65 results about "Dna transfection" 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 relates to high-strength temperature-sensitive hydrogel as well as a preparation method and application thereof. The high-strength temperature-sensitive hydrogel is prepared by copolymerizing 2-vinyl-4,6-diamino-1,3,5-triazine and N-isopropyl acrylamide as raw materials in the presence of a cross-linking agent and an initiator, wherein the mass ratio of the 2-vinyl-4,6-diamino-1,3,5-triazine as a monomer to the N-isopropyl acrylamide is 0.2-2:1. The high-strength temperature-sensitive hydrogel has high stretching and compression resistance and favorable biocompatibility and optical property, and the surface of the hydrogel has the capacities of adsorbing DNA (Deoxyribonucleic Acid) transfection cells and regulating the attaching and disattaching behaviors of the cells through temperature. The preparation method of the hydrogel is simple, and the hydrogel is easy to store for a long term and transport for a long distance.

The invention discloses high-strength photosensitive hydrogel as well as a preparation method and application thereof. The high-strength photosensitive hydrogel is formed by free radical polymerization copolymerization of polyethylene glycol methacrylic ester, 2-vinyl-4,6-diamino-1,3,5-triazine and a double-bond-containing monomer with a spiropyrane structure; the three monomers and an initiator are dissolved in a solvent, unsaturated bonds on the molecules of the monomers are initiated by the initiator, and the hydrogel is prepared through free radical polymerization reaction. The photosensitive hydrogel disclosed by the invention has higher stretching resistance and compression resistance, and has good biocompatibility. Moreover, the hydrogel has the capability of adsorbing DNA (Deoxyribonucleic Acid) transfection cells as well as adjusting attaching and un-attaching behaviors of the cells by illumination.

The present invention discloses a method for assembling foot-and-mouth disease virus empty capsids in insect cells via the alteration of acid-resistance. The method for assembling foot-and-mouth disease virus empty capsids in insect cells includes the following steps: (1) the altered P12A gene and the non-structural protein gene 3C of foot-and-mouth disease virus are introduced into bacteria via baculovirus vectors for recombination to produce recombinant rhabdovirus A; (2) the DNA of the recombinant rhabdovirus A is used to transfect the insect cells, so that the foot-and-mouth disease virus empty capsids are obtained. The method assembles the integral foot-and-mouth disease virus empty capsids in the insect cells for the first time, lays a foundation for the research and the development of gene-engineered subunit vaccines and novel diagnostic reagents.

The invention relates to the use of DNA-loaded nanoparticles for a transfection of DNA into cells in vitro or in vivo.

The present invention relates to the field of molecular cloning and to the field of expression cloning in higher, eukaryotic cells. In particular, the present invention relates to a method for fast and reliable identification of vectors and vector DNA which will be capable of providing a desired expression product if a eukaryotic host cell is transfected with the vector DNA.

The invention discloses a building method of a silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system, comprising: taking silkworm wild type BmNPV genome DNA as the template; respectively taking P10-upF / P10-upB and P10-downF / P10-down B as a primer PCR for amplification to obtain p10-up and p10-down; after processed, inserting into BamHI-PstI-HindIII locus in pUC 19 to obtain recombinant plasmid pUC19-p10-up-down; using pUC-19-p10-up-polh-down, liposome lipofectin and MilliQ H2O to prepare DNA-Lipofectin mixed liquor; adding the mixed liquor into BmN cells for cultivating; collecting transfection cell supernatant; inoculating BmN cells, and recovering a polyhedral body; extracting virus DNA from the polyhedral body and electrically converting DH10 beta competent cells; screening locus ceruleus and cultivating; after cultivating PCR positive bacterial plaque, extracting macro-molecular DNA to transfect to the BmN cells; separating to obtain helper plasmids from DH10Bac culture bacteria of AcMNPV Bac-to-Bac; converting the helper plasmids into E.coli DH10 beta containg Ploh+BmBacmid; and screening the DH10 beta bacterial strain of the helper plasmid containg Ploh+BmBacmid. The invention can produce recombinant virus capable of infecting by eating with mouth, and recombinant virus does not need to infect by intracutaneous inoculation, thus improving the production efficiency of the silkworm rhabdovirus expression system.

