克隆新基因及获得其 c DNA全长是现代生命科学中研究基因功能的重要前提。随着分子生物学技术的迅速发展 ,克隆新基因的技术也不断得到发展和完善 ,尤其是生物技术与信息技术的结合和应用 ,计算机克隆基因开始兴起。本文介绍几种近年常...克隆新基因及获得其 c DNA全长是现代生命科学中研究基因功能的重要前提。随着分子生物学技术的迅速发展 ,克隆新基因的技术也不断得到发展和完善 ,尤其是生物技术与信息技术的结合和应用 ,计算机克隆基因开始兴起。本文介绍几种近年常用的克隆新基因 c DNA全长的方法。展开更多
Early diagnosis of leptospirosis of pulmonary diffuse hernorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific methed for diagnosis, a genomic library of the main pathoge...Early diagnosis of leptospirosis of pulmonary diffuse hernorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific methed for diagnosis, a genomic library of the main pathogen of PDH, L. interrogans serovar lai strain 017, was constructed with the plasmid vector PUC9. Recombinant plasmids which have hornologous fragments of pathogenic leptospires were screened from the bank. A recombinant plasmid,designated PCX7, could detect 1.7 kb fragment of strain 017, 9. 0 kb of strain 601 and 30. 0 kb of strain Hebdomadis, respectively, without cross hybridization with nonpathogenic leptospires such as L. biflexa strain Patoc I and hoptonema illini. The recombinant plasmid PCX7 could detect pathogenic leptospires which are the main pathogens endemic to Sichuan Province.展开更多
Plasmid DNA, an effective vaccine vector, can induce both cellular and humoral immune responses. However, plasmid DNA raises issues concerning potential genomic integration after injection. This issue should be consid...Plasmid DNA, an effective vaccine vector, can induce both cellular and humoral immune responses. However, plasmid DNA raises issues concerning potential genomic integration after injection. This issue should be considered in preclinical studies. Tiantan vaccinia virus (TV) has been most widely utilized in eradicating smallpox in China. This virus has also been considered as a successful vaccine vector against a few infectious diseases. Potent T cell responses through T-cell receptor (TCR) could be induced by three injections of the DNA prime vaccine followed by a single injection of recombinant vaccinia vaccine. To develop a safer immunization strategy, a single DNA prime followed by a single recombinant Tiantan vaccinia (rTV) AIDS vaccine was used to immunize mice. Our data demonstrated that one DNA prime/rTV boost regimen induced mature TCR activation with high functional avidity, preferential T cell Vβ receptor usage and high sensitivity to anti-CD3 antibody stimulation. No differences in T cell responses were observed among one, two or three DNA prime/rTV boost regimens. This study shows that one DNA prime/rTV boost regimen is sufficient to induce potent T cell responses against HIV.展开更多
To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3...To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region. The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyle-neimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis. The results showed that expression of pEGFP-Nl was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the β-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.展开更多
随着蛋白质融合技术的蓬勃发展,蛋白标签(proteintag)技术迅速兴起,其利用DNA体外重组技术(DNA in vitro recombination Technology),与目的蛋白融合,共同表达的一种多肽或者蛋白质。随着技术的不断发展,研究人员相继开发出了诸如TrxHis...随着蛋白质融合技术的蓬勃发展,蛋白标签(proteintag)技术迅速兴起,其利用DNA体外重组技术(DNA in vitro recombination Technology),与目的蛋白融合,共同表达的一种多肽或者蛋白质。随着技术的不断发展,研究人员相继开发出了诸如TrxHis、Flag、MBP、GST等具有各种不同功能的蛋白标签,其对于目的蛋白的表达、检测、示踪和纯化等有着广泛的应用前景。本文将介绍TrxHis、Flag、GST、GFP四种常见蛋白质标签,并进行一定的总结与展望。展开更多
Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene ...Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.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
Meiosis comprises two rounds of nuclear division following a single phase of DNA replication, leading to the production of haploid gametes and is essential for sexual reproduction in eukaryotes. Unlike mitosis, meiosi...Meiosis comprises two rounds of nuclear division following a single phase of DNA replication, leading to the production of haploid gametes and is essential for sexual reproduction in eukaryotes. Unlike mitosis, meiosis involves homologous chromosome pairing, synapsis, and recombination during prophase I. Meiotic recombination not only ensures the accurate segregation of homologs, but also redistributes alleles among offspring. DNA synthesis is a critical process during meiotic recombination, but our understanding of the proteins that execute and regulate it is limited. This review summarizes the recent advances in defining the role of DNA synthesis in meiotic recombina- tion through analyses of DNA synthesis genes, with specific emphasis on DNA polymerases （e.g., Pole and PolS）, replication processivity factor RFC1 and translesion polymerases （e.g., Pol~）. We also present a new double strand break repair model for meiotic recombination, which includes lagging strand DNA synthesis and leading strand elongation. Finally, we propose that DNA synthesis is one of critical factors for discriminating meiotic recombination pathways and that this differentiation may be conserved among eukaryotes.展开更多
文摘Early diagnosis of leptospirosis of pulmonary diffuse hernorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific methed for diagnosis, a genomic library of the main pathogen of PDH, L. interrogans serovar lai strain 017, was constructed with the plasmid vector PUC9. Recombinant plasmids which have hornologous fragments of pathogenic leptospires were screened from the bank. A recombinant plasmid,designated PCX7, could detect 1.7 kb fragment of strain 017, 9. 0 kb of strain 601 and 30. 0 kb of strain Hebdomadis, respectively, without cross hybridization with nonpathogenic leptospires such as L. biflexa strain Patoc I and hoptonema illini. The recombinant plasmid PCX7 could detect pathogenic leptospires which are the main pathogens endemic to Sichuan Province.
