57 results about "Dual promoter" patented technology

The present invention is based on the development of a dual promoter system (preferably a RNA pol I-pol II system) for the efficient intracellular synthesis of viral RNA. The resultant minimal plasmid-based system may be used to synthesize any RNA virus, preferably viruses with a negative single stranded RNA genome. The viral product of the system is produced when the plasmids of the system are introduced into a suitable host cell. One application of the system is production of attenuated, reassortant influenza viruses for use as antigens in vaccines. The reassortant viruses generated by cotransfection of plasmids may comprise genes encoding the surface glycoproteins hemagglutinin and neuramimidase from an influenza virus currently infecting the population and the internal genes from an attenuated influenza virus. An advantageous property of the present invention is its versatility; the system may be quickly and easily adapted to synthesize an attenuated version of any RNA virus. Attenuated or inactivated RNA viruses produced by the present invention may be administered to a patient in need of vaccination by any of several routes including intranasally or intramuscularly.

The present invention is based on the development of a dual promoter system (preferably a RNA pol I-pol II system) for the efficient intracellular synthesis of viral RNA. The resultant minimal plasmid-based system may be used to synthesize any RNA virus, preferably viruses with a negative single stranded RNA genome. The viral product of the system is produced when the plasmids of the system are introduced into a suitable host cell. One application of the system is production of attenuated, reassortant influenza viruses for use as antigens in vaccines. The reassortant viruses generated by cotransfection of plasmids may comprise genes encoding the surface glycoproteins hemagglutinin and neuraminidase from an influenza virus currently infecting the population and the internal genes from an attenuated influenza virus. An advantageous property of the present invention is its versatility; the system may be quickly and easily adapted to synthesize an attenuated version of any RNA virus. Attenuated or inactivated RNA viruses produced by the present invention may be administered to a patient in need of vaccination by any of several routes including intranasally or intramuscularly.

The invention discloses a method for targeted knockout of a specific gene of a Suqin yellow chicken embryonic stem cell. The method comprises the following steps: firstly, checking out an exon sequence of a gene, cloning the gene, sequencing to obtain a complete exon sequence, designing a CRISPR / Cas9 knockout target site on the sequence as gRNA, and constructing a CRISPR / Cas9 dual-promoter knockout vector; performing SSA activity detection on the constructed Cas9 vector, transfecting a control group with an empty vector, detecting a luciferase signal, and obtaining a result that the more the luciferase activity is increased relative to the control group, the higher the gRNA shearing activity is shown; transfecting the CRISPR / Cas9 vector which is high in SSA activity with ESCs, using flow cytometry to screen a GFP-positive cell, extracting genome DNA, and designing a primer.

The invention discloses a method for preparing (R)-2-hydroxy-4-phenyl ethyl butyrate by catalyzing recombinant carbonyl reductase, which belongs to the technical field of biological engineering. The method comprises the following steps of: cloning gene segments of carbonyl reductase (IolS) and glucose dehydrogenase (GDH) from bacillus subtilis CGMCC NO.1.1508, expressing an IolS gene and a GDH gene in series by adopting a dual-starter method to construct a recombinant plasmid pET24a-G-T7-I, and introducing the plasmid into escherichia coli BL21(DE3); and under the condition of not adding or adding a small amount of NADP+cofactors, performing biotransformation by taking a cell-free extract of the escherichia coli recombinant plasmid as a catalyst, 2-oxo-4-phenyl ethyl butyrate as a substrate and glucose as a substrate to obtain (R)-2-hydroxy-4-phenyl ethyl butyrate, wherein the enantiomeric excess value of the product is higher than 99.5 percent. In the method, IolS and GDH are co-expressed, so that efficient regeneration of an intra-cellular cofactor NADP(H) is realized, production cost is lowered, and a good industrial application prospect is achieved.

