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Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene 被引量:10
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作者 Chunling Ma Kun Yao +1 位作者 Feng Zhou Minsheng Zhu 《Cellular & Molecular Immunology》 SCIE CAS CSCD 2006年第6期459-465,共7页
In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus (SARS-CoV)-N gene recombinant replication-defective adenovir... In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus (SARS-CoV)-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ (IFN-γ) secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd-N was significantly stronger than that of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific (IFN-γ) secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirns boosting could be used as a potential SARS-CoV vaccine. 展开更多
关键词 SARS-COV DNA vaccine nucleocapsid protein adenovirus vector
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Construction of Recombinant Adenovirus Vector Containing CEVB2L Gene 被引量:2
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作者 邵洪泽 毛文智 +4 位作者 宋阳 李琳 程荣华 孙健 孙强 《Agricultural Science & Technology》 CAS 2010年第3期94-97,共4页
[Objective] Sheep contagious ecthyma virus B2L gene recombinant adenovirus was built by adenovirus vector system.[Method] Genome DNA extracted from sheep contagious ecthyma virus strain JLSY04 as a template,Gene fragm... [Objective] Sheep contagious ecthyma virus B2L gene recombinant adenovirus was built by adenovirus vector system.[Method] Genome DNA extracted from sheep contagious ecthyma virus strain JLSY04 as a template,Gene fragments obtained from B2L by PCR amplification;B2L gene cloning was cloned into PDNR-CMD vector,screening positive clones and plasmid CTC572-6 was obtained;CTC572-6 plasmid for homologous was recombined with the adenoviral vector.Screening positive clones and bacilli PCR,digestion and sequencing and so on were identified.[Result] After identified by enzyme digestion and gene sequencing,recombinant adenovirus vector CTC572Ade-30 of carrying sheep contagious ecthyma virus B2L gene was constructed successfully.[Conclusion] Which laid the foundation for sheep contagious ecthyma genetically engineered vaccine. 展开更多
关键词 CEV B2L gene adenovirus vector
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Recombinant adenovirus vector Ad-hIL-10 protects grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats 被引量:11
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作者 Si, Zhong-Zhou Li, Jie-Qun +3 位作者 Qi, Hai-Zhi He, Zhi-Jun Hu, Wei Li, Yi-Ning 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2010年第2期144-148,共5页
BACKGROUND: Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of ade... BACKGROUND: Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of adenovirus-mediated human IL-10 (Ad-hIL-10) gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats. METHODS: Adenoviruses encoding hIL-10 or beta-galactosidase (Ad-lacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48 hours after transduction, and stored for 12 hours at 4 degrees C in lactated Ringer's solution prior to transplantation. The rats were divided into saline, Ad-lacZ, and Ad-hIL-10 groups. Liver function test, histopathological examination, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were performed at 24 hours after transplantation in the three groups. RESULTS: Liver function (ALT and AST) was significantly improved, and the Suzuki score was significantly decreased in the Ad-hIL-10 group. The levels of hepatic TNF-alpha, MIP-2, ICAM-1 mRNA, and NF-kappa B protein in the Ad-hIL-10 group were significantly decreased. The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ. CONCLUSIONS: Donor pretreatment with Ad-hIL-10 down-regulates the expression of proinflammatory cytokines TNF-alpha, MIP-2, and ICAM-1 mRNA. hIL-10 protects against hepatic cold ischemia-reperfusion injury, at least in part, by suppressing NF-kappa B activation and subsequent expression of proinflammatory mediators. (Hepatobiliary Pancreat Dis Int 2010; 9: 144-148) 展开更多
关键词 adenovirus vector interleukin 10 ischemia-reperfusion injury gene transfer
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Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B 被引量:6
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作者 Xue, Xin-Bo Chen, Kun +4 位作者 Wang, Cong-Jun Zheng, Jian-Wei Yu, Yuan Peng, Zhi-Hai Wu, Zai-De 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2008年第5期509-514,共6页
BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of ... BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modified Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was confirmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and flow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. RESULT S: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P<0.01). In addition, cell block in G2/M was identified by exposure of HCC cells to secreted MDA-7 protein, but this was not found in L-02. The gene expression of Bcl-2 was markedly decreased in Hep3B but not in L-02. CONCLUSIONS: mda-7 selectively induces growth inhibition and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the antiapoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC. 展开更多
