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1.
Vaccine ; 39(8): 1241-1247, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33516600

RESUMO

Without approved vaccines and specific treatments, COVID-19 is spreading around the world with above 26 million cases and approximately 864 thousand deaths until now. An efficacious and affordable vaccine is urgently needed. The Val308 - Gly548 of spike protein of SARS-CoV-2 linked with Gln830 - Glu843 of Tetanus toxoid (TT peptide) (designated as S1-4) and without TT peptide (designated as S1-5) were expressed and renatured. The antigenicity and immunogenicity of S1-4 were evaluated by Western Blotting (WB) in vitro and immune responses in mice, respectively. The protective efficiency was measured preliminarily by microneutralization assay (MN50). The soluble S1-4 and S1-5 protein was prepared to high homogeneity and purity. Adjuvanted with Alum, S1-4 protein stimulated a strong antibody response in immunized mice and caused a major Th2-type cellular immunity supplemented with Th1-type immunity. Furthermore, the immunized sera could protect the Vero E6 cells from SARS-CoV-2 infection with neutralizing antibody titer 256. Recombinant SARS-CoV-2 RBD with a built in T helper epitope could stimulate both strong humoral immunity supplemented with cellular immunity in mice, demonstrating that it could be a promising subunit vaccine candidate.


Assuntos
Anticorpos Antivirais/imunologia , Epitopos de Linfócito T/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Glicoproteína da Espícula de Coronavírus/genética
2.
Vaccine ; 38(51): 8238-8246, 2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33187763

RESUMO

BACKGROUND: To analyze the epidemiological distribution of Hepatitis B virus (HBV) genotype in the mainland of China following the implementation of effective preventive measures. METHODS: Five hundred and seventeen HBsAg-positive subjects aged 1-29 years surveyed in the 2014 national HBV sero-survey in the mainland of China were enrolled in the study. The full-length HBV genome was obtained by PCR amplification and sequencing. The HBV genotype was determined by phylogenetic analysis. Combined with questionnaire information, HBV genotype distribution was analyzed. RESULTS: Of the 517 HBsAg-positive subjects, 369 (71.4%) were included in the analysis. HBV genotypes found were B (45.0%), C (36.6%), D (6.0%), C/D (9.8%), B/C (2.2%), and I (0.5%). Geographic differences in HBV genotype were significant for seven regions. Three serotypes were found: adw (47.2%), adr (35.5%), and ayw (17.3%). B2 (43.9%) and C2 (25.2%) were the two major subgenotypes. The predominant genotypes differed between the Han group and the other ethnic groups. No statistical differences in genotype distribution were found by gender, age group, or hepatitis B (HepB) vaccination history. CONCLUSION: The prevalence of HBV genotype B was higher than that of genotype C with subgenotypes B2 and C2 endemic in 1-29-year-olds in the mainland of China, after HBV prevalence has reduced significantly due to the implementation of preventive measures. HepB vaccination or other factors did not interfere with HBV genotype distribution. The surveillance of HBV genotype was essential for responding to the potential changes and impact on the preventive policies in the future.

3.
Vaccine ; 38(32): 5071-5075, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32513514

RESUMO

SARS-CoV-2 is the cause of the worldwide outbreak of COVID-19 that has been characterized as a pandemic by the WHO. Since the first report of COVID-19 on December 31, 2019, 179,111 cases were confirmed in 160 countries/regions with 7426 deaths as of March 17, 2020. However, there have been no vaccines approved in the world to date. In this study, we analyzed the biological characteristics of the SARS-CoV-2 Spike protein, Pro330-Leu650 (SARS-CoV-2-SPL), using biostatistical methods. SARS-CoV-2-SPL possesses a receptor-binding region (RBD) and important B (Ser438-Gln506, Thr553-Glu583, Gly404-Aps427, Thr345-Ala352, and Lys529-Lys535) and T (9 CD4 and 11 CD8 T cell antigenic determinants) cell epitopes. High homology in this region between SARS-CoV-2 and SARS-CoV amounted to 87.7%, after taking the biological similarity of the amino acids into account and eliminating the receptor-binding motif (RBM). The overall topology indicated that the complete structure of SARS-CoV-2-SPL was with RBM as the head, and RBD as the trunk and the tail region. SARS-CoV-2-SPL was found to have the potential to elicit effective B and T cell responses. Our findings may provide meaningful guidance for SARS-CoV-2 vaccine design.


