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1.
Vaccine ; 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38796326

RESUMO

We conducted a phase I, randomized, double-blind, placebo-controlled trial including healthy adults in Sui County, Henan Province, China. Ninety-six adults were randomly assigned to one of three groups (high-dose, medium-dose, and low-dose) at a 3:1 ratio to receive one vaccine dose or placebo. Adverse events up to 28 days after each dose and serious adverse events up to 6 months after all doses were reported. Geometric mean titers and seroconversion rates were measured for anti-rotavirus neutralizing antibodies using microneutralization tests. The rates of total adverse events in the placebo group, low-dose group, medium-dose group, and high-dose group were 29.17 % (12.62 %-51.09 %), 12.50 % (2.66 %-32.36 %), 50.00 % (29.12 %-70.88 %), and 41.67 % (22.11 %-63.36 %), respectively, with no significant difference in the experimental groups compared with the placebo group. The results of the neutralizing antibody assay showed that in the adult group, the neutralizing antibody geometric mean titer at 28 days after full immunization in the low-dose group was 583.01 (95 % confidence interval [CI]: 447.12-760.20), that in the medium-dose group was 899.34 (95 % CI: 601.73-1344.14), and that in the high-dose group was 1055.24 (95 % CI: 876.28-1270.75). The GMT of serum-specific IgG at 28 days after full immunization in the low-dose group was 3444.26 (95 % CI: 2292.35-5175.02), that in the medium-dose group was 6888.55 (95 % CI: 4426.67-10719.6), and that in the high-dose group was 7511.99 (95 % CI: 3988.27-14149.0). The GMT of serum-specific IgA at 28 days after full immunization in the low-dose group was 2332.14 (95 % CI: 1538.82-3534.45), that in the medium-dose group was 4800.98 (95 % CI: 2986.64-7717.50), and that in the high-dose group was 3204.30 (95 % CI: 2175.66-4719.27). In terms of safety, adverse events were mainly Grades 1 and 2, indicating that the safety of the vaccine is within the acceptable range in the healthy adult population. Considering the GMT and positive transfer rate of neutralizing antibodies for the main immunogenicity endpoints in the experimental groups, it was initially observed that the high-dose group had higher levels of neutralizing antibodies than the medium- and low-dose groups in adults aged 18-49 years. This novel inactivated rotavirus vaccine was generally well-tolerated in adults, and the vaccine was immunogenic in adults (ClinicalTrials.gov number, NCT04626856).

2.
World J Gastroenterol ; 24(45): 5109-5119, 2018 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-30568388

RESUMO

AIM: To establish a rotavirus (RV)-induced diarrhea model using RV SA11 in neonatal rhesus monkeys for the study of the pathogenic and immune mechanisms of RV infection and evaluation of candidate vaccines. METHODS: Neonatal rhesus monkeys with an average age of 15-20 d and an average weight of 500 g ± 150 g received intragastric administration of varying doses of SA11 RV ( 107 PFUs/mL, 106 PFUs/mL, or 105 PFUs/mL, 10 mL/animal) to determine whether the SA11 strain can effectively infect these animals by observing their clinical symptoms, fecal shedding of virus antigen by ELISA, distribution of RV antigen in the organs by immunofluorescence, variations of viral RNA load in the organs by qRT-PCR, histopathological changes in the small intestine by HE staining, and apoptosis of small intestinal epithelial cells by TUNEL assay. RESULTS: The RV monkey model showed typical clinical diarrhea symptoms in the 108 PFUs SA11 group, where we observed diarrhea 1-4 d post infection (dpi) and viral antigen shed in the feces from 1-7 dpi. RV was found in jejunal epithelial cells. We observed a viral load of approximately 5.85 × 103 copies per 100 mg in the jejunum at 2 dpi, which was increased to 1.09 × 105 copies per 100 mg at 3 dpi. A relatively high viral load was also seen in mesenteric lymph nodes at 2 dpi and 3 dpi. The following histopathological changes were observed in the small intestine following intragastric administration of SA11 RV: vacuolization, edema, and atrophy. Apoptosis in the jejunal villus epithelium was also detectable at 3 dpi. CONCLUSION: Our results indicate that we have successfully established a RV SA11 strain diarrhea model in neonatal rhesus monkeys. Future studies will elucidate the mechanisms underlying the pathogenesis of RV infection, and we will use the model to evaluate the protective effect of candidate vaccines.