关键词 replication-incompetent adenovirus vector melanoma differentiation associated-7 gene carcinoma hepatocellular Bcl-2
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Immunological tolerance of human hepatocyte xenograft induced by adenovirus vector-mediated CTLA4Ig gene transfer 被引量:2
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作者 Yao-Kai Chen,Xiao-Cong Liu,Jun-Gang Li,Guo-Dong Liu,Yan Guo,Ling Cheng and Yu-Ming Wang Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing 400038, China and Department of Digestive Diseases, General Hospital of Chengdu Military Command, Chengdu 610083, China 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2012年第2期148-153,共6页
BACKGROUND:Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants,but has potential for systemic immune inhibition.This study was designed to induce hepatocyte i... BACKGROUND:Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants,but has potential for systemic immune inhibition.This study was designed to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation.METHODS:A normal human liver cell line(L02) was transfected with adenovirus vector containing the CTLA4Ig gene(Ad-CTLA4Ig-EGFP) in vitro,and the expression of CTLA4Ig by transfected cells was assessed by fluorescent imaging and immunocytochemical staining.Transfected cells then were injected into the spleen of Sprague-Dawley rats,the survival of cells was determined by immunohistochemistry,and the immune status was examined through CD4 + and CD69 + T cellcounts and ELISA detection of IL-2 in peripheral blood.RESULTS:L02 cells expressed CTLA4Ig in the cytoplasm for >4 weeks.Surviving L02 cells were observed in the experimental group at 3 and 4 weeks post-transplantation,while none was detected in the control group.Furthermore,the percentages of CD4 + and CD4 + CD69 + T cells in the CTLA4-transfected group were 24.5% and 45.1%,markedly lower than those in the control group at 4 weeks post-transplantation(P<0.01).Furthermore,the IL-2 level was also lower in the CTLA4transfected group than in the control group.CONCLUSION:Adenovirus-mediated CTLA4Ig gene transfer into human hepatocytes has the potential to become an effective method of inducing immunological tolerance in hepatocyte transplantation. 展开更多
关键词 CTLA4IG adenovirus vectors hepatocyte transplantation immune tolerance graft rejection
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In situ tumor vaccination with adenovirus vectors encoding measles virus fusogenic membrane proteins and cytokines 被引量:4
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作者 Dennis Hoffmann Wibke Bayer Oliver Wildner 《World Journal of Gastroenterology》 SCIE CAS CSCD 2007年第22期3063-3070,共8页
AIM: To evaluate whether intratumoral expression of measles virus fusogenic membrane glycoproteins H and F (MV-FMG), encoded by an adenovirus vector Ad.MV-HI F, alone or in combination with local coexpression of cy... AIM: To evaluate whether intratumoral expression of measles virus fusogenic membrane glycoproteins H and F (MV-FMG), encoded by an adenovirus vector Ad.MV-HI F, alone or in combination with local coexpression of cytokines (IL-2, IL-12, IL-18, IL-21 or GM-CSF), can serve as a platform for inducing tumor-specific immune responses in colon cancer.METHODS: We used confocal laser scanning microscopy and flow cytometry to analyze cell-cell fusion after expression of MV-FMG by dye colocalization. In a syngeneic bilateral subcutaneous MC38 and Colon26 colon cancer model in C57BL/6 and BALB/c mice, we assessed the effect on both the directly vector-treated tumor as well as the contralateral, not directly vector- treated tumor. We assessed the induction of a tumorspecific cytotoxic T lymphocyte (CTL) response with a lactate dehydrogenase (LDH) release assay.RESULTS: We demonstrated in vitro that transduction of MC38 and Colon26 cells with Ad.MV-H/F resulted in dye colocalization, indicative of cell-cell fusion, in addition, in the syngeneic bilateral tumor model we demonstrated a significant regression of the directly vector-inoculated tumor upon intratumoral expression of MV-FMG alone or in combination with the tested cytokines. We observed the highest anti-neoplastic efficacy with MV-FMG and lL-21 coexpression. The degree of tumor regression of the not directly vector-treated tumor correlated with the anti-neoplastic response of the directly vector-treated tumor. This regression was mediated by a tumor-specific CTL response.CONCLUSION: Our data indicate that intratumoral expression of measles virus fusogenic membrane glycoproteins is a promising tool both for direct tumor treatment as well as for tumor vaccination approaches that can be further enhanced by cytokine coexpression. 展开更多
