Since its discovery in the 1980s,the insect cell-baculovirus expression vector system(IC-BEVS)has been widely used in biomedical applications,such as recombinant protein expression,drug screening,vaccine development,g...Since its discovery in the 1980s,the insect cell-baculovirus expression vector system(IC-BEVS)has been widely used in biomedical applications,such as recombinant protein expression,drug screening,vaccine development,gene therapy and so on[1].As a eukaryotic system,IC-BEVS has great development prospects due to its advantages such as high safety,simple operation,simultaneous expression of multi-subunit proteins,and suitability for large-scale cultivation[2].展开更多
In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-39...In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.展开更多
[Objective]Foxtail mosaic virus(FoMV)infects gramineous and dicotyledonous plants.In this study,we sought to construct a viral vector based on FoMV to express exogenous proteins in plants.[Method]A recombinant viral e...[Objective]Foxtail mosaic virus(FoMV)infects gramineous and dicotyledonous plants.In this study,we sought to construct a viral vector based on FoMV to express exogenous proteins in plants.[Method]A recombinant viral expression vector was constructed by inserting the promotor of Potato virus X(PVX)and exogenous gene sequences into the 3’non-coding region of the FoMV coat protein gene.[Results]The plasmid pCB301-FoMV-CP-PVXprom-GFP expressed green fluorescent protein in inoculated Nicotiana benthamiana leaves.[Conclusion]A recombinant viral expression vector was constructed successfully.展开更多
Insect baculovirus(IBV) is a biological insecticide in agriculture and forestry, butits slow speed of killing pests and narrow range of host make its application quitelimited. Hence, polyhedrin (ocu) gene of IBV was c...Insect baculovirus(IBV) is a biological insecticide in agriculture and forestry, butits slow speed of killing pests and narrow range of host make its application quitelimited. Hence, polyhedrin (ocu) gene of IBV was chosen for construction of展开更多
The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here,we establi...The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here,we established a simple method to construct a milk expression vector, by a combination of homologous recombination and digestion-ligation. Our methodology is expected to have the advantages of both plasmid and bacterial artificial chromosome(BAC) vectors. The BAC of mouse whey acidic protein gene(mWAP) was modified twice by homologous recombination to produce a universal expression vector, and the human lysozyme gene(hLZ) was then inserted into the vector by a digestionligation method. The final vector containing the 8.5 kb mWAP 5′ promoter, 4.8 kb h LZ genomic DNA, and 8.0 kb m WAP 3′ genomic DNA was microinjected into pronuclei of fertilized mouse embryos, to successfully generate two transgenic mouse lines that expressed recombinant human lysozyme(rhLZ) in milk. The highest expression level of rhLZ was 0.45 g$L–1, and rhLZ exhibited the same antibacterial activity as native h LZ. Our results have provided a simple approach to construct a universal milk expression vector, and demonstrated that the resulting vector regulates the expression of hLZ in milk.展开更多
文摘Since its discovery in the 1980s,the insect cell-baculovirus expression vector system(IC-BEVS)has been widely used in biomedical applications,such as recombinant protein expression,drug screening,vaccine development,gene therapy and so on[1].As a eukaryotic system,IC-BEVS has great development prospects due to its advantages such as high safety,simple operation,simultaneous expression of multi-subunit proteins,and suitability for large-scale cultivation[2].
基金Shanghai Medical Development grant No. ZD99001 and aGrant (SFB-542) from the Deutsche Forschungsgemeinschaft.
文摘In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.
基金Supported by Key Laboratory Open Foundation Project of Hunan Education Department(18K100)Graduate Research Innovation Project of Hunan Province(CX2018B800)~~
文摘[Objective]Foxtail mosaic virus(FoMV)infects gramineous and dicotyledonous plants.In this study,we sought to construct a viral vector based on FoMV to express exogenous proteins in plants.[Method]A recombinant viral expression vector was constructed by inserting the promotor of Potato virus X(PVX)and exogenous gene sequences into the 3’non-coding region of the FoMV coat protein gene.[Results]The plasmid pCB301-FoMV-CP-PVXprom-GFP expressed green fluorescent protein in inoculated Nicotiana benthamiana leaves.[Conclusion]A recombinant viral expression vector was constructed successfully.
文摘Insect baculovirus(IBV) is a biological insecticide in agriculture and forestry, butits slow speed of killing pests and narrow range of host make its application quitelimited. Hence, polyhedrin (ocu) gene of IBV was chosen for construction of
基金supported by High-Level Talent Project of Foshan University (GG040965)Science and Technology Project of Foshan City (GS54207)Science Foundation of Jiangsu Province (BK20160206)
文摘The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here,we established a simple method to construct a milk expression vector, by a combination of homologous recombination and digestion-ligation. Our methodology is expected to have the advantages of both plasmid and bacterial artificial chromosome(BAC) vectors. The BAC of mouse whey acidic protein gene(mWAP) was modified twice by homologous recombination to produce a universal expression vector, and the human lysozyme gene(hLZ) was then inserted into the vector by a digestionligation method. The final vector containing the 8.5 kb mWAP 5′ promoter, 4.8 kb h LZ genomic DNA, and 8.0 kb m WAP 3′ genomic DNA was microinjected into pronuclei of fertilized mouse embryos, to successfully generate two transgenic mouse lines that expressed recombinant human lysozyme(rhLZ) in milk. The highest expression level of rhLZ was 0.45 g$L–1, and rhLZ exhibited the same antibacterial activity as native h LZ. Our results have provided a simple approach to construct a universal milk expression vector, and demonstrated that the resulting vector regulates the expression of hLZ in milk.