Assuntos
Betacoronavirus/química , Desenho de Fármacos , Modelos Imunológicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/química , Vacinas Virais/imunologia , Sequência de Aminoácidos , Antígenos Virais/química , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Humanos , Modelos Moleculares , Pandemias/prevenção & controle , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Alinhamento de Sequência , Vacinas de Subunidades/química , Vacinas de Subunidades/imunologia
4.
Hum Vaccin Immunother ; 16(1): 16-20, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31339432

RESUMO

Background: To assess the long-term protection conferred by plasma-derived hepatitis B vaccine at 20-31y after primary immunization during infancy in Chinese rural community.Method: Participants born between 1986 and 1996, who received a full course of primary vaccination with plasma-derived hepatitis B vaccine and had no experience with booster vaccination were enrolled. An epidemiological investigation was performed, and blood samples were collected to detect hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), and hepatitis B core antibody (anti-HBc). The positive rate of HBsAg, anti-HBs, and anti-HBc were calculated to evaluate the long-term protection of the plasma-derived hepatitis B vaccine.Results: A total of 949 participants were enrolled in the final analysis. Six subjects were detected to be HBsAg-positive, resulting in a HBsAg carrier rate of 0.63% (6/949). A total of 468 (52.41%) participants maintained a level of anti-HBs antibody ≥10 mIU/mL, with a GMC of 112.20 mIU/mL (95%CI: 97.72 ~ 128.82 mIU/mL). A significant downtrend was observed in the anti-HBs positive rate (P < .001). The average anti-HBc positive rate was 5.90% (56/949), increased with prolongation of immunization (P < .001).Conclusions: The plasma-derived hepatitis B vaccine maintained satisfactory protection at 20-31 y after primary immunization. These results indicate that a booster dose is not necessary. Further studies on the immune memory induced by the plasma-derived hepatitis B vaccine are needed.

5.
Biomed Environ Sci ; 32(7): 531-540, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31331437

RESUMO

OBJECTIVE: To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine. METHODS: Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 µg/mouse) alone, or CTA1-DD (5 µg/mouse) alone, or H3N2 split vaccine (3 µg/mouse) plus CTA1-DD (5 µg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 µg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured. RESULTS: H3N2 split vaccine formulated with CTA1-DD could elicit higher IgM, IgG and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory IgA titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus. CONCLUSION: CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines.


Assuntos
Adjuvantes Imunológicos , Toxina da Cólera , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza , Proteínas Recombinantes de Fusão , Administração Intranasal , Animais , Feminino , Imunidade Humoral , Camundongos Endogâmicos BALB C , Mucosa Nasal/imunologia , Distribuição Aleatória
6.
BMC Infect Dis ; 19(1): 482, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31146699

RESUMO

BACKGROUND: To assess the immune persistence conferred by a Chinese hamster ovary (CHO)-derived hepatitis B vaccine (HepB) 17 to 20 years after primary immunization during early life. METHODS: Participants born between 1997 and 1999 who received a full course of primary vaccination with HepB (CHO) and who had no experience with booster vaccination were enrolled. Blood samples were required from each participant for measurement of hepatitis B surface antibody (anti-HBs), surface antigen and core antibody levels. For those who possessed an anti-HBs antibody < 10 mIU/mL, a single dose of HepB was administered, and 30 days later, serum specimens were collected to assess the booster effects. RESULTS: A total of 1352 participants were included in this study. Of these, 1007 (74.5%) participants could retain an anti-HBs antibody ≥10 mIU/mL, with a geometric mean concentration (GMC) of 57.4 mIU/mL. HBsAg was detected in six participants, resulting in a HBsAg carrier rate of 0.4% (6/1352). Of those participants with anti-HBs antibodies < 10 mIU/mL, after a challenge dose, 231 (93.1%) presented an anti-HBs antibody ≥10 mIU/mL, with a GMC of 368.7 mIU/mL. A significant increase in the anti-HBs positive rate (≥ 10 mIU/mL) after challenge was observed in participants with anti-HBs antibodies between 2.5 and 10 mIU/mL and participants boosted with HepB (CHO), rather than those with anti-HBs antibodies < 2.5 mIU/mL and those boosted with HepB (SC). CONCLUSION: Since satisfactory immune protection against HBV infection conferred by primary vaccination administered 17-20 years ago was demonstrated, there is currently no urgent need for booster immunization.