Assuntos
Diarreia/imunologia , Modelos Animais de Doenças , Macaca mulatta , Infecções por Rotavirus/imunologia , Rotavirus/patogenicidade , Animais , Animais Recém-Nascidos , Diarreia/diagnóstico , Diarreia/virologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Fezes/virologia , Humanos , Intestino Delgado/citologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Intestino Delgado/virologia , RNA Viral/isolamento & purificação , Rotavirus/genética , Rotavirus/imunologia , Infecções por Rotavirus/diagnóstico , Infecções por Rotavirus/virologia , Eliminação de Partículas Virais
3.
World J Clin Cases ; 6(11): 426-440, 2018 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-30294607

RESUMO

AIM: To determine the distribution of rotavirus VP7 gene in hospitalized children in Yunnan, China. METHODS: A total of 366 stool specimens were collected from hospitalized children in hospitals in Yunnan Province from September 2010 to December 2013. The genomic RNA electropherotypes and the G genotypes of the rotaviruses were determined. A phylogenetic analysis of the VP7 gene was performed. Rotavirus isolation was performed, and characterized by plaque, minimum essential medium, and all genes sequence analysis. Quantification of antibodies for inactivated vaccine prepared with ZTR-68 was examined by enzyme-linked immunosorbent assay and microneutralization assay. RESULTS: Group A human rotavirus was detected in 177 of 366 (48.4%) stool samples using a colloidal gold device assay. The temporal distribution of rotavirus cases showed significant correlation with the mean air temperature. Rotaviruses were isolated from 13% of the rotavirus-positive samples. The predominant genotype was G1 (43.5%), followed by G3 (21.7%), G9 (17.4%), G2 (4.3%), G4 (8.7%), and mixed (4.3%) among a total of 23 rotavirus isolates. A rotavirus strain was isolated from a rotavirus-positive stool sample of a 4-month-old child in The First People's Hospital of Zhaotong (2010) for use as a candidate human inactivated rotavirus vaccine strain and for further research, and was designated ZTR-68. The genotype of 11 gene segments of strain ZTR-68 (RVA/Human-wt/CHN/ZTR-68/2010/G1P[8]) was characterized. The genotype constellation of strain ZTR-68 was identified as G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. The VP7 and VP4 genotypes of strain ZTR-68 were similar to Wa-like strains.CONCLUSIONSA high prevalence of the G1, G2, and G3 genotypes was detected from 2010 to 2012. However, a dominant prevalence of the G9 genotype was identified as the cause of gastroenteritis in children in Yunnan, China, in 2013. A candidate human inactivated rotavirus vaccine strain, designated ZTR-68 was isolated, characterized, and showed immunogenicity. Our data will be useful for the future formulation and development of a vaccine in China.

4.
Curr Stem Cell Res Ther ; 10(2): 109-20, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25300822

RESUMO

Bone marrow-derived mesenchymal stem cells hold great potential for cytotherapeutics of neurodegenerative disorders, including Parkinson's disease. The neurotrophic factor neurturin can rescue dopaminergic neurons damaged during the disease process. Lmx1α can promote mesencephalic dopaminergic differentiation during embryogenesis. In this study, we tested a cytotherapeutic strategy combining NTN/Lmx1α gene therapy and cell transplantation to ameliorate disease progression in hemiparkinsonian rhesus. Rhesus BMSCs were prepared for autologous grafting by transfection with recombinant adenoviral vectors expressing secreted NTN and Lmx1α,and cultured in the presence of induce factors, particularly the Lmx1α regulatory factor sonic hedgehog, to guide dopaminergic differentiation. These induced rh-BMSCs exhibited gene/protein expression phenotypes resembling nigral dopaminergic neurons. They survived and retained dopaminergic function following stereotaxic injection into the MPTP-lesioned right-side substantia nigra as indicated by SPECT measurement of DAT activity. Injected cells preserved and supplemented the remaining endogenous population of dopamine neurons (TH-positive cell ipsilateral/contralateral ratio was 56.81% ± 7.28% vs. 3.86%±1.22% in vehicle-injected controls; p<0.05). Cell injection also partially restored motor function and reduce apomorphine-evoked rotation (p<0.05). Moreover, function recovery occurred earlier than in previous studies on injected BMSCs. Our findings demonstrate a promising strategy for restoration of PD-associated motor dysfunction by transplantation of autologous BMSCs overexpressing NTN/Lmx1α.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Proteínas com Homeodomínio LIM/biossíntese , Células-Tronco Mesenquimais/fisiologia , Neurogênese , Neurturina/biossíntese , Doença de Parkinson Secundária/terapia , Fatores de Transcrição/biossíntese , Animais , Embrião de Galinha , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Expressão Gênica , Humanos , Proteínas com Homeodomínio LIM/genética , Macaca mulatta , Masculino , Transplante de Células-Tronco Mesenquimais , Neurturina/genética , Doença de Parkinson Secundária/fisiopatologia , Fatores de Transcrição/genética , Transplante Autólogo
5.
Nan Fang Yi Ke Da Xue Xue Bao ; 30(4): 681-5, 2010 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-20423825

