BACKGROUND： Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeu...BACKGROUND： Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene. OBJECTIVE： To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN： Gene directed cloning. SETTING： Central Laboratory of Northern China Coal Medical College. MATERIALS： DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University. METHODS： NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid （pAAV-NT-3）. pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10：1 and the mixture was used to infect HT 1080 cells. X-gal stain was used to measure virus titer. MAIN OUTCOME MEASURES： Size of targeted gene fragments, validity of vehicle construction and virus titer. RESULTS： Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified. β-galactoside staining in situ indicated that LacZ genes were expressed in human fibrosar展开更多
Nuclear factor κB （NF-κB） overactivation, requiring phosphorylation and degradation of its inhibitor IκBα, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-Iκ...Nuclear factor κB （NF-κB） overactivation, requiring phosphorylation and degradation of its inhibitor IκBα, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IκBα, carrying an IκBα gene from human placenta, we optimized a novel IκBα mutant （IκBα） gene, constructed and characterized its replication-deficient recombinant adenovirus （AdIκBαM）, and tested whether AdIκBαM-mediated overexpression of IκBαM could inhibit the NF-κB activation in endothelial cells.展开更多
Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persist...Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persistent Epstein-Barr virus (EBV) infection.NPC vaccine studies have focused on enhancing specific antiviral CTL responses.In this study,three vaccines capable of expressing the EBV-latent membrane protein 2 (LMP2) (a DNA vector,an adeno-associated virus (AAV) vector,and a replication-defective adenovirus serotype 5 (Ad5) vector) were respectively used to immunize female Balb/c mice (4-6 weeks old) at weeks 0,2 and 4,either alone or in combination.Our results suggest that combined immunization with DNA,AAV,and adenovirus vector vaccines induced specific cellular immunity more effectively than any of these vectors alone or a combination of two of the three,constituting a sound vaccine strategy for the prevention and treatment of NPC.展开更多
BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment o...BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment of multidrug resistance gene 1 (mdr1) gene cDNA sequence. METHODS: The fragment of the mdr1 gene from the plasmid pHaMDRI-1 carrying the whole human mdr1 cDNA sequence was inserted reversely into the shuttle plasmid pAdTrack-CMV of adenoviral vector system AdEasy. The homologous recombination process was taken place in E. coli BJ5183 with the backbone plasmid pAdEasy-1. After packaging in 293 cells, recombinant adenoviral plasmid was generated. The recombinant adenoviral plasmid was identified by polymerase chain reaction (PCR), restriction endonucleases digest, DNA sequence analysis and fluorescence microscopic photograph, respectively. RESULTS: The recombinant adenovirus pAdEasy-GFPASmdr1 was successfully constructed and identified by PCR, restriction digest, and sequencing with strong green fluorescence expression in fluorescence microscopic photograph. CONCLUSIONS: The recombinant adenoviral mdr1 vector would introduce the antisense mdr1 gene into the human multidrug resistance hepatocellular cell fine effectively, which would provide an experimental basis to study the multidrug resistance in human hepatocellular carcinoma.展开更多
Rat calcineurin （CAN） A a isoform （Ppp3ca） cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was...Rat calcineurin （CAN） A a isoform （Ppp3ca） cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rht, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG- FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment （3.97 kb） obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect （CPE） appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A a （Ppp3ca） cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.展开更多
Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these...Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus （PRRSV） and the E2 protein of classical swine fever virus （CSFV）. Foot-and-mouth disease virus （FMDV） 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1：128 and a PRRSV-neutralizing antibody titer of 1：16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.展开更多
文摘BACKGROUND： Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene. OBJECTIVE： To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN： Gene directed cloning. SETTING： Central Laboratory of Northern China Coal Medical College. MATERIALS： DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University. METHODS： NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid （pAAV-NT-3）. pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10：1 and the mixture was used to infect HT 1080 cells. X-gal stain was used to measure virus titer. MAIN OUTCOME MEASURES： Size of targeted gene fragments, validity of vehicle construction and virus titer. RESULTS： Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified. β-galactoside staining in situ indicated that LacZ genes were expressed in human fibrosar
基金This study was supported by grants from the National Youth NaturalScience Foundation of China(No.30400191)and the Key Subjectof’135’Project of Jiangsu Province(No.20013102).
文摘Nuclear factor κB （NF-κB） overactivation, requiring phosphorylation and degradation of its inhibitor IκBα, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IκBα, carrying an IκBα gene from human placenta, we optimized a novel IκBα mutant （IκBα） gene, constructed and characterized its replication-deficient recombinant adenovirus （AdIκBαM）, and tested whether AdIκBαM-mediated overexpression of IκBαM could inhibit the NF-κB activation in endothelial cells.
基金supported by the National High Technology Research and Development Program of China(Grant No. 2006AA02A229)
文摘Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persistent Epstein-Barr virus (EBV) infection.NPC vaccine studies have focused on enhancing specific antiviral CTL responses.In this study,three vaccines capable of expressing the EBV-latent membrane protein 2 (LMP2) (a DNA vector,an adeno-associated virus (AAV) vector,and a replication-defective adenovirus serotype 5 (Ad5) vector) were respectively used to immunize female Balb/c mice (4-6 weeks old) at weeks 0,2 and 4,either alone or in combination.Our results suggest that combined immunization with DNA,AAV,and adenovirus vector vaccines induced specific cellular immunity more effectively than any of these vectors alone or a combination of two of the three,constituting a sound vaccine strategy for the prevention and treatment of NPC.
文摘BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment of multidrug resistance gene 1 (mdr1) gene cDNA sequence. METHODS: The fragment of the mdr1 gene from the plasmid pHaMDRI-1 carrying the whole human mdr1 cDNA sequence was inserted reversely into the shuttle plasmid pAdTrack-CMV of adenoviral vector system AdEasy. The homologous recombination process was taken place in E. coli BJ5183 with the backbone plasmid pAdEasy-1. After packaging in 293 cells, recombinant adenoviral plasmid was generated. The recombinant adenoviral plasmid was identified by polymerase chain reaction (PCR), restriction endonucleases digest, DNA sequence analysis and fluorescence microscopic photograph, respectively. RESULTS: The recombinant adenovirus pAdEasy-GFPASmdr1 was successfully constructed and identified by PCR, restriction digest, and sequencing with strong green fluorescence expression in fluorescence microscopic photograph. CONCLUSIONS: The recombinant adenoviral mdr1 vector would introduce the antisense mdr1 gene into the human multidrug resistance hepatocellular cell fine effectively, which would provide an experimental basis to study the multidrug resistance in human hepatocellular carcinoma.
文摘Rat calcineurin （CAN） A a isoform （Ppp3ca） cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rht, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG- FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment （3.97 kb） obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect （CPE） appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A a （Ppp3ca） cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.
文摘Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus （PRRSV） and the E2 protein of classical swine fever virus （CSFV）. Foot-and-mouth disease virus （FMDV） 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1：128 and a PRRSV-neutralizing antibody titer of 1：16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.
