The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been...The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recom-binant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with α-32P labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cyto-toxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.展开更多
By employing the pUC19 as a backbone,the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19. The recombinant E. Coli-my...By employing the pUC19 as a backbone,the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19. The recombinant E. Coli-mycobacteria shuttle expression plasmid PBCG-8000 was constructed. The PBCG-8000 was able to replicate in both E. Coli and mycobacteria (including BCG) systems, and to confer stable kanamycin resistance upon transformants. The study should facilitate the development of BCG and other mycobacteria into multivalent vaccine vectors.展开更多
[Objective] pSCM201 is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea.For gene expression and function analysis of specific genes in model strain Haloarcula hispa...[Objective] pSCM201 is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea.For gene expression and function analysis of specific genes in model strain Haloarcula hispanica,a novel shuttle expression vector based on pSCM201 was developed.[Methods] By combining the minimal replicon of pSCM201,the pUC19 replicon of Escherichia coli,and two antibiotic genes,a new shuttle vector between haloarchaea and E.coli was established.Furthermore,the hsp5 core promoter,multiple clone sites and a C-terminal His·Tag sequence were added to construct the expression vector pSCM307.With the help of reporter gene bgaH,the replication ability and gene expression of pSCM307 were tested in H.hispanica AS2049 by PCR identification and β-galactosidase activity assay.[Results] pSCM307 could stably self-replicate in AS2049,and bgaH was successfully expressed under the control of hsp5 promoter.[Conclusions] A user-friendly expression vector was successfully constructed in haloarchaea.展开更多
基金Project supported by the National Natural Science Foundation of China (Grant No. 39280016).
文摘The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recom-binant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with α-32P labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cyto-toxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.
文摘By employing the pUC19 as a backbone,the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19. The recombinant E. Coli-mycobacteria shuttle expression plasmid PBCG-8000 was constructed. The PBCG-8000 was able to replicate in both E. Coli and mycobacteria (including BCG) systems, and to confer stable kanamycin resistance upon transformants. The study should facilitate the development of BCG and other mycobacteria into multivalent vaccine vectors.
文摘[Objective] pSCM201 is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea.For gene expression and function analysis of specific genes in model strain Haloarcula hispanica,a novel shuttle expression vector based on pSCM201 was developed.[Methods] By combining the minimal replicon of pSCM201,the pUC19 replicon of Escherichia coli,and two antibiotic genes,a new shuttle vector between haloarchaea and E.coli was established.Furthermore,the hsp5 core promoter,multiple clone sites and a C-terminal His·Tag sequence were added to construct the expression vector pSCM307.With the help of reporter gene bgaH,the replication ability and gene expression of pSCM307 were tested in H.hispanica AS2049 by PCR identification and β-galactosidase activity assay.[Results] pSCM307 could stably self-replicate in AS2049,and bgaH was successfully expressed under the control of hsp5 promoter.[Conclusions] A user-friendly expression vector was successfully constructed in haloarchaea.