基金supported by the China Comprehensive Integrated Programs for Research on AIDS(CIPRA, U19AI51915)by the National Key Projects on Major Infectious Diseases (Grant No. 2008ZX10001-010,2012ZX10001-008)by the National Natural Science Foundation of China (31000413)
文摘Plasmid DNA, an effective vaccine vector, can induce both cellular and humoral immune responses. However, plasmid DNA raises issues concerning potential genomic integration after injection. This issue should be considered in preclinical studies. Tiantan vaccinia virus (TV) has been most widely utilized in eradicating smallpox in China. This virus has also been considered as a successful vaccine vector against a few infectious diseases. Potent T cell responses through T-cell receptor (TCR) could be induced by three injections of the DNA prime vaccine followed by a single injection of recombinant vaccinia vaccine. To develop a safer immunization strategy, a single DNA prime followed by a single recombinant Tiantan vaccinia (rTV) AIDS vaccine was used to immunize mice. Our data demonstrated that one DNA prime/rTV boost regimen induced mature TCR activation with high functional avidity, preferential T cell Vβ receptor usage and high sensitivity to anti-CD3 antibody stimulation. No differences in T cell responses were observed among one, two or three DNA prime/rTV boost regimens. This study shows that one DNA prime/rTV boost regimen is sufficient to induce potent T cell responses against HIV.
文摘To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region. The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyle-neimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis. The results showed that expression of pEGFP-Nl was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the β-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.
文摘随着蛋白质融合技术的蓬勃发展,蛋白标签(proteintag)技术迅速兴起,其利用DNA体外重组技术(DNA in vitro recombination Technology),与目的蛋白融合,共同表达的一种多肽或者蛋白质。随着技术的不断发展,研究人员相继开发出了诸如TrxHis、Flag、MBP、GST等具有各种不同功能的蛋白标签,其对于目的蛋白的表达、检测、示踪和纯化等有着广泛的应用前景。本文将介绍TrxHis、Flag、GST、GFP四种常见蛋白质标签,并进行一定的总结与展望。
文摘Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.
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.
基金Acknowledgments We apologize to colleagues whose work could not be cited owing to space constraints. J.H., H.M. and Y.W. are supported by the Ministry of Science and Technology of China （2011CB944603）, the National Natural Science Foundation of China （31370347）, and by funds from Fudan University and Rijk Zwaan. G.P.C. is supported by the US National Science Foundation （MCB- 1121563） and Rijk Zwaan.
文摘Meiosis comprises two rounds of nuclear division following a single phase of DNA replication, leading to the production of haploid gametes and is essential for sexual reproduction in eukaryotes. Unlike mitosis, meiosis involves homologous chromosome pairing, synapsis, and recombination during prophase I. Meiotic recombination not only ensures the accurate segregation of homologs, but also redistributes alleles among offspring. DNA synthesis is a critical process during meiotic recombination, but our understanding of the proteins that execute and regulate it is limited. This review summarizes the recent advances in defining the role of DNA synthesis in meiotic recombina- tion through analyses of DNA synthesis genes, with specific emphasis on DNA polymerases （e.g., Pole and PolS）, replication processivity factor RFC1 and translesion polymerases （e.g., Pol~）. We also present a new double strand break repair model for meiotic recombination, which includes lagging strand DNA synthesis and leading strand elongation. Finally, we propose that DNA synthesis is one of critical factors for discriminating meiotic recombination pathways and that this differentiation may be conserved among eukaryotes.