The invention relates to expression systems useful for regulated expression of a gene of interest based on the constitutive expression of the original TetR repressor and the expression of the polynucleotide driven by a constitutive promoter operably linked to an operator sequence for a tetracycline operator sequence. The system can be provided as two different polynucleotides or as an all-in-one vector. The invention also relates to vectors, host cells and viral particles according to the invention as well as to the uses thereof for in vitro and in vivo production of products of interest or for therapy.

The invention discloses expression of human insulin and analogues thereof in a methanol yeast containing dual promoters and a high-efficient preparation method of the human insulin and the analogues thereof, and the method is characterized in that recombinant human insulin and the analogues thereof are prepared by constructing an expression vector containing the dual promoters and engineering cells in host cells of the methanol yeast, and performing the optimal fermentation technology and the high-efficient purification technology, so that the production cost of the recombinant human insulin and the analogues thereof can be effectively reduced.

The invention discloses a method for conveniently and efficiently constructing a recombinant baculovirus. The method mainly comprises the following steps: a donor plasmid which contains dual-promoter and can directionally and rapidly clone PCR product is constructed; (2) an I-SceI cassette is introduced into the genome of E.coliDH10B, simultaneously two I-Scel sites and a resistance gene are introduced into the baculovirus Bacmid, and the plasmid which can express Red-Gam recombinase is transformed into E.coliDH10B to construct a new receptor strain CTRDH10B / Bacmid / PML104; (3) the recombined donor plasmid is introduced into the CTRDH10B / Bacmid / pML104 to induce the expression of I-SceI endonuclease and the Red-Gam recombinase, and the recombinant Bacmid is obtained by homologous recombination in the bacterium; (4) the recombinant Bacmid DNA transfection insect cells are prepared by the conventional method and analyzed. The method needs no complex PCR verification and fussy plaque purification, and is really efficient, rapid and convenient. The method is especially suitable for constructing high-quality baculovirus cDNA library with large storage capacity.

The present invention provides a method for preparing and screening a cell line expressing bispecific antibody. The method comprises: constructing two plasmids expressing bispecific antibody, wherein the two plasmids contain double promoters and respectively express different fluorescent proteins; and carrying out transformation, culture and extraction on the two recombinant plasmids, co-transfecting the two recombinant plasmids into host cells, screening the positive monoclonal cell line expressing the double fluorescence, and evaluating the yield and the stability according to the fluorescence intensity. With the method of the present invention, the high yield cell line can be conveniently and rapidly screened in advance, the test period can be shortened, the manpower and material resource investment can be reduced, and the accuracy is high.

The invention relates to a high-expression recombination protein carrier pRELPN. The high-expression recombination protein carrier pRELPN is artificially synthesized ELP fusion protein(RELPN) consisting of 9 polyarginine (R9)-elastin polypeptide (ELP) n-10 poly asparagine (N10), when multiple clone sites cloned in pronucleus or eukaryotic expression vectors (p) are not directly expressed as exogenous genes, the effect of a second promoter can be achieved. The pRELPN carrier can promote independent high expression of an exogenous target gene behind start cipher ATG cloned behind ELP fusion protein. The fusion protein is seldom expressed, and self expression of hosts of escherichia coli or eukaryocyte and the like can be slightly restrained, so that recombination protein accumulation and high expression of the exogenous target gene can be promoted. The high expression vector pRELPN containing dual promoters is suitable for independent high expression of exogenous genes of antibodies, antigens, enzymes, recombination protein, polypeptide, ELP fusion protein and the like, so as to contribute to solving of the problem of disindustrialization caused by that a common expression vector islow or non in expression on an exogenous gene an exogenous gene or a fusion gene consisting of the exogenous gene and ELP.

The invention discloses a method which is feasible in pharmaceutical industry for efficient secretion expressing, preparing and purifying of gene recombinant human metallothionein or analogues thereof. The method is characterized by introducing exogenous genes containing dual-promoters into relevant host yeast cells in transformation or double crossover recombination manner by using plasmid vector pPICZ alpha-MT of exogenous metal sulfur protein genes which contain methanol response element AOX and contain or not contain metal response element MRE to construction expression engineering cells containing the dual-promoters in the methanol yeast host cells. According to the invention, several problems with traditional preparation method of metallothionein, such as high production cost, slaughter and organ harvesting to large number of animals, virus pollution derived from animal, endotoxin pollution derived from bacterial, immunogen of non-humanized protein and the like, are avoided effectively,.