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 invention provides a polyethyleneimine derivative and a preparation method thereof. The polyethyleneimine derivative is a cationic polymer and can be used as a gene delivery carrier. The polyethyleneimine derivative provided by the invention can be effectively combined with DNA (Deoxyribonucleic Acid), and nanoparticles with particle diameter potential can be obtained. With the polymer, the plasmid DNA of the green fluorescent protein and firefly luciferase protein can be effectively transfected into HEK293 cells; extremely high transfection efficiency is shown; and even the transfection efficiency is higher than that of the commercial polyethyleneimine. The derivative is easy and practical in synthesis method and can be subjected to mass production.

The invention relates to a cyprinid herpesvirus II DNA vaccine based on a baculovirus vector as well as a building method and application thereof. Codons of regional sequences 1-186, 993-1197, 603-783and 85-186 nt of cyprinid herpesvirus II ORF72, ORF66, ORF81 and ORF82 are optimized; then, ORF72-ORF66-ORF81-ORF82 fusion sequences are chemically synthesized; next, the promoter control is performed; the clone into the baculovirus transfer vector pFSATBacTMDual is performed to build recombinant plasmids pFSATBacTMDual-FA-D4ORF; after the plasmids are used for converting DH10 / Bac competent cells, Bacmid-FA-D4ORF is obtained; after the Bacmid-FA-D4ORF DNA is used for transfecting silkworm culture cells, BmNPV-FA-D4ORF is obtained and is the cyprinid herpesvirus II DNA vaccine based on the baculovirus vector. The DNA vaccine can realize the immune on carassius auratus gibelio by using an injection or oral administration or soaking method; the gill bleeding diseases of the carassius auratusgibelio can be reduced.

The invention provides a recombinant sequence containing H7N9 virus HA gene, a recombinant baculovirus and application of the virus in vaccine preparation. The recombinant sequence SP-HA-TM includes a gene coding H7N9 influenza virus HA protein, a gp64 signal peptide sequence and a transmembrane region sequence, wherein the signal peptide sequence is connected to the 5' end of HA protein gene, and the gp64 transmembrane region sequence is connected to the 3' end of the HA protein gene. The recombinant baculovirus Bm-gp64-HA is inserted into a donor plasmid by the recombinant nucleotide sequence SP-HA-TM and is subjected to homologous recombination with shuttle vector Bacmid's genome by transposition so as to obtain the recombinant baculovirus genome DNA, then the recombinant baculovirus genome DNA transfects silkworm cells, and packaging is carried out in silkworm cells to obtain the recombinant baculovirus. The virus is purified and a heat source is removed so as to obtain a vaccine for human immunization and prevention of avian influenza infection among people.

The present invention is directed to a method for inactivation of expression of a gene in a eucaryotic cell comprising (i) transfection of a eucaryotic cell with DNA comprising an expression cassette for expression of a cell surface protein and an expression cassette for expression of a RNAi compound, said compound being capable of inactivating expression of said gene, wherein said expression cassette for expression of a cell surface protein and said expression cassette for expression of a RNAi compound are located on the same vector DNA, and (ii) enrichment and / or selection of cells which express said cell surface protein.

The invention discloses an establishment method and application of a novel rat hypertension model. The disclosed establishment method of the novel rat hypertension model includes the following steps that 1, a human-body intron-derived 27nt-microRNA sequence is provided to construct its high expression plasmids; 2, the high expression plasmids in the step 1 is mixed with an X-treme GENEHP DNA transfection reagent in a certain proportion, after dilution with normal saline, the reagent is injected instantaneously in a SD rat at high speed from the caudal vein of the rat, the blood pressure of therat is increased to a high level, and the novel rat hypertension model is formed. The rat hypertension model is an effective animal disease model, can be used for verifying the influence of human-body intron-derived 27nt-MicroRNA on the blood pressure and vasodilator factors of the normal rat and screening antihypertensive drugs, and has wide application prospects.