RESUMO

OBJECTIVE: To construct a recombinant adenovirus for carry tyrosine hydroxylase (TH) gene and expressing bioactive TH protein in the animal model of Parkinson disease. METHODS: The TH gene was inserted into the shuttle plasmid, which was transformed into E.coli BJ-5183 for homologous recombination with the adenovirus genome. 293 cells were transfected with the recombinant adenovirus genome to obtain the recombinant virus, and the transcription and expression of TH were determined by RT-PCR and immunofluorescence assay, respectively. The production of L-DOPA in the in vitro reaction system was determined using capillary electrophoresis. RESULTS: We have successfully constructed the recombinant adenovirus. The TH mRNA and the corresponding protein were detected by RT-PCR and immunofluoresence assay in 293 cells. L-DOPA was also detected in the reaction system. CONCLUSION: The adenovirus constructed allows efficient expression of bioactive TH protein in vitro, which provides a basis for future study of gene therapy of Parkinson disease in animal models.


Assuntos
Adenoviridae/metabolismo , Vetores Genéticos/genética , Tirosina 3-Mono-Oxigenase/biossíntese , Adenoviridae/genética , Linhagem Celular , Eletroforese Capilar , Escherichia coli/genética , Escherichia coli/metabolismo , Terapia Genética , Humanos , Levodopa/análise , Levodopa/biossíntese , Levodopa/genética , Doença de Parkinson/terapia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Tirosina 3-Mono-Oxigenase/genética
6.
Bing Du Xue Bao ; 25(5): 344-8, 2009 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-19954110

RESUMO

To study the effect of simian vacuolating virus 40 (SV40) on development and differentiation of dendritic cells (DC) from rhesus macaque, the peripheral blood-derived dendritic cells from rhesus monkey were pulsed with inactivated SV40 and infective SV40, respectively at the 5th day post DC cultivation. Expressions of CD1a, HLA-DR, CD86 and CD83 on the cell surface at the 7th, 9th day post DC cultivation were analyzed by flow cytometry (FCM). The results showed that expressions of CD1a, HLA-DR, CD86 and CD83 on the cell surface in the inactivated SV40-pulsed experimental group were higher than those in the infective SV40-pulsed experimental group (P < 0.05). These cell surface molecules represented characteristic development and differentiation phase of DC. Down-regulation of expressions of these cell surface molecules indicated that infective SV40 might hamper differentiation and maturation of dendritic cells from rhesus monkey.


Assuntos
Diferenciação Celular , Células Dendríticas/citologia , Células Dendríticas/virologia , Vírus 40 dos Símios/fisiologia , Animais , Antígenos CD/metabolismo , Antígenos CD1/metabolismo , Antígeno B7-2/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Citometria de Fluxo , Antígenos HLA-DR/metabolismo , Imunoglobulinas/metabolismo , Macaca mulatta , Glicoproteínas de Membrana/metabolismo , Infecções por Polyomavirus/fisiopatologia , Antígeno CD83
7.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 23(10): 894-7, 2007 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-17908492