The invention is an double-starter DNA vaccine expression carrier pCMVnir and its preparing method. The expression carrier pCMVnir is double stranded closed-loop molecule, a gene cloning carrier, and the length is 5.1Kb, used to express to prepare genetic engineering product and produce DNA vaccine, containing two starters, one is an eukaryoti cell starter (HCMV IE), and the other is an escherichia coli nitratase gene starter nirB, the two starters are activated in cell and prokaryotic cell to express the corresponding proteins, respectively, it contains polyclonal restriction site where the external source is inserted, and the carrier has the sequence shown in SEQ ID NO 1.It can be widely used to high-efficacy expression of external source gene and need not add inducer to the culturing system.

The invention relates to a preparation method of a novel PD1 FAB monoclonal antibody in a DG44 expression system. The method comprises the steps of (a) cloning a gene of an amino acid sequence with a light chain and a heavy chain of an Fab fragment of signal peptide at N end separately to an expression vector with dual promoters to build a recombinant expression vector; (b) transposing the obtained recombinant expression vector into a competent cell, screening recombinant bacterial plaques, extracting recombinant plasmids, transfecting the recombinant plasmids to a DG44 cell, further amplifying the recombinant cell, expressing Fab protein by using the amplified and cultured cell, and centrifugally collecting a supernatant product containing the Fab protein; and (c) expressing the supernatant by the obtained cell, carrying out ultrafiltration concentration by using a membrane coating, replacing a buffer solution, and then carrying out chromatographic column purification to obtain a fully human monoclonal antibody Fab fragment of the monoclonal antibody PDI.

The invention provides a double starter cascade regulation gene expression vector and a construction method and application thereof. The double starter cascade regulation gene expression vector carries a nucleic acid construct, the nucleic acid construct comprises two gene expression cassettes with cascade regulation relation, the first expression cassette is driven by an inducible starter, and the second expression cassette is driven by a strong starter; wherein the inducible starter is induced and expressed by a specific substance, and a downstream gene coding of the inducible starter can regulate a regulatory element capable of regulating and controlling the expression of the strong starter; and the downstream gene of the strong starter is a target gene. The successful establishment ofthe double starter cascade regulation and control mode not only provides a new method and a new idea for improving the sensitivity of genetic engineering bacteria to environmental pollutants, but alsoenables the high-efficiency amplification effect of the cascade control mode on reporter gene expression to be applied to research of screening analysis of starters or interaction of proteins and thelike, and new technical convenience for gene expression control research is provided.

The invention provides a yeast recombinant human III type triple helix collagen and a preparation method thereof. The preparation method comprises the following steps: S1, constructing a pPICZ alpha B plasmid containing a human III type collagen alpha 1 chain; s2, constructing a double-promoter expression vector of the alpha subunit and the beta subunit of the human P4H; s3, co-transforming the double-promoter expression vector and the pPICZ alpha B plasmid into saccharomycetes, so as to obtain a co-expression strain; s4, screening a yeast recombination strain containing a P4H alpha subunit and a beta subunit from the co-expression strain; and S5, carrying out induced expression on the yeast recombinant strain to obtain the yeast recombinant human III-type triple helix collagen. According to the preparation method disclosed by the invention, the problem of pyrogen does not exist, the generation of immunogenicity is effectively avoided, and the interaction force, the thermal stability and the anti-enzymolysis level among collagen molecules are effectively improved.