关键词 adenovirus vectors Measles virus fusogenic membrane glycoproteins Colorectal cancer INTERLEUKINS
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Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology 被引量:1
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作者 Gui-Fang Wang Bing Qi +3 位作者 Lei-Lei Tu Lian Liu Guo-Cheng Yu Jing-Xiang Zhong 《International Journal of Ophthalmology(English edition)》 SCIE CAS 2016年第9期1271-1275,共5页
AIM: To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS: Gateway recombinant cloning technology was used to co... AIM: To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS: Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown- multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Desld yielding the final expression constructs pAV.Exld-CMV〉wt-lumican/IRES/ EGFP and pAV.Exld-cytomegalovirus (CMV) 〉mutlumican/IRES/EGFP, respectively.RESULTS: The adenovirus plasmids pAV.Exld-CMV〉 wt-lumican/IRES/EGFP and pAV.Exld-CMV 〉mutlumican/IRESlEGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION: We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumicangene in the pathogenesis of high myopia. 展开更多
关键词 MYOPIA LUMICAN adenovirus vector gateway recombinant cloning technology
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Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector 被引量:1
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作者 Ma, Chun-Ling Wang, Gui-Bin +1 位作者 Gu, Run-Guo Wang, Fang 《World Journal of Gastroenterology》 SCIE CAS CSCD 2010年第24期3078-3082,共5页
AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HB... AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy. 展开更多
关键词 CALRETICULIN Hepatitis B virus Hepatitis B surface antigen adenovirus expression vector Fusion protein Therapeutic vaccine
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Gene therapy for allergic rhinitis with recombinant adenovirus vector containing CTLA4Ig in mice 被引量:1
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作者 朱瑾 吴军 +3 位作者 陈希炜 易绍萱 罗高兴 贺伟峰 《Journal of Medical Colleges of PLA(China)》 CAS 2003年第1期15-18,共4页
Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and chall... Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and challenging with ovalbumin(OVA).Ad-CTLA4Ig was intraperitoneally injected 30 min before OVA challenge.Adenovirus vector without inserted CTLA4Ig cDNA served as the control.The symptoms and morphological changes of nasal mucosa of each group were observed, and the serum levels of IgE against OVA were detected with ELISA.Results: There were no obvious symptoms and pathological changes in Ad-CTLA4Ig treated group, in which the serum OVA-specific IgE levels were significantly lower than that in control groups(P< 0.05).Conclusion: Ad-CTLA4Ig prevents and treats allergic rhinitis of mice,implying the possibility of the usage of Ad-CTLA4Ig against allergic rhinitis in clinic in future. 展开更多
关键词 recombinant CTLA4Ig adenovirus vector allergic rhinitis IGE
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CONSTRUCTION OF THE DICISTRONIC ADENOVIRUS VECTOR EXPRESSING BIOACTIVE HUMAN INTERLEUKIN-12
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作者 章卫平 曹雪涛 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 1997年第4期67-71,共5页
The full-length cDNA encoding the subunits p40 and p35 of human interleukin12(hIL12) were cloned separately by RTPCR, linked together by internal ribosomal entry site (IRES) of encephalomyocarditis virus which initiat... The full-length cDNA encoding the subunits p40 and p35 of human interleukin12(hIL12) were cloned separately by RTPCR, linked together by internal ribosomal entry site (IRES) of encephalomyocarditis virus which initiates capindependent translation to form a dicistronic gene fragment. The dicistronic fragment was placed between the cytomegalovirus (CMV) promoter and SV40 polyA signal to form a dicistronic expression cassette. Subsequently, the dicistronic expression cassette was inserted into E1 region of Ad5 genome in cosmid vector pAx1cw of E1substitution type. By homologous recombination with EcoT22Idigested Ad5 DNATPC in 293 cells, the replicationdeficient recombinant adenoviruses of hIL12 were generated efficiently. After infected with hIL12 recombinant adenoviruses in vitro, 293 cells, human hepatocellular carcinoma cells HepG2, and primary human skin fibroblasts expressed and secreted hIL12 at comparable levels (30~60ng/ 106cells/24hr), which could stimulate the proliferation and IFNγ production of human lymphoblasts. These suggest that the dicistronic adenovirus vector of hIL12 could effectively mediate the expression of bioactive hIL12 and might be used in cancer gene therapy. 展开更多
关键词 Interleukin12 Dicistronic vector adenovirus vector Internal ribosomal entry site Geneexpression.