Disclosed is a novel formulation for the production of a lyophilized plasmid / DNA transfection reagent complex capable of serving as a carrier for additional free plasmids. Upon rehydration, this plasmid / DNA transfection reagent carrier can be used to introduce simultaneously the complexed plasmid and the additional free plasmids into animal cells. This novel formulation can be useful for viral particle production, gene transfer experiments like gene silencing experiments, reporter gene, or integration / selection experiments.

A single embedded polyhedronvirus shuttle expression carrier of bollworm is configured through inserting the low-copy bacterium F replicon, kanamycine resistant gene and beta galactosidase gene to the polyhedrom gene site of the virus genom. The doner plasmid is used to transfer the exogenous gene to the insetion site of transposon in the said shuttle expression carrier. After bollworm cell is transfected by said DNA, the infections recombinant virus particles are formed to express exogenous gene under control of polyhedron promoter and P10 promoter.

The invention relates to proteins, notably SAP-2 and SAP-3, having an antibiotic action. The invention also relates to a method for purifying certain antimicrobial proteins, as well as to a use of said antimicrobial proteins for antibiotic therapy or to a use of cells which were transfected with a DNA which codes for the proteins provided for in the invention.

The invention belongs to the field of biological medicine, and discloses production of a novel cholesterol-modified human-derived cell penetrating peptide hPP-chol, and a cell penetrating peptide hPP-chol mediated plasmid DNA transfection method. The sequence of the novel cholesterol-modified human-derived cell penetrating peptide hPP-chol is Chol-KIPLPRFKLKCIFCKKRRKR. The C terminal or the N terminal of the novel cholesterol-modified human-derived cell penetrating peptide hPP-chol is connected with a marker or a carried molecule via covalence connection or non-covalence connection; and the marker or the carried molecule is carried by the novel cholesterol-modified human-derived cell penetrating peptide hPP-chol to enter into cells via penetrating cell film. The novel cholesterol-modified human-derived cell penetrating peptide hPP-chol is obtained based on improvement and modification of human-derived cell penetrating peptide, is safe, and is low in immunogenicity and toxicity, is obtained via solid phase synthesis, is low in cost, is excellent in cell penetrating effect, and can be widely used in the fields of drug, health care product, skin care product, transfection reagent, and diagnostic reagent practical production; and quality control is convenient to realize.

The invention discloses a recombined rhabdovirus for expressing a porcine bocavirus VP2 protein and application thereof. A porcine bocavirus VP2 gene is amplified by PCR (polymerase chain reaction), and the nucleotide sequence of the porcine bocavirus VP2 gene is represented by SEQ ID NO.1. The amplified porcine bocavirus VP2 gene is inserted into BamHI and XhoI locuses of a rhabdovirus transmissible plasmid pFastBacTMHTB to construct a recombined transmissible plasmid pFastBac HTB-VP2; the recombined transmissible plasmid is converted into a DH10Bac competent cell comprising a rhabdovirus framework plasmid; the VP2 gene is integrated into the rhabdovirus framework plasmid Bacmid through homologous recombination; an sf9 cell is transfected by extracting the DNA of the recombined Bacmid to obtain the recombined rhabdovirus rBV-VP2 of which the preservation number is CCTCC V201401. A large quantity of VP2 protein and virus sample particles are expressed and produced in insect cells, and an effective PBoV VLPs subunit vaccine is developed.