The invention belongs to the field of biological genetic engineering, and provides a dual-promoter inducible secretable shuttle plasmid. The shuttle plasmid consists of an enzyme cutting Escherichia coli pET-28a plasmid and an enzyme cutting bacillus subtilis pE194 plasmid, wherein a lysozyme-antibacterial peptide functional fragment and an antibacterial peptide gene are inserted into the enzyme cutting Escherichia coli pET-28a plasmid. The invention also provides a method for constructing the dual-promoter inducible secretable shuttle plasmid. The shuttle plasmid is transferred to a prokaryote to express proteins such as toxalbumin, cecropin and the like in a fusion mode without toxicity to a host. The shuttle plasmid constructed by the method can be induced by IPTG or lactose to improve gene expression level; the shuttle plasmid is also suitable for exocrine expression, and a target protein for the exocrine expression is convenient to process and is not polluted by endotoxin; and because the shuttle plasmid has dual functional promoters, different genes can be inserted in appropriate correct directions for gene expression.

The invention provides a method for constructing dual-promoter DNA anti-caries vaccine plasmid pCN-SSIE, and a preparation method of the anti-caries vaccine by the plasmid pCN-SSIE. Antigen gene SBR is inserted in the vector pCMVnir by the molecular cloning technology, green fluorescent protein reporter gene is added in the downstream of the SBR, and the SBR and the reporter gene pass through the interior of the ribosome and enter the site gene for connection so as to prevent the fusion expression thereof; and finally, a section of signal peptide sequence is fused at the 5' end of the SBR. The anti-caries vaccine improves the immunogenicity of the anti-caries vaccine, enhances the immune effect, has simple and convenient, effective and economical immune method with long duration, and can carry out immunity repeatedly with little side effects.

The invention discloses a high-modulus and high-temperature-resistant bismaleimide resin composition. The bismaleimide resin composition is prepared from bismaleimide monomer, cyanide resin, aminostyrene, allyl compound, an oxime promoter and an imidazole promoter. A synergistic promotive special effect can be realized by adopting the oxime and imidazole dual-promoter system, the curing temperature is reduced to 200 DEG C, the glass transition temperature of the glass fiber composite material can achieve 370 DEG C without performing post-curing processing, and the use advantage of high-temperature using the aspects of composite material, the adhesive, the paint and the like by the bismaleimide at low-temperature curing is enlarged. Furthermore, the tensile modulus of a casting body thereofcan achieve 5.3GPa, the compression strength of the composite material is improved, and the new material is provided for torpedo, submarine oil pipeline and like applications needing the high modulusresin.

Through cloning to obtain the specific fruit promoters E82.2 and E81.1 gene of excellent pure tomato breed Zhongshu No.5, driving the vaccine molecule with the promoter E82.2 and the compound double promoter E35S-E81.1 and constituting plant expression operon unit with NOS terminator, and introducing to Agrobacterium tumefaciens LBA4404, engineering bacterium is obtained. By means of optimizing the complete technological system, recombinant vaccine molecule protein with efficient and stable expression and its tomato transforming strain are obtained. The present invention constitutes technological platform of efficient and stable tomato expression for exogenous vaccine molecule and medicinal protein production and provides excellent technological platform for developing oral tomato vaccine and its medicinal protein.

The invention discloses recombinant arthrobotrys oligospora of Aozl gene dual promoters, which is used for preventing and controlling ruminant animal digestive tract nematode diseases caused by nemathelminthes nematode, and a preparation method of the recombinant arthrobotrys oligospora. The Aozl gene promoters of arthrobotrys oligospora are cloned by adopting a chromosome DNA (Deoxyribonucleic Acid) walking technology, and the recombinant arthrobotrys oligospora efficiently expressing the Aozl gene dual promoters is constructed by utilizing an agrobacterium tumefaciens-mediated fungus transformation technology. Compared with a traditional genetic transformation method, the method provided by the invention has the advantages of wide recipient material range, high transformation efficiency,stable inheritance transformant and the like, and the bottleneck of a traditional transformation method is overcome. Compared with a non-recombinant maternal arthrobotrys oligospora strain, the strain obtained by the method has good genetic stability; the Aozl gene transcriptional level is enhanced and the nematode-trapping activity is improved; an efficient gene engineering strain is provided for researches and development of a biological prevention and control preparation for the ruminant animal digestive tract nematode diseases.