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Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector
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作者 陈庆永 王春友 +3 位作者 陈道达 陈剑英 蒋春舫 郑海 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2006年第3期325-328,共4页
The recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cD... The recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector Ad- PTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2. 5 × 10^10 pfu/mL, and about 70 % breast cancer cells were infected with Ad-PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer. 展开更多
关键词 adenovirus vector PTEN breast carcinoma homologeous recombination
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THE ROLE OF RECOMBINANT Rb GENE ADENOVIRUS VECTOR IN THE GROWTH OF LUNG ADENOCARCINOMA CELLS
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作者 黎健 蒋雷 +4 位作者 夏永静 李红霞 胡亚军 胡师学 徐洪基 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 1998年第4期7-10,共4页
Objective: To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene, on the growth of lung adenocarcinoma cell line GLC 82 and explore a gene therapy approach for lung adenoca... Objective: To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene, on the growth of lung adenocarcinoma cell line GLC 82 and explore a gene therapy approach for lung adenocarcinoma Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method Infection effects were detected by biochemical staining of β gal and immunohistochemical analysis of Rb protein The Rb cDNA of infected cells were determined by PCR The cell growth rate and cell cycle were observed by cell counting and flow cytometry Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein The transfection of wild type Rb gene could suppress GLC 82 cell proliferation and decrease the cellular DNA synthesis Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer 展开更多
关键词 adenovirus vector Retinoblastoma gene GLC 82 cell
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CONSTRUCTION OF A RECOMBINANT ADENOVIRUS VECTOR OF HUMAN PAPILLOMAVIRUS TYPE 16 L1_E7C
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作者 卞继峰 于修平 +8 位作者 王芸 赵蔚明 张丽华 董杰德 贾继辉 周亚滨 栾怡 齐眉 陈华波 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2000年第1期21-24,共4页
Objective: Human papillomaviruses are closely associated with human cervical cancer, especially HPV types 16 and 18. At present, HPV can not be produced in large quantity; it also has tumorgenicity and these propertie... Objective: Human papillomaviruses are closely associated with human cervical cancer, especially HPV types 16 and 18. At present, HPV can not be produced in large quantity; it also has tumorgenicity and these properties of HPV have seriously hampered the development of HPV vaccine. HPV type 16 LI proteins can assembled into virus-like particles (VLP), which are morphologically identical to the nature virion. In order to develop the recombinant adenovirus vectors of HPV, we constructed a recombinant adenovirus shuttle plasmid pCA14 L1-E7C. Methods: Human papillomavirus type 16 LI open reading frame without terminator codon (TAA) (5559–7152) and E7C (682–855) were amplified using PCR. The L1 and E7C fragments were inserted into pGEM-T easy vectors by T-A strategy, named pTAL1 and pTAE7C. pTAL1 was cut with Hind III and BgIII, the pTAE7C with BamHI and ClaI. The L1 DNA fragment, E7C and pBluesscript SK were ligated together using T4 DNA ligase. pBSL1-E7C and pBSL1-E7C was digested with Hind III and Xhol. The L1-E7C fragment was inserted into adenovirus shuttle plasmids pCA14, named pCA14L1-E7C. DNA sequence results indicated that The L1-E7C DNA fragment can encode the HPV16L1-E7 fusion protein correctly. Results: The L1 and E7C DNA fragments were amplified by PCR and recombinant plasmid pTAL1, pTAE7C, pBSL1-E7C and pCA14L1-E7C were constructed correctly. The pCA14L1-E7C can be used in the further research work, cotransfected the 293 cell with the parent adenovirus pBHG10. Conclusion: Our results indicated that we have constructed a HPV16L1-E7 fusion DNA fragments and the adenovirus shuttle plasmids pCAL1-E7C for the further research. 展开更多
关键词 Human papillomavirus adenovirus vector VACCINE
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Preparation and identification of monoclonal antibodies against the adenovirus vector
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作者 Chunyan Zhang Jinsong Gong Yan Chen Fanghe Li 《The Chinese-German Journal of Clinical Oncology》 CAS 2007年第4期411-414,共4页
