm3 ± 4.14 cm3 and control 2:8.0 cm3 ± 4.44 cm3 28 d after tumor cell injection).The tumor volume did not differ significantly in the control groups(P = 0.13).As single agents MS-275 and PTK/ZK reduced tumor volume by 58.6% ± 2.6% and 48.7% ± 3.2% vs control group 1,which was significant only for MS-275(P = 0.025).The combination of MS-275 and PTK/ZK induced a nearly complete and highly significant tumor shrinkage by 90.3% ± 1%(P = 0.005).Addition of TAM showed no further efficacy,while quadruple therapy with retinoic acid increased antitumoral efficacy(tumor reduction by 93 ± 1%) and side effects.PCNA positive cells were not significantly reduced by the single agents,while dual therapy(MS-275 and PTK/ZK) and quadruple therapy reduced the PCNA-positive cell fraction significantly by 9.1 and 20.6% vs control 1(P < 0.05).The number of TUNEL-positive cells,markers for ongoing apoptosis,was increased significantly by the single agents(control 1:6.9%,PTK/ZK:11.4%,MS-275:12.2% with P < 0.05 vs control 1).The fraction of TUNEL-positive cells was upregulated highly significantly by dual therapy(18.4%) and quadruple therapy(24.8%,P < 0.01 vs control 1).For the proliferating(PCNA positive) and apoptotic cell fraction,quadruple therapy was significantly superior to dual therapy(P = 0.01).CONCLUSION:Combined PTK/ZK and MS-275 were highly effective in this hepatoma model.Quadruple therapy enhanced the effects microscopically,but not macroscopically.These results should be investigated further.展开更多
AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MA...AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.展开更多
Objective:The aim of our study was to investigate the relationship between cell apoptosis and dephosphorylated RB protein and proliferating cell nuclear antigen(PCNA) in human breast cancer.Methods:MTT colorimetric as...Objective:The aim of our study was to investigate the relationship between cell apoptosis and dephosphorylated RB protein and proliferating cell nuclear antigen(PCNA) in human breast cancer.Methods:MTT colorimetric assay was applied to examine the growth inhibition,and the apoptosis was determined by flow cytometry(FCM).The expressing quantity of dephosphorylated RB protein and PCNA pre-and post the action of ADR were detected with immunocytochemistry.Results:MTT assay revealed that ADR inhibited proliferation of MCF-7/S cells in a dose dependent manner,the 50% inhibition concentration(IC50) value was 0.128 mg/L.Tumor cell apoptotic rate(AR) in ADR group(χ= 0.259) was significantly higher than that in the control group(χ = 0.045)(P < 0.01).The expressive levels of dephosphorylated RB protein in ADR group(MOD × area = 986.8 ± 207.4) was significantly higher than that in control group(MOD × area =131.7 ± 31.9)(P < 0.01).PCNA positive expression rate in ADR group(χ = 0.3371) was significantly lower than that in the control group(χ = 0.5152)(P < 0.01).Conclusion:In ADR group,there was significant positive correlation between AR and the expressing quantity of dephosphorylated RB protein,but there was significant negative correlation between AR and PCNA.展开更多
Objective To investigate the changes of neural stem cells (NSCs) in the rat hippocampus after cerebral infarction (CI) and to evaluate the neurogenesis caused by the activation of NSCs. Methods CI models of rats were ...Objective To investigate the changes of neural stem cells (NSCs) in the rat hippocampus after cerebral infarction (CI) and to evaluate the neurogenesis caused by the activation of NSCs. Methods CI models of rats were made and rats were assigned to 6 groups: sham-operated, 1 day, 3 days, 7 days, 14 days, and 28 days after CI. The dynamic expression of bromodeoxyuridine (BrdU), polysialylated neural cell adhesion molecule (PSA-NCAM), glial fibrillary acidic protein (GFAP), and neuronal nuclear antigen (NeuN) were determined by immunohistochemistry and immunofluorescence staining. BrdU was used to mark the proliferated NSCs. PSA-NCAM was used to mark the plasticity of activated NSCs. GFAP and NeuN were used to mark the differentiated NSCs. Results Compared with the controls, the number of BrdU+ cells in the hippocampus increased significantly at 1 day after CI (P<0.05), reached peak at 7 days after CI (P<0.05), decreased but still elevated compared with the controls at 14 days after CI (P<0.05), and nearly unchanged at 28 days after CI. The number of BrdU+/PSA-NCAM+ cells increased significantly at 7 days after CI (P<0.05), reached peak at 14 days after CI (P<0.05), and decreased but still elevated compared with the controls at 28 days after CI (P<0.05). The number of BrdU+/PSA-NCAM+ cells was equal to 60% of the number of BrdU+ cells in all the same period. The number of BrdU+/NeuN+ cells in the hippocampus increased significantly at 14 days after CI (P<0.05) and reached peak at 28 day after CI (P<0.05). The number of BrdU+/GFAP+cells in the hippocampus nearly unchanged after CI. Conclusion CI can stimulate the proliferation of inherent NSCs, and most proliferated NSCs may differentiate into neurons and represent neural plasticity.展开更多
基金Supported by Special Fund for Agro-scientific Research in the Public Interest(201303034-8)~~