The invention discloses a gene engineering bacterium for efficiently reducing azo dyes, and a construction method and application thereof. An NADH-azo reductase gene and a forward promoter sequence thereof are connected with an expression vector pET-22b(+), and transformed into Escherichia coli E.coli BL21(DE3) to obtain the gene engineering bacterium E.coli BL21(DE3) / azoR with double promoters for efficiently reducing azo dyes. The NADH-azo reductase gene is disclosed as a nucleotide sequence of which the Genebank registration number is EF198254, and the forward promoter of the NADH-azo reductase gene is disclosed as a nucleotide sequence of which the Genebank registration number is EF647586. The gene engineering bacterium E.coli BL21(DE3) / azoR can implement decolorization of azo dyes within wider temperature range, has higher decolorizing active and long-term survival capacity, and has wider application prospects in dye wastewater treatment.

The invention discloses a rhamnolipid producing plasmid, a construction method thereof, engineered escherichia coli and an application. Known rhamnosyltransferase genes rhlA, rhlB and rhlC are optimized and synthesized according to preference of an escherichia coli codon and named SyrhlA, SyrhlB and SyrhlC respectively; synthetic escherichia coli RBS (ribosome bind site) is connected to upstream SyrhlB, SyrhlA is connected to upstream RBS, and a gene cluster SyrhlA-RBS-SyrhlB is constructed; SyrhlA-RBS-SyrhlB and SyrhlC are constructed in plasmid pACYCDuet-1 containing double promoters, the plasmid is introduced into escherichia coli BL21(DE3) for expression, so that rhamnolipid is synthesized from escherichia coli with glucose as a carbon source, and the yield of rhamnolipid synthesized from escherichia coli is increased greatly.

The invention discloses a recombinant plasmid, including vectors and gene fragments. The gene fragments include pyridine nucleotide transhydrogenase gene containing promoters and isocitric dehydrogenase genes containing promoters. Compared with the prior art, the present invention has simple expression and purification process, the double promoter expression vector can not only provide possibility of right expression of protein difficult to express, but also can improve the output of the expressed protein with economic value, including pharmaceutical proteins and industrial enzymes, thereby reducing production costs and saving resources.

The present invention features vectors that contain a promoter effective for expression in bacterial cells and a promoter effective for expression in insect cells. The dual promoter system allows use of the same vector in both host cell systems so that construction of only a single vector is needed to express a polynucleotide inserted at a downstream cloning site. In preferred embodiments the vector is used to derive a recombinant baculovirus that is used to infect host cells. In particular vectors the promoters are a baculovirus polh promoter and a T7lac promoter. In particular vectors the promoter effective for expression in bacteria is positioned between the promoter effective for expression in insect cells and a cloning site. The invention also features various high throughput screening methods.

The invention provides a porcine circovirus type 3 virus-like particle as well as a preparation method and application thereof. According to the invention, an insect-baculovirus expression system is utilized to express a PCV3 Cap protein variant so as to prepare virus-like particles. According to the invention, double promoters of baculovirus are used to start Cap protein expression, on the basis of keeping the N terminal of the Cap protein rich in basic amino acid, the first to 22th amino acids are deleted, and a sequence for coding the basic protein P6.9 N terminal rich in basic amino acid of the baculovirus is inserted, so that the Cap protein is expressed in a fusion manner. Results show that the expressed fusion protein not only can form the virus-like particles, but also can be expressed at a relatively high level, and a basis is provided for further development of PCV3 vaccines and realization of industrial production of the PCV3 virus-like particles.