RESUMO

AIM: To express two fusion forms of hepatitis B virus surface antigen (HBsAg-s and s-HBsAg) in the Pichia pastoris expression system, and compare immunogenicity of the two fusion proteins. METHODS: Was fused GM-CSF to 5' or 3' terminal of HBsAg by inserting the gene fragment of connecting peptide (Gly(4)Ser)(3) to linker gene of GM-CSF and HBsAg. The two fusion proteins were expressed by secreting type expression plasmid pPIC9K in the Pichia pastoris, then the expressed products were detected by SDS-PAGE, Western blot and purified by DEAE-Sepharose Fast Flow ion exchange columns. Mice were inoculated with the two purified HBsAg/GM-CSF fusion proteins and HBsAg respectively in each, and the levels of anti-HBsAg in mice sera were tested by ELISA. RESULTS: Two HBsAg/GM-CSF fusion proteins were successfully expressed in the form of secretion in Pichia pastoris strain GS115, and exhibited specific reaction with both anti-HBsAg and anti-GM-CSF antibodies in Western blot. ELISA results showed after the inoculation the levels of anti-HBsAg induced by the two HBsAg/GM-CSF fusion proteins was higher than by HBsAg alone (P<0.05). Furthermore, the effect by fusing GM-CSF to C terminal of HBsAg was better than by fusing GM-CSF to N terminal of HBsAg. CONCLUSION: The immunogenicity of HBsAg could be enhanced by fusing GM-CSF.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Antígenos de Superfície da Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/imunologia , Pichia/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos/análise , Anticorpos/imunologia , Especificidade de Anticorpos , Western Blotting , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Antígenos de Superfície da Hepatite B/isolamento & purificação , Humanos , Camundongos , Pichia/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação
9.
J Chromatogr A ; 1139(2): 228-35, 2007 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-17118380

RESUMO

In the present study, we compared the performances of size-exclusion chromatography for the purification of plasmid DNA when different concentrations (0.5M, 1M, 2M, respectively) of two types of salt (NaCl and (NH(4))(2)SO(4)) are present in running buffers. Our experiment results displayed that it is not only the resolution of RNA but also those of supercoiled plasmid DNA and host's genomic DNA were increased greatly in the presence of high concentration of water-structure salt. We deduce that two separation modes may be involved in the process: The supercoiled plasmid DNA is influenced mainly by compaction effect and eluted in the size-exclusion mode; whereas, RNA and genomic DNA are influenced mainly by hydrophobic effect due to their stretched and loose structures and eluted in the interaction mode. This method led to an improved efficiency of size-exclusion chromatography.


Assuntos
Cromatografia em Gel/métodos , DNA/isolamento & purificação , Plasmídeos/genética , Cloreto de Sódio/química
10.
Nan Fang Yi Ke Da Xue Xue Bao ; 26(12): 1806-11, 2006 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-17259126

RESUMO

OBJECTIVE: To prepare small interfering RNA (siRNA) targeting survivin for inhibition of endogenous survivin gene expression in Hela cell line and evaluate its effect on promoting Hela cell apoptosis. METHODS: The recombinant plasmid pshRNA-survivin-1 and pshRNA-survivin-2 were constructed and transfected into Hela cells, in which the expression level of survivin was determined by immunofluorescence staining and survivin gene transcription detected by semi-quantitative RT-PCR. RESULTS: Introduction of the plasmids pshRNA-survivin-1 and pshRNA-survivin-2 into Hela cells resulted in efficient and specific inhibition of survivin expression as demonstrated by immunofluorescence staining. Semi-quantitative RT-PCR showed that mRNA transcription of survivin gene was reduced. In contrast, the control plasmid did not exhibit any inhibitory effect on the protein expression and mRNA transcription of survivin gene. PI-Annexin V staining indicated an apoptosis rate of the transfected Hela cells of (36.02-/+2.12)% (P<0.01) and (35.29-/+2.02)% (P<0.01), respectively. CONCLUSION: The prepared siRNA targeting survivin gene is capable of inducing marked inhibitions of survivin protein expression and RNA transcription and significant enhancement of apoptosis in Hela cells, which shed light on a new strategy in gene silence therapy targeting survivin.


Assuntos
Vetores Genéticos/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Apoptose , Citometria de Fluxo , Imunofluorescência , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Survivina , Transfecção
11.
Protein Expr Purif ; 42(2): 278-85, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15935697