Assuntos
Anticorpos Anti-Hepatite B/sangue , Vacinas contra Hepatite B/administração & dosagem , Hepatite B/prevenção & controle , Imunização Secundária , Prevenção Primária , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/uso terapêutico , Adolescente , Adulto , Animais , Células CHO , Cricetinae , Cricetulus , Feminino , Seguimentos , Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Humanos , Recém-Nascido , Masculino , Prevenção Primária/métodos , Estudos Retrospectivos , Fatores de Tempo , Adulto Jovem
7.
Vaccine ; 36(41): 6053-6060, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30195490

RESUMO

Ebola virus (EBOV) disease (EVD) leads to lethal hemorrhagic fever with a case fatality rate as high as 90%, thus posing a serious global public health concern. However, while several vaccines based on the EBOV glycoprotein have been confirmed to be effective in animal experiments, no licensed vaccines or effective treatments have been approved since the first outbreak was reported in 1976. In this study, we prepared the extracellular domain of the EBOV GP protein (designated as N20) by prokaryotic expression and purification via chromatography. Using CTA1-DD (designated as H45) as a mucosal adjuvant, we evaluated the immunogenicity of N20 by intranasal administration and the associated protective efficacy against mouse-adapted EBOV challenge in mice. We found that intranasal vaccination with H45-adjuvanted N20 could stimulate humoral immunity, as supported by GP-specific IgG titers; Th1 cellular immunity, based on IgG subclasses and IFN-γ/IL-4 secreting cells; and mucosal immunity, based on the presence of anti-EBOV IgA in vaginal lavages. We also confirmed that the vaccine could completely protect mice against a lethal mouse-adapted EBOV (MA-EBOV) challenge with few side effects (based on weight loss). In comparison, mice that received N20 or H45 alone succumbed to lethal MA-EBOV challenge. Therefore, mucosal vaccination with H45-adjuvanted N20 represents a potential vaccine candidate for the prevention of EBOV in an effective, safe, and convenient manner.


Assuntos
Aminoácidos/imunologia , Vacinas contra Ebola/administração & dosagem , Vacinas contra Ebola/uso terapêutico , Ebolavirus/imunologia , Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Vacinação/métodos , Administração Intranasal , Animais , Feminino , Imunidade Celular/fisiologia , Imunidade Humoral/fisiologia , Camundongos , Camundongos Endogâmicos BALB C
8.
Diagn Microbiol Infect Dis ; 92(2): 112-117, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29941366

RESUMO

In this study, a real-time reverse transcription-polymerase chain reaction (real time RT-PCR) assay targeting 2 genetic segments was established to detect HDV RNA. Utilizing the World Health Organization International Standard for Hepatitis D Virus RNA, the lower limit of detection was 575 IU/mL, and the linearity of quantification ranged from 575,000 IU/mL to 575 IU/mL. 384 HBsAg-positive samples collected from China were tested by this method and HDV antibody detection. Eleven samples were positive for anti-HDV IgG which may persist after HDV resolution, 6 samples were HDV RNA positive, and 5 samples were positive for anti-HDV IgM. This assay showed more sensitivity than the detection of anti-HDV IgM. These data demonstrate that the real-time RT-PCR assay for HDV RNA could be implemented in the clinical detection of HDV infection in chronic HBV-infected patients in China.


Assuntos
Anticorpos Anti-Hepatite/imunologia , Hepatite D/diagnóstico , Vírus Delta da Hepatite/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adulto , China , Feminino , Genótipo , Hepatite D/virologia , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/imunologia , Humanos , Masculino , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Carga Viral
9.
Biomed Environ Sci ; 31(5): 343-350, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29866216

RESUMO

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 HBcAg 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, IgG 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 IgG subtypes were mainly IgG1 and IgG2b 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.