Objective: To prepare and identity monoclonal antibodies (McAbs) against the capsid proteins of adenovirus vector. Methods: BALB/c mice were immunized with a mixture of the purified adenovirus vector (Adv) and A... Objective: To prepare and identity monoclonal antibodies (McAbs) against the capsid proteins of adenovirus vector. Methods: BALB/c mice were immunized with a mixture of the purified adenovirus vector (Adv) and AI(OH)3. McAbs were produced using cell fusion technique in a conventional way. The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay (ELISA), immunocytochemical staining and Western blotting. Results: Six strains of hybridoma cells (A4H11, A8C7, F1H5, G1D2, G4E3 and H2G8) that can stably secrete the IgG1 McAb against Adv were obtained. After 3 months subculture and low concentration of serum adapting culture, six strains retained their stability to secrete McAb. The ascites titers were between 1:10^6 and 1:10^8. Western blot analysis demonstrated that all the McAbs reacted with one protein (about 114 kDa) which is present in wild type 3 adenovirus (wtAd3), wild type 5 adenovirus (wtAd5), wild type 7 adenovirus (wtAd7) and adenovirus vector. Conclusion: Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector, and provided the substantial foundation of preclinical research of adenovirus vectors. 展开更多
关键词 gene therapy adenovirus adenovirus vector monoclonal antibodies
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Construction and expression of SET gene and siRNA recombinant adenovirus vectors
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作者 许波群 陆品红 +6 位作者 李瑛 薛凯 李梅 马翔 刁飞扬 崔毓桂 刘嘉茵 《生殖医学杂志》 CAS 2010年第A02期64-72,共9页
Objective:To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA(SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels. Methods:The cDNA sequence of SET w... Objective:To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA(SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels. Methods:The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction(RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET.The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells.The expression of SET in AD293 cells was detected by Western blot.In addition,we constructed SET gene SiRNA recombinant adenovirus vector(Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results:The recombinant adenovirus vectors,both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET,were proven to be constructed successfully by the evidence of endonulease digestion and sequencing.AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP.The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector.On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion:The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells.It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases. 展开更多
关键词 重组腺病毒载体 SIRNA 基因片段 逆转录聚合酶链反应 绿色荧光蛋白 巨细胞病毒 RNA干扰 293细胞
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Construction of the recombinant adenovirus vectors of CALB_2 gene and small interfering RNA,and application in testicular Leydig cells
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作者 罗建 王菁 +6 位作者 刘姗 孙雪萍 高超 高莉 杨晓玉 刘嘉茵 崔毓桂 《生殖医学杂志》 CAS 2011年第B12期57-65,共9页
Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in t... Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in testicular Leydig cells. Methods:The cDNA sequence of CALB_2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB_2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB_2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB_2.The recombinant AdCMV-CALB_2 was further packaged and amplificated in AD293 cells.The expression of CALB_2 protein in AD293 cells was detected by Western blotting.CALB_2 protein was over-expressed in mouse Leydig cell line(MLTC-1 cells) by the constructed AdCMV-CALB_2. CALB_2 gene siRNA recombinant adenovirus vector(Ad-H1-siRNA/CALB_2 was also constructed simultaneously. Its efficacy was detected in AD293 cells by Western blotting. Results:The CALB_2 gene recombinant adenovirus vector AdCMV-CALB_2 and the CALB_2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB_2 were constructed successfully by endonulease digestion and sequencing. AD293 cells infected with AdCMV-CALB_2 or Ad-H1-SiRNA/CALB_2 significantly expressed GFP protein. The expression of CALB_2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB_2 plasmids, while the expression of CALB_2 protein was down-regulated by 60%in the CALB_2 cells infected with Ad-H1-SiRNA/CALB_2. MLTC-1 cells did not markedly express CALB_2 protein,while MLTC-1 cells infected with AdCMV-CALB_2 expressed CALB_2 protein at a high level. Conclusions:The recombinant adenovirus vectors of AdCMV-CALB_2 and Ad-H1-SiRNA/CALB_2 were successfully constructed.Both vectors effectively expressed in AD293.CALB_2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB_2.These vectors of CALB_2 gene and Leydig cell line are useful tools for investigating the testicular function. 展开更多
关键词 重组腺病毒载体 睾丸间质细胞 小干扰RNA 基因片段 WESTERN印迹法 逆转录聚合酶链反应 siRNA 293细胞
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A quadrivalent norovirus vaccine based on a chimpanzee adenovirus vector induces potent immunity in mice
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作者 Yihua Jiang Lingjin Sun +6 位作者 Nan Qiao Xiang Wang Caihong Zhu Man Xing Hui Liu Ping Zhou Dongming Zhou 《Virologica Sinica》 SCIE CAS CSCD 2024年第4期675-684,共10页
