[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid ...[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid FG12-T7 RNAP plasmid was obtained via identification with double enzymes digestion and gene sequencing.The transient expressed T7 RNAP protein was determined by WB in 293T cells transfected with FG12-T7 RNAP plasmid.The recombinant FG12-RNAP lenti-virus was packaged up by transfecting the 293 cells with the recombinant vector FG12-RNAP and the helper plasmids via lipofectamine-2000,which was then used to infect BHK-21 cells.The positive cell clones were obtained after continuous screening by GFP.The expression of T7 RNAP gene was confirmed by Western blot.[Result]The cell line stably expressing the T7 RNAP gene was established by Western blot.[Conclusion]T7 RNAP gene could be stably expressed in eukaryotic cells and it provided a good platform to rescue RNA virus in vivo.展开更多
Chinese hamster ovary(CHO)cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications.However,toxic proteins and membrane proteins are often ...Chinese hamster ovary(CHO)cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications.However,toxic proteins and membrane proteins are often difficult-to-express in living cells.Alternatively,cell-free protein synthesis can be employed.This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both,cell-based and cell-free approaches.A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis.This resulted in elevated yields,while eliminating the necessity for exogenous additions during cell-free production,thereby substantially enhancing efficiency.Additionally,we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression.These findings provide promising advancements in bioproduction technologies,offering flexibility to switch between cell-free and cell-based protein production as needed.展开更多
A coupled expression system for plants was established in this study. The 5’-terminal of T7 RNA poly-merase gene was modified by addition of the coding sequence of nuclear location signal from SV40 large T antigen. P...A coupled expression system for plants was established in this study. The 5’-terminal of T7 RNA poly-merase gene was modified by addition of the coding sequence of nuclear location signal from SV40 large T antigen. Plant expression vector pBBT7 was constructed with the modified T7 RNA polymerase gene under the control of CaMV35S promoter. Another expression vector pBTG contained cassette of gusA controlled by T7 promoter. The two vectors were co-transformed into tobacco via the Agrobecte-rium -mediated method. Results of GUS activity indicated that the co-transformed plant with pBBT7 and pBTG showed a high level of GUS activity. The results demonstrated that the coupled expression system of T7 polymerase and T7 promoter was workable in plants.展开更多
Small non-coding RNAs(sRNAs)have received much attention in recent years due to their unique biological properties,which can efficiently and specifically tune target gene expressions in bacteria.Inspired by natural sR...Small non-coding RNAs(sRNAs)have received much attention in recent years due to their unique biological properties,which can efficiently and specifically tune target gene expressions in bacteria.Inspired by natural sRNAs,recent works have proposed the use of artificial sRNAs(asRNAs)as genetic tools to regulate desired gene that has been applied in several fields,such as metabolic engineering and bacterial physiology studies.However,the rational design of asRNAs is still a challenge.In this study,we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs.T7 expression system was one of the most useful recombinant protein expression systems.However,it was deeply limited by the formation of inclusion body.To settle this problem,we designed a series of asRNAs to inhibit the T7 RNA polymerase(Gene1)expression to balance the rate between transcription and folding of recombinant protein.Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E.coli,the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.展开更多
基金Supported by National Transgenic Major Program of China(2009ZX08007-006B)the National Natural Science Foundation of China(31072160)+2 种基金Science and Technique Foundation of Shandong Province(2009GG20002032)Natural Science Foundation of Shandong Province(Y2008D20)an Open Issue of State Key Laboratory of Veterinary Biotechnology Fund(SKLVBF200806)~~
文摘[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid FG12-T7 RNAP plasmid was obtained via identification with double enzymes digestion and gene sequencing.The transient expressed T7 RNAP protein was determined by WB in 293T cells transfected with FG12-T7 RNAP plasmid.The recombinant FG12-RNAP lenti-virus was packaged up by transfecting the 293 cells with the recombinant vector FG12-RNAP and the helper plasmids via lipofectamine-2000,which was then used to infect BHK-21 cells.The positive cell clones were obtained after continuous screening by GFP.The expression of T7 RNAP gene was confirmed by Western blot.[Result]The cell line stably expressing the T7 RNAP gene was established by Western blot.[Conclusion]T7 RNAP gene could be stably expressed in eukaryotic cells and it provided a good platform to rescue RNA virus in vivo.
基金supported by the European Regional Development Fund(EFRE)and the German Ministry of Education and Research(BMBF 031B0831C).
文摘Chinese hamster ovary(CHO)cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications.However,toxic proteins and membrane proteins are often difficult-to-express in living cells.Alternatively,cell-free protein synthesis can be employed.This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both,cell-based and cell-free approaches.A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis.This resulted in elevated yields,while eliminating the necessity for exogenous additions during cell-free production,thereby substantially enhancing efficiency.Additionally,we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression.These findings provide promising advancements in bioproduction technologies,offering flexibility to switch between cell-free and cell-based protein production as needed.
基金This work was supported by the National Natural Science Foundation of China (Grant Nos. 39989001, 39980024 and 39880023) the High Technology Research and Development Program of China (Grant Nos. 2001AA212041, 2001AA222251 and 101-06-01-06)the Nationa
文摘A coupled expression system for plants was established in this study. The 5’-terminal of T7 RNA poly-merase gene was modified by addition of the coding sequence of nuclear location signal from SV40 large T antigen. Plant expression vector pBBT7 was constructed with the modified T7 RNA polymerase gene under the control of CaMV35S promoter. Another expression vector pBTG contained cassette of gusA controlled by T7 promoter. The two vectors were co-transformed into tobacco via the Agrobecte-rium -mediated method. Results of GUS activity indicated that the co-transformed plant with pBBT7 and pBTG showed a high level of GUS activity. The results demonstrated that the coupled expression system of T7 polymerase and T7 promoter was workable in plants.
基金The author would like to acknowledge the National Science Fund for Distinguished Young Scholars(21425624)the National Natural Science Foundation of China(21476026,21376028).
文摘Small non-coding RNAs(sRNAs)have received much attention in recent years due to their unique biological properties,which can efficiently and specifically tune target gene expressions in bacteria.Inspired by natural sRNAs,recent works have proposed the use of artificial sRNAs(asRNAs)as genetic tools to regulate desired gene that has been applied in several fields,such as metabolic engineering and bacterial physiology studies.However,the rational design of asRNAs is still a challenge.In this study,we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs.T7 expression system was one of the most useful recombinant protein expression systems.However,it was deeply limited by the formation of inclusion body.To settle this problem,we designed a series of asRNAs to inhibit the T7 RNA polymerase(Gene1)expression to balance the rate between transcription and folding of recombinant protein.Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E.coli,the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.