Assuntos
Enterovirus Humano A/genética , Infecções por Enterovirus/imunologia , Epitopos/imunologia , Imunidade Celular , Imunidade Humoral , Proteínas Recombinantes de Fusão/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/sangue , Infecções por Enterovirus/virologia , Epitopos/metabolismo , Escherichia coli/metabolismo , Feminino , Camundongos
10.
Biomed Environ Sci ; 29(11): 790-801, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27998385

RESUMO

OBJECTIVE: To determine the hepatitis B immunoprophylactic failure rate in infants born to hepatitis B virus (HBV) infected mothers and to characterize HBV genes. METHODS: HBV-serological testing was conducted for pregnant women and infants. The complete genomes of 30 HBV isolates were sequenced, and genetic characteristics were analyzed using MEGA 5 software. RESULTS: The immunoprophylactic failure rate for infants who had completed the scheduled hepatitis B vaccination program was 5.76% (32/556). High sequence homology (99.8%-100%) was observed in 8 of the 10 mother-infant pairs. We identified 19 subgenotype C2 strains, 9 subgenotype B2 strains, and 2 subgenotype C1 strains. Three serotypes were detected: adr (19/30), adw (9/30), and ayw (2/30). The frequency of amino acid mutation of the 'a' determinant region was 16.67% (5/30), including that of Q129H, F134Y, S136Y, and G145E. We detected 67 amino acid mutations in the basal core promoter, precore, and core regions of the genome. CONCLUSION: The immunoprophylactic failure rate in infants born to HBV-infected mothers is low in the regions of China examined during this study. Moreover, HBV mutation in the 'a' determinant region could not account for immunoprophylactic failure for all infants.


Assuntos
Vacinas contra Hepatite B/uso terapêutico , Vírus da Hepatite B/genética , Hepatite B/congênito , Adulto , Animais , Células CHO , China/epidemiologia , Cricetinae , Cricetulus , Feminino , Hepatite B/epidemiologia , Hepatite B/prevenção & controle , Hepatite B/transmissão , Humanos , Recém-Nascido , Transmissão Vertical de Doença Infecciosa , Mutação , Filogenia , Gravidez , Falha de Tratamento , Adulto Jovem
11.
Biomed Environ Sci ; 29(6): 417-23, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27470102

RESUMO

OBJECTIVE: Hepatitis Delt a Virus (HDV) antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose. METHODS: Reconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method. RESULTS: The expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified (98%) by immobilized metal ion affinity chromatography (IMAC) using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity (97% for IgM and 100% for IgG) and specificity (100% for IgG and IgM) for the detection of anti-HDV antibodies. CONCLUSION: Expression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy.


Assuntos
Ensaio de Imunoadsorção Enzimática , Hepatite D/diagnóstico , Vírus Delta da Hepatite/imunologia , Antígenos da Hepatite delta/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Hepatite D/imunologia , Hepatite D/virologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Int J Infect Dis ; 33: 156-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25625179

RESUMO

OBJECTIVES: Hepatitis A virus (HAV), transmitted mainly through the fecal-oral route, is one of the major causes of acute viral hepatitis worldwide. HAV is endemic in China. This study performed genetic and evolutionary analysis of HAV isolates circulated in the country. METHODS: Clinical samples were collected and HAV nucleotide and deduced amino acid sequences were analyzed. 70 representative sequences of HAV VP3-VP1-2A regions sampled from 1988 to 2014 were compared and characterized using the Bayesian Markov Chain Monte Carlo approach (BEAST software, Version1.7.5). RESULTS: All isolates from China in this study belonged to genotype I, with most of the samples clustering in subgenotype IA, while several unique amino acid variants were observed. The estimated mean substitution rate was 5.56×10(-4) substitutions / site / year, the time to the most recent common ancestor of genotype I isolates in China was calculated to be around 180 years ago. Skyline plots showed the incidence of HAV went down gradually from the mid-1990s. CONCLUSIONS: The evolution estimations were consistent with the laboratory and epidemiological results. Several isolates from China showed amino acid changes close to the immunodominant sites, which needs to be further analyzed. The study results have indicated the effectiveness of improving economic and sanitation levels together with HAV vaccination to control HAV-related infections in China.


Assuntos
Vírus da Hepatite A/genética , Sequência de Aminoácidos , Teorema de Bayes , China , Evolução Molecular , Genótipo , Vírus da Hepatite A/classificação , Vírus da Hepatite A/isolamento & purificação , Humanos , Filogenia , RNA Viral/química
13.
Bing Du Xue Bao ; 30(6): 605-13, 2014 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-25868273

RESUMO

The highly-pathogenic EV71 strain is the primary cause of mortality in hand-foot-and-mouth disease (HFMD) associated with neurological symptoms, for which no clinically effective drugs or vaccines exist. This study aimed to construct infectious cDNA clones of the EV71 highly-pathogenic strain and the cell-culture adapted strain using a reverse genetics approach. The genomic RNAs of EV71 parent strains were used as the templates for RT-PCR amplification, and then the PCR products that overlapped the full-length genome were connected into the pBR322 vector to produce infectious clones of pEV71 (HP) and pEV71 (CCA), respectively. The results showed that the HP strain propagated much more quickly than the CCA strain. The rescued viruses derived from the infectious clones not only maintained their consistency with their parent strains in terms of genomic sequences, but also retained their respective biological phenotypes. This research will contribute to our understanding of EV71 pathogenesis and the development of novel vaccines against HFMD.