文摘[Objective] This study aimed to develop an indirect ELISA to detect the antibodies against Actinobacil us pleuropneumoniae （APP） using the recombinant ApxⅡA1 protein expressed in prokaryotic cells as the antigen. [Method] The major epi-tope domain of ApxⅡ was cloned into the prokaryotic expression vector pET-28a （＋） to obtain the recombinant plasmid pET-ApxⅡA1, which was then transformed into E. coli BL21 （DE3） for expression. The immunogenicity of the recombinant pro-tein was analyzed by western-blotting. After that, the purified recombinant protein was used as the coating antigen in the indirect ELISA for detecting the antibodies against APP. Final y, the concentration of coated antigen and the dilution of serum were optimized. [Result] Proved by enzyme digestion and sequencing, the recombi-nant plasmid pET-ApxⅡA1 was constructed successful y. The recombinant protein was highly expressed in prokaryotic cells, and Western-blotting analysis showed that it was recognized specifical y by positive serum of APP. The indirect ELISA could detect the antibody against APP with the purified recombinant protein as the coating antigen. The optimal concentration of coated antigen was 1.23 μg/ml and the opti-mal dilution of serum was 1：100. Compared with a commercial ELISA kit detecting antibody against ApxⅣ, the coincidence rate of the indirect ELISA was 90.4%. [Conclusion] Our results indicated that the indirect ELISA is sensitive and specific, and suitable for evaluating the effect of APP vaccine and epidemiological surveys.
基金supported by the grant of Shanghai Science and Technology Committee(No.03DZ19113)National Key Basic Research Program of China(No.2001CB510006)+1 种基金863 project(No.2001AA231011)a specific project against SARS from Chinese Academy of Sciences.
文摘The nueleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (Nl and N2) of the N protein of SARS-CoV were predicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodies were isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-induced polyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SAR,S-CoV. Furthermore, it was confirmed that Nl peptide-specific IgG antibodies were detectable in the sera of severe acute respiratory syndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified and N protein specific Abs were produced by peptide immunization, which will be useful for the study of SARS-CoV.
基金National Natural Science Foundation of China (No.30070686)Chinese Educational Deputy Fund for skeleton teachers
文摘Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods: The recombinant baculovirus bac-S0.7 with the 700 bp fragment of S gene 5' terminal of Hantaan virus was constructed, and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay, micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0.7 infecting insect cells, specific antibody with the highest titer of 1∶1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0.7 gene fragment in insect cells is immunogenic.
基金Supported by The Schering AG,Berlin (Germany) which friendly provided PTK787/ZK222584 and MS-275
文摘AIM:To evaluate the antitumoral effect of combined inhibitors of angiogenesis and histone deacetylases in an experimental rat hepatoma model.METHODS:MH7777A hepatoma cells were injected into the liver of male Buffalo rats.After 7 d treatment with the vascular endothelial growth factor receptor antagonist PTK787/ZK222584(PTK/ZK),the histone deacetylase inhibitor MS-275,tamoxifen(TAM) and/or retinoic acid was initiated(n ≥ 8 animals/group).Natural tumor development was shown in untreated control groups(control 1 with n = 12,control 2 with n = 8).The control groups were initiated at different time points to demonstrate the stability of the hepatoma model.For documentation of possible side effects,we documented any change in body weight,loss of fur and diarrhea.After 21 d treatment,the rats were euthanized.Main target parameters were tumor size and metastasis rate.Additionally,immunohistochemistry for the proliferating cell nuclear antigen(PCNA) and TdT-mediated dUTP-biotin nick end labeling(TUNEL) assay were performed.RESULTS:The control groups developed large tumor nodules with extrahepatic tumor burden in the lung and abdominal organs(control 1:6.18 cm3 ± 4.14 cm3 and control 2:8.0 cm3 ± 4.44 cm3 28 d after tumor cell injection).The tumor volume did not differ significantly in the control groups(P = 0.13).As single agents MS-275 and PTK/ZK reduced tumor volume by 58.6% ± 2.6% and 48.7% ± 3.2% vs control group 1,which was significant only for MS-275(P = 0.025).The combination of MS-275 and PTK/ZK induced a nearly complete and highly significant tumor shrinkage by 90.3% ± 1%(P = 0.005).Addition of TAM showed no further efficacy,while quadruple therapy with retinoic acid increased antitumoral efficacy(tumor reduction by 93 ± 1%) and side effects.PCNA positive cells were not significantly reduced by the single agents,while dual therapy(MS-275 and PTK/ZK) and quadruple therapy reduced the PCNA-positive cell fraction significantly by 9.1 and 20.6% vs control 1(P < 0.05).The number of TUNEL-positive cells,markers for ongoing apoptosis,was increased significantly by the single agents(control 1:6.9%,PTK/ZK:11.4%,MS-275:12.2% with P < 0.05 vs control 1).The fraction of TUNEL-positive cells was upregulated highly significantly by dual therapy(18.4%) and quadruple therapy(24.8%,P < 0.01 vs control 1).For the proliferating(PCNA positive) and apoptotic cell fraction,quadruple therapy was significantly superior to dual therapy(P = 0.01).CONCLUSION:Combined PTK/ZK and MS-275 were highly effective in this hepatoma model.Quadruple therapy enhanced the effects microscopically,but not macroscopically.These results should be investigated further.