Norovirus(NoV)infection is a major cause of gastroenteritis worldwide.The virus poses great challenges in developing vaccines with broad immune protection due to its genetic and antigenic diversity.To date,there are n... Norovirus(NoV)infection is a major cause of gastroenteritis worldwide.The virus poses great challenges in developing vaccines with broad immune protection due to its genetic and antigenic diversity.To date,there are no approved NoV vaccines for clinical use.Here,we aimed to develop a broad-acting quadrivalent NoV vaccine based on a chimpanzee adenovirus vector,AdC68,carrying the major capsid protein(VP1)of noroviral GI and GII genotypes.Compared to intramuscular(i.m.),intranasal(i.n.),or other prime-boost immunization regimens(i.m.t i.m.,i.m.t i.n.,i.n.t i.m.),AdC68-GI.1-GII.3(E1)-GII.4-GII.17(E3),administered via i.n.t i.n.induced higher titers of serum IgG antibodies and higher IgA antibodies in bronchoalveolar lavage fluid(BALF)and saliva against the four homologous VP1s in mice.It also significantly stimulated the production of blocking antibodies against the four genotypes.In response to re-stimulation with virus-like particles(VLP)-GI.1,VLP-GII.3,VLP-GII.4,and VLP-GII.17,the quadrivalent vaccine administered according to the i.n.t i.n.regimen effectively triggered specific cell-mediated immune responses,primarily characterized by IFN-γsecretion.Furthermore,the preparation of this novel quadrivalent NoV vaccine requires only a single recombinant adenovirus to provide broad preventive immunity against the major GI/GII epidemic strains,making it a promising vaccine candidate for further development. 展开更多
关键词 Norovirus(NoV) Quadrivalent vaccine adenovirus vector
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Inhibition of Metastatic Progression of SSTR2 Gene Transfection Mediated by Adenovirus in Human Pancreatic Carcinoma Cells 被引量:7
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作者 冯延平 黄涛 +2 位作者 高军 常青 秦仁义 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2006年第1期68-71,共4页
The inhibition of metastatic progression of Somatostatin receptor type 2 (SSTR2) gene transfection mediated by adenovirus in human pancreatic carcinoma cells and the mechanisms involved in this effect were studied. ... The inhibition of metastatic progression of Somatostatin receptor type 2 (SSTR2) gene transfection mediated by adenovirus in human pancreatic carcinoma cells and the mechanisms involved in this effect were studied. The full-length human SSTR2 cDNA was introduced into the pancreatic cancer cell line BXPC-3 by adenovirus-mediated transfection. Stable expression of mRNAs and protein of SSTR2 was detected by RT-PCR and Western-blot. The Matrigel-coated Transwell was used to detect the migratory and invasive ability of SSTR2-expressing cells, Adv-GFP control cells and mock control cells. Furthermore, the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) was detected by RT-PCR in these cells. The stable expression of SSTR2 was detected in BXPC-3 transfected by Adv-GFP-SSTR2. A dramatic decrease of BXPC-3 expressing sst2 cells migrating through a Matrigel-coated filter was observed, as compared with Adv-GFP control and mock control cells (P〈0. 01). Moreover, the expression of MMP-2 mRNA was significantly reduced in the SSTR2-expressing cells and converse- ly the expression of TIMP-2 mRNA was significantly increased in the SSTR2-expressing cells when compared with the Adv-GFP control and mock control (P〈0. 01). The expression of reintroduced human SSTR2 gene in BXPC-3 cells by Adv-GFP-SSTR2 had the anti-migratory and anti-invasive effects, and the mechanisms involved in this effect may be due to the down-regulated expression of MMP-2 and up-regulated expression of TIMP-2. 展开更多
关键词 pancreatic carcinoma adenovirus vector somatostatin receptor type 2 metalloproteinase-2 tissue inhibitor of metalloproteinase-2
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Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein 被引量:6
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作者 Yong FU Shen-qing WANG +3 位作者 Ying-peng LIU Guo-peng WANG Jian-ting WANG Shu-sheng GONG 《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 SCIE CAS CSCD 2008年第4期299-305,共7页
Objective: To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of ... Objective: To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP. Methods: The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. Results: After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. Conclusion: We have success- fully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies. 展开更多
关键词 Recombinant adenovirus vector Viral infection Fetal neural stem cells Green fluorescent protein
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Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor 被引量:3
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作者 Hongyu Yang Hong Qi +1 位作者 Junjie Zou Xi-wei Zhang 《Journal of Nanjing Medical University》 2008年第1期52-56,共5页
Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods: Rat VEGF was obtained by using RT-PCR... Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods: Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3 × 10^9 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a ufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches. 展开更多
关键词 vascular endothelial growth factor adenovirus vector genetic transfection
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