Assuntos
Enterovirus Humano A/genética , Doença de Mão, Pé e Boca/virologia , Animais , Chlorocebus aethiops , DNA Complementar , Enterovirus Humano A/crescimento & desenvolvimento , Enterovirus Humano A/isolamento & purificação , Enterovirus Humano A/patogenicidade , Humanos , Filogenia , Células Vero , Cultura de Vírus
14.
Artigo em Chinês | MEDLINE | ID: mdl-24044208

RESUMO

OBJECTIVE: To express the recombinant D protein in prokaryotic expression system solubly and make preparation for producing D-carrier conjugate vaccine next step. METHODS: The hpd gene fragment removed of signal peptide from genomic DNA of Hib CMCC was inserted into pET43. 1a. The recombinant plasmid was transformed to competent E. coli BL21 (DE3) for expression under induction of IPTG. The expressed recombination protein was precipitated with ammonium sulfate, purified by DEAE anion exchange column chromatography and identified for reactogenicity by Western Blot. RESULTS: The expressed recombination protein, in a soluble form, constained about 50% of total somatic protein and showed specific reaction with the HIB antisera after preliminary purification. CONCLUSION: The D protein recombined expression plasmid was constructed successfully and expressed D protein in prokaryotic cells in a solube form.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Haemophilus influenzae tipo b/genética , Imunoglobulina D/genética , Lipoproteínas/genética , Western Blotting , Escherichia coli/genética , Plasmídeos , Proteínas Recombinantes/biossíntese , Solubilidade
15.
Bing Du Xue Bao ; 29(2): 106-11, 2013 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-23757838

RESUMO

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.


Assuntos
Infecções por Coronavirus/virologia , Coronavirus Humano NL63/genética , Escherichia coli/genética , Proteínas do Envelope Viral/genética , Animais , Infecções por Coronavirus/metabolismo , Coronavirus Humano NL63/química , Coronavirus Humano NL63/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Engenharia de Proteínas , Estrutura Terciária de Proteína , Receptores Virais/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo
16.
Zhonghua Liu Xing Bing Xue Za Zhi ; 34(3): 214-7, 2013 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-23759223

RESUMO

OBJECTIVE: To investigate the risk of HBV infection among the spouses of hepatitis B virus surface antigen (HBsAg) carriers and to provide a reference for developing strategies on hepatitis B control and prevention. METHODS: A case-control study including HBsAg carriers aged 20 - 45 years-old from the nationwide sero-epidemiological survey for Hepatitis B in both Guangdong and Jiangxi provinces in 2006, together with their spouses were selected as case group, while. HBsAg negative persons and their spouses were among the control groups, under the same residential areas, gender, age and age of marriage to the HBsAg carriers. Questionnaire survey and hepatitis B serological markers detection were carried out, together with the HBV genotype detection among the HBsAg positive couples between husband and wife by PCR. RESULTS: Among the spouses of HBsAg carriers, the positive rate of HBsAg was 13.21%, while the rate was 6.29% for the spouse of HBsAg negative population, with difference statistically significant (χ² = 4.23, P < 0.05). HBsAg positive rate among spouses of the case group was higher than that in the control group. Among the spouses of HBsAg carriers, the HBsAg rate was positively correlated with the age of marriage, frequency of sexual intercourse and condom use. There were 21 pairs of HBsAg carriers between husband and wife, and HBV were isolated among 13 pairs, and there were 11 pairs carrying the same HBV genotype, accounting for 84.62%. HBV genotypes would include 8 pairs of type B and 3 pairs of type C. However, only 2 pairs were infected with different HBV genotype. CONCLUSION: High risks of HBV infection existed in the spouses of HBsAg carriers. It was important to ask the HBsAg carriers to take the initiative in informing their spouses, and carrying out the appropriate measures, such as safe sex or timely hepatitis B vaccination for the spouse of HBsAg carriers etc., so as to reduce the HBV transmission between husband and wife.