RESUMO

As HuGM-CSF and huIL-6 seem to have synergistic and complementary actions, researchers have proposed that fusion proteins incorporating these two cytokines could show increased biological activity, especially in terms of hematopoietic function. Here, we sought to obtain a functional GM-CSF/IL-6 fusion protein and to investigate its biological activities in vitro. A novel construct encoding a fusion protein of huGM-CSF (9-127) and IL-6 (29-184) was generated in the pBV220 expression vector by step-by-step cloning. Amino acids 1-8 of huGM-CSF and amino acids 1-28 of huIL-6 were deleted by PCR. The mutant huGM-CSF (9-127) and huIL-6 (29-184) cDNAs were linked via a linker sequence encoding 15 amino acid residues (G-G-S-G-S)3. Direct sequencing was used to confirm the validity of the desired construct, and the fusion protein was expressed in Escherichia coli host strain BL21 (DE3) in the form of inclusion bodies (IBs). The expression level was more than 25% of the total cell lysate, and a novel purification and refolding strategy was used to isolate the fusion protein product. Inclusion bodies were purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding by Sephacryl S-200. The renatured fusion proteins were obtained at a purity of >95%, and the strategy of refolding on the gel filtration column was found to be efficient, with a relative refolding rate of 80%. This entire refolding and purification procedure could be performed within one day and may prove applicable to large-scale purification and refolding of recombinant proteins from IBs in E. coli. This new method was used to obtain huGM-CSF (9-127)/IL-6 (29-184) fusion protein with high purity and biological activity. MTT assays in TF-1 and B9 cell lines showed that the specific biological activity of huGM-CSF was 1.14+/-0.10 x 10(8) U/mg, and that for huIL-6 was 1.89+/-0.11 x 10(7) U/mg. The fusion protein exhibited enhanced huGM-CSF, but similar huIL-6 biological activities compared with those of either GM-CSF or IL-6 alone. This suggests that our novel huGM-CSF (9-127)/IL-6 (29-184) fusion protein may hold future promise as a therapeutic agent.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Interleucina-6/biossíntese , Fragmentos de Peptídeos/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Bases , Escherichia coli , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Humanos , Interleucina-6/genética , Interleucina-6/isolamento & purificação , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação
12.
Di Yi Jun Yi Da Xue Xue Bao ; 24(8): 913-6, 2004 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-15321760

RESUMO

OBJECTIVE: To efficiently express and identify recombinant human survivin in E. coli. METHODS: Survivin cDNA was amplified by reverse transcriptional (RT)-PCR and cloned into the prokaryotic expression vector pBV220, followed by expression of the recombinant plasmid in E.coli strain BL21 (Gold). To obtain survivin protein, DEAE-Sepharose Fast-Flow ion exchange chromatography and Sephacryl S-200 gel filtration were performed. Western blot analysis was used for detecting the expressed product. RESULTS: Survivin protein was expressed in E.coli in the form of inclusion body at the expression level over 30% of the total cell protein. After ion exchange chromatography and gel filtration, the recombinant protein reached a purity over 95% and exhibited specific reaction with mouse anti-human antibody. CONCLUSION: Survivin protein with high purity can be obtained by the method described above to facilitate further study of the anti-apoptosis function of survivin.


Assuntos
Escherichia coli/metabolismo , Vetores Genéticos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/biossíntese , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Escherichia coli/genética , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Células Procarióticas/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Survivina
13.
Chin Med Sci J ; 19(2): 78-83, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15250239

RESUMO

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). METHODS: The 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 Pichia pastoris under 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 visualization. RESULTS: The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with 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 immunogenicity 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.


Assuntos
Antígenos de Hepatite/biossíntese , Antígenos de Superfície da Hepatite B/biossíntese , Vírus da Hepatite B/imunologia , Vírus da Hepatite E/imunologia , Pichia/genética , Epitopos , Engenharia Genética , Antígenos de Hepatite/genética , Antígenos de Hepatite/imunologia , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite E/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Vacinas Sintéticas
14.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 20(1): 104-8, 2004 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-15182634

RESUMO

AIM: To construct and express the gene encoding hIL-6/GM-CSF fusion protein. METHODS: The genes encoding the two hIL-6/GM-CSF fusion proteins were constructed in pBV220 expression vector. Fusion proteins were expressed in E.coli BL-21 and purified through Q Sepharose HP ion exchange chromatography and Sephacryl S-200 gel filtration columns. The biologic activities of the fusion proteins were detected by proliferation of hIL-6 dependent cell line B9 and hGM-CSF dependent cell line TF1 with MTT assay. RESULTS: Both fusion proteins were expressed in E.coli BL-21 in the form of inclusion body. The expression levels were more than 25% of the total cell lysates. Both fusion proteins were obtained with high purity which had both hIL-6 and hGM-CSF biologic activities. CONCLUSION: Two hIL-6/GM-CSF fusion proteins with high purity and bilolgic activities have been acquired successfully.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Interleucina-6/genética , Proteínas Recombinantes de Fusão/biossíntese , Western Blotting , Linhagem Celular , Escherichia coli/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-6/farmacologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia
15.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 25(3): 324-8, 2003 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-12905749