基金Supported by A PhD grant from the French Ministry of Foreign Affairs (French Embassy in Beijing) to Ren-Yong Linby a project grant from the "Foundation Transplantation" (2005-2006)+1 种基金by a grant from NSFC, No. 30860253 and 30760239by the Xinjiang Key-Lab project grants on Echinococcosis, No. XJDX0202-2005-01 and XJDX0202-2007-04
文摘AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.
文摘Objective:The aim of our study was to investigate the relationship between cell apoptosis and dephosphorylated RB protein and proliferating cell nuclear antigen(PCNA) in human breast cancer.Methods:MTT colorimetric assay was applied to examine the growth inhibition,and the apoptosis was determined by flow cytometry(FCM).The expressing quantity of dephosphorylated RB protein and PCNA pre-and post the action of ADR were detected with immunocytochemistry.Results:MTT assay revealed that ADR inhibited proliferation of MCF-7/S cells in a dose dependent manner,the 50% inhibition concentration(IC50) value was 0.128 mg/L.Tumor cell apoptotic rate(AR) in ADR group(χ= 0.259) was significantly higher than that in the control group(χ = 0.045)(P < 0.01).The expressive levels of dephosphorylated RB protein in ADR group(MOD × area = 986.8 ± 207.4) was significantly higher than that in control group(MOD × area =131.7 ± 31.9)(P < 0.01).PCNA positive expression rate in ADR group(χ = 0.3371) was significantly lower than that in the control group(χ = 0.5152)(P < 0.01).Conclusion:In ADR group,there was significant positive correlation between AR and the expressing quantity of dephosphorylated RB protein,but there was significant negative correlation between AR and PCNA.
基金Supported by the Advanced College Research Project from the Education Department of Liaoning province (05L094)Natural Science Foundation of Liaoning province (20072171)
文摘Objective To investigate the changes of neural stem cells (NSCs) in the rat hippocampus after cerebral infarction (CI) and to evaluate the neurogenesis caused by the activation of NSCs. Methods CI models of rats were made and rats were assigned to 6 groups: sham-operated, 1 day, 3 days, 7 days, 14 days, and 28 days after CI. The dynamic expression of bromodeoxyuridine (BrdU), polysialylated neural cell adhesion molecule (PSA-NCAM), glial fibrillary acidic protein (GFAP), and neuronal nuclear antigen (NeuN) were determined by immunohistochemistry and immunofluorescence staining. BrdU was used to mark the proliferated NSCs. PSA-NCAM was used to mark the plasticity of activated NSCs. GFAP and NeuN were used to mark the differentiated NSCs. Results Compared with the controls, the number of BrdU+ cells in the hippocampus increased significantly at 1 day after CI (P<0.05), reached peak at 7 days after CI (P<0.05), decreased but still elevated compared with the controls at 14 days after CI (P<0.05), and nearly unchanged at 28 days after CI. The number of BrdU+/PSA-NCAM+ cells increased significantly at 7 days after CI (P<0.05), reached peak at 14 days after CI (P<0.05), and decreased but still elevated compared with the controls at 28 days after CI (P<0.05). The number of BrdU+/PSA-NCAM+ cells was equal to 60% of the number of BrdU+ cells in all the same period. The number of BrdU+/NeuN+ cells in the hippocampus increased significantly at 14 days after CI (P<0.05) and reached peak at 28 day after CI (P<0.05). The number of BrdU+/GFAP+cells in the hippocampus nearly unchanged after CI. Conclusion CI can stimulate the proliferation of inherent NSCs, and most proliferated NSCs may differentiate into neurons and represent neural plasticity.