The invention discloses a dual-promoter universal plasmid for expressing T4 ligase of a domestic silkworm middle silk gland bioreactor as well as application and a method of the dual-promoter universal plasmid. The plasmid takes piggy Bac transposons as a foundation and carries an Amp resistance gene; the plasmid comprises function expression frames of a T4 ligase gene used as an exogenous gene and a green fluorescence EGFP (Enhanced Green Fluorescent Protein) gene used as a marker gene; the plasmid is constructed by utilizing a molecular biology method and two special restriction enzyme cutting sites containing ApaI and NheI are formed between DDDDK and a fibroin light-chain gene polyA; the universal plasmid is subjected to dual-enzyme digestion by adopting the ApaI and the NheI; after the universal plasmid is connected with the T4 ligase gene, the universal plasmid and an auxiliary plasmid are commonly injected into a domestic silkworm fertilized ovum; the green fluorescence protein gene and the T4 ligase gene are transferred into a domestic silkworm genome through utilizing properties of the transposons and are stably inherited and expressed to obtain transgenic domestic silkworms. The transgenic domestic silkworms are screened with the help of a fluorescence marker gene and domestic silkworm silk gland cells are used for specifically synthesizing and secreting T4 ligase protein.

The invention relates to expression systems useful for regulated expression of a gene of interest based on the constitutive expression of the original TetR repressor and the expression of the polynucleotide driven by a constitutive promoter operably linked to an operator sequence for a tetracycline operator sequence. The system can be provided as two different polynucleotides or as an all-in-one vector. The invention also relates to vectors, host cells and viral particles according to the invention as well as to the uses thereof for in vitro and in vivo production of products of interest or for therapy.

The invention provides a yeast recombinant human type I triple helix collagen and a preparation method thereof. The preparation method comprises the following steps: S1, constructing a pPICZ alpha B plasmid containing a human type I collagen alpha 1 chain; s2, constructing a double-promoter expression vector of the alpha subunit and the beta subunit of the human P4H; s3, transforming the double-promoter expression vector into saccharomycetes; s4, the pPICZ alpha B plasmid is transformed into saccharomycetes containing the double promoter expression vector, and a co-expression strain is obtained; s5, screening a yeast recombination strain containing a P4H alpha subunit and a beta subunit from the co-expression strain; and S6, carrying out induced expression on the yeast recombinant strain to obtain the yeast recombinant human I-type triple helix collagen. According to the preparation method disclosed by the invention, the problem of pyrogen does not exist, the generation of immunogenicity is effectively avoided, and the interaction force, the thermal stability and the anti-enzymolysis level among collagen molecules are effectively improved.

The invention belongs to the technical field of genetic engineering, and particularly relates to a double-promoter expression vector capable of simultaneously expressing target genes in prokaryotic and mammalian cells and a construction method of the double-promoter expression vector. The vector takes a eukaryotic expression vector as a starting vector, and a genome contains a prokaryotic promoterT7 sequence, a ribosome binding site and a T7 termination sequence. The constructed double-promoter expression vector can simultaneously express the same target gene in the prokaryotic and eukaryoticcells, can overcome the defect that steps are complicated as the existing same target protein needs two expression systems in the mammalian and prokaryotic cells, and also lays a certain technical foundation for expression of related target genes in different expression systems.

The invention discloses a Bombyx mori posterior silk gland bioreactor dual-promoter universal plasmid for expressing T4 ligase and application and a method thereof. The plasmid takes a piggyBac transposon as a basis, carries an Amp resistant gene and contains a T4 ligase gene serving as an exogenous gene and a function expression box of a green fluorescent EGFP gene serving as a marker gene. The plasmid is constructed by using a molecular biology method, and two restriction enzyme cutting sites unique to ApaL and NheI are contained between DDDDK and a fibroin light chain gene poly. After the ApaL and NheI double restriction enzyme cutting universal plasmid is connected with the T4 ligase gene, the plasmid and an auxiliary plasmid are jointly injected into Bombyx mori zygote, and characteristics of the transposon are utilized to guide the green fluorescent protein gene and the T4 ligase gene into a Bombyx mori genome and to be stably inherited and expressed to obtain transgenic Bombyx mori. The transgenic Bombyx mori is screened with the help of the fluorescent marker gene, and a Bombyx mori silk gland cell is utilized to specifically synthesize T4 ligase protein.