RESUMO

OBJECTIVE: To construct a replication-defective recombinant adenovirus expressing the ORF2 (112-660aa) antigen of hepatitis E virus (HEV) and evaluate its immunization effect in BALB/c mice by mucosal inoculation. METHODS: The HEV ORF2 gene encoding for 112-660aa was amplified from plasmid pUC-HEV and inserted into the transfer vector pTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E. coli strain BJ5183. Taking the advantage of the high efficient homologous recombination machinery presented in bacteria, the recombinant adenovirus backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant Adenoviruses were propagated in 293 cells with high titers. 8-week-old BALB/c mice were inoculated intraperitoneally and intranasally with 10(7) pfu recombinant adenovirus each on weeks 0, 3, 5, 7, 10. RESULTS: Both groups of mice induced humoral IgG immune response with the highest titers 1:1,000 and 1:10,000 each. Only the group inoculated intranasally could induce mucosal IgA immune response. CONCLUSIONS: The adenoviral recombinant can stimulate specific humoral and mucosal immune response in mice and is potentially to be used as a candidate vaccine for the treatment of HEV infection.


Assuntos
Adenovírus Humanos/genética , Antígenos de Hepatite/imunologia , Mucosa Nasal/imunologia , Proteínas Virais/imunologia , Animais , Antígenos de Hepatite/genética , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peritônio/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas contra Hepatite Viral , Proteínas Virais/biossíntese , Proteínas Virais/genética
16.
Sheng Wu Gong Cheng Xue Bao ; 18(3): 291-4, 2002 May.
Artigo em Chinês | MEDLINE | ID: mdl-12192859

RESUMO

The huGM-CSF(9-127)-IL-6(29-184) fusion protein was precipitated on column when being purified by Q Sepharose H.P. ion exchange chromatography after renaturation by dilution. To solve this problem, a novel purification and refolding strategy was adopted. Inclusion bodies was first purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding on column by Sephacryl S-200. Renatured fusion protein was obtained in a purity of more than 95%. It was showed that the method of refolding on gel filtration column is efficient, with relative refolding rate at 80%. By the whole procedure, refolding and purification of recombinant protein can be performed within one day. This strategy is also promising to be applied in large scale purification and refolding of recombinant protein from inclusion bodies in E. coli.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Interleucina-6/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Interleucina-6/química , Fragmentos de Peptídeos/química , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química
17.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 24(6): 592-5, 2002 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-12905686

RESUMO

OBJECTIVE: To observe anti-HEV IgG response to vaccination of recombinant antigen fragments and evaluate its protection from Hepatitis E Virus infection in rhesus monkeys (Macaca mulatta). METHODS: Twelve monkeys were divided into three groups and immunized respectively with three different recombinant antigens: namely Ag1 (carboxyl terminal 431 amino acids of ORF2), Ag2 (128aa fragment at the carboxyl terminal of ORF2), and Ag3 (full length ORF3 ligated with two ORF2 fragments encoded by 6743-7126nt and 6287-6404nt). The monkeys were challenged intravenously with fecal suspension from experimentally infected rhesus monkeys, and the other three monkeys served as the placebo group for challenge with HEV. The dynamic changes of the levels of ALT and anti-HEV IgG were examined. Pathological changes of liver tissue were observed by light microscope. Excretion of virus was detected by RT-nPCR. RESULTS: Hepatic histopathology of two monkeys in the placebo group was consistent with acute viral hepatitis, and ALT was elevated 3-4 weeks after inoculated with virus, up to 10-20 times higher than normal level. The liver tissue of monkeys immunized with antigen kept normal, ALT in several monkeys elevated mildly, and anti-HEV IgG conversation occurred at 1-2 weeks after vaccination, with the titer reaching 1:12,800. The virus RNA could be detected by RT-nPCR from days 7 to 50 in monkeys of control group, and from days 7 to 21 in vaccinated monkeys after challenged with virus. CONCLUSIONS: The recombinant antigens could induce the production of anti-HEV IgG, which protected rhesus monkeys from acute Hepatitis symptoms related to HEV infection.


Assuntos
Antígenos Virais/imunologia , Vírus da Hepatite E/imunologia , Hepatite E/prevenção & controle , Vacinas contra Hepatite Viral/imunologia , Animais , Imunoglobulina G/imunologia , Macaca mulatta , RNA Viral/sangue , Proteínas Recombinantes/imunologia , Vacinação
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