Assuntos
Portador Sadio , Hepatite B/epidemiologia , Cônjuges , Adulto , Portador Sadio/sangue , Portador Sadio/virologia , Estudos de Casos e Controles , Feminino , Genótipo , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/genética , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
17.
Artigo em Chinês | MEDLINE | ID: mdl-24645316

RESUMO

OBJECTIVE: To construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity. METHODS: PreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg (aa 1-144), between the 78th (Asp) and 79th (Pro). The genes synthesized after the codon optimization were ligated to the pET43. 1a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E. coli expression system. The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting. RESULTS: The morphology of the virus-like particles were confirmed by electron microscope. H2 were solid particles with a diameter of (31.61 +/- 1.27) nm, while H3 were hollow particles with a diameter of (28.46 +/- 1.16) nm. Statistical analysis showed that H2 is larger than H3 in the diameter (P < 0.01). The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting. CONCLUSION: Chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed, purified and identified, which lays the foundation for its application in vaccine research.


Assuntos
Epitopos/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Precursores de Proteínas/imunologia , Epitopos/química , Epitopos/genética , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/química , Vírus da Hepatite B/genética , Humanos , Testes de Neutralização , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia
18.
Artigo em Chinês | MEDLINE | ID: mdl-24579478

RESUMO

OBJECTIVE: To overexpress hepatitis B virus S gene in CHO cells cultured in serum-free media. METHOD: Plasmid was constructed by cloning of HBV S gene and then it was transfected into CHO cells. After cell screen, the positive clones were identified and isolated into a serum-free media followed by the serological and morphological characterization of the expression product. RESULT: CHO cell strains which can express HBsAg efficiently and stably were obtained. Spherical and filamentous HBsAg could be detected under electronic microscope. The titer of the expression product was up to 1:5000. CONCLUSION: Serum-free media cultured CHO cell strain for overexpression of HBsAg was successfully constructed and the expression product was high antigenic.


Assuntos
Expressão Gênica , Antígenos de Superfície da Hepatite B/genética , Animais , Células CHO , Cricetinae , Cricetulus , Antígenos de Superfície da Hepatite B/imunologia , Transfecção
19.
Bing Du Xue Bao ; 28(4): 324-9, 2012 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-22978154

RESUMO

A transient four-plasmid cotransfection system was used to construct avian influenza A (H5N1) pseudotyped viral particle (H5N1Pp) by incorporating hemagglutinin (HA) protein and neuraminidase (NA) protein from H5N1 avian influenza virus onto Murine leukemia virus pseudotyped viral particles, the transmission electron microscopy, infectivity titer assay, hemagglutination assay, neutralization assay of H5N1Pp were studied. We established a pseudotyped H5N1 viral particle at a high titer of 10(8) Pp/mL, the morphology,the hemagglutination activity and neutralization specificity of H5N1Pp is simililar to wild H5N1 virus. The research result sets a platform for studying this virus, including its receptors, the functional analysis of HA and NA, neutralizing antibodies and anti-H5N1 drug development.


Assuntos
Engenharia Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Aviária/virologia , Carga Viral/genética , Vírion/genética , Animais , Aves , Cricetinae , Células HEK293 , Hemaglutinação , Humanos , Testes de Neutralização , Transfecção
20.
Artigo em Chinês | MEDLINE | ID: mdl-23002540

RESUMO

OBJECTIVE: To prepare HDAg with biological activities as a candidate of diagnostic reagent. METHODS: To synthesize HDV gene fragment after codon optimization. To construct a thio-fused recombinant plasmid based on M48 expression vector. To express in E. coli induced by IPTG. To purify the protein by affinity chromatography followed by characterization in ELISA: RESULTS: Plasmid construction was verified by enzyme digestion. SDS-PAGE indicated the molecular weight of the protein was the same as we expectation. ELISA proved its affinity with HDV antibodies. CONCLUSION: HDAg was obtained successfully and it will pave the road to the research of HDV diagnostic reagent.


Assuntos
Hepatite D/diagnóstico , Antígenos da Hepatite delta/imunologia , Ensaio de Imunoadsorção Enzimática , Antígenos da Hepatite delta/genética , Antígenos da Hepatite delta/isolamento & purificação , Humanos , Plasmídeos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
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