The present invention relates to the field of biomedicine, and discloses a novel dendritic human cell-penetrating peptide hPP7K and a production method thereof, and an hPP7K mediated plasmid DNA transfection method. According to the present invention, hPP7K is a novel dendritic human cell-penetrating peptide, is covalently or non-covalently linked to a marker or vector molecule at the C or N terminal, and carries a marker or cargo molecule to penetrate through the cell membrane so as to enter the cell; the cell-penetrating peptide hPP7K belongs to the improvement and modification of the human cell-penetrating peptide hPP7K, has characteristics of low immunogenicity, safety, low toxicity and high cell-penetrating effect, wherein the efficiency is higher than hPP10; the cell-penetrating peptide hPP7K is subjected to solid phase synthesis, such that the cost is low, and the quality control is easy to achieve; and the cell-penetrating peptide hPP7K can carry markers or cargo molecules to enter a variety of cells in the transmembrane manner, such that the cell-penetrating peptide hPP7K is the transmembrane transport carrier having the development prospects.

The invention discloses a preparation method of virus-like particles of a muscovy duck parvovirus. The method comprises the following steps: cloning a gene VP3 of the muscovy duck parvovirus into a baculovirus transfer vector to obtain a recombinant transfer vector pFastBacl-VP3; converting a recombinant plasmid of the pFastBacl-VP3 into DH10Bac competent cells, and performing coated plate cultivation till blue-white spots appear; picking white single colonies, extracting a recombinant Bacmid DNA by an alkaline cracking method, and performing transfection on the recombinant Bacmid DNA to enable an insect cell Sf9 to grow to prepare a rcombinant bculovirus, wherein SDS-PAGE, Western blot and IFA results show that a recombinant protein VP3 is successfully expressed in the insect cell; infecting the insect cell with P3 replacing the virus, collecting cell cracking supernate, and performing sucrose density gradient centrifugal purification to obtain spherical virus-like particles with the diameter of about 20 to 25 nm, wherein the spherical virus-like particles are highly similar to natural DPVs (Duck Parvovirus).

A process of producing a protein of interest by expression of said protein of interest from a sequence of interest in a plant or in plant leaves, comprising: (a) transfecting said plant or said plant leaves by infiltrating said plant or said plant leaves with an Agrobacterium strain in the presence of a complementing factor, said Agrobacterium strain containing in T-DNA a heterologous DNA sequence having a sequence portion encoding a replicon, wherein said sequence encoding a replicon contains sequences necessary for replicon function of said replicon, said sequences being derived from a plant virus, and said sequence of interest to be expressed from said replicon, (b) optionally isolating said protein of interest from said plant or said plant leaves infiltrated in step (a), wherein said Agrobacterium strain is provided with a first genetic modification rendering said Agrobacterium strain defective for transfecting organisms with said T-DNA in the absence of said complementing factor.

The present invention provides a new method for gene point mutation repair. Main steps are as follows: in a target genome sequence, cell gene mutation sites are selected as repaired gene sites; according to the selected gene repair sites, sgRNA of a CRISPR / Cas 9 system is designed; the sgRNA is constructed to an expression vector carrying CRISPR / Cas9 proteins; a segment of correct sequence is selected near upstream or downstream of the mutation sites, and a designed Donor DNA is used as a template for the gene mutation repair; in vitro culture of cells carrying the mutation site genes is conducted; an electrotransfection method is used to transfect the sgRNA, CRISPR / Cas9 expression vector and Donor DNA into the cells. The method fully mixes a Cas9 expression vector, the sgRNA and the DonorDNA with mouse fibroblast cell line carrying Msh5 point mutation established in vitro, then the cell line is electrically transfected to increase transfection efficiency of the cells, and the CRISPR / Cas9 system is used to conduct the repair of the mutation sites, thereby providing a scientific basis for treatment of primary amenorrhea in women.

The invention relates to infectious clone construction, rescuing and application of two porcine reproductive and respiratory syndrome virus intensity strains highly homologous in genomes, and belongsto the technical field of bioengineering. The two constructed infectious clone viruses are an XJ17-5 virulent strain and a JSTZ1712-12 natural weak strain which are highly homologous but are significantly different in virulence based on an HP-PRRSV genome. A method for constructing infectious clone is simple and convenient, DNA transfection is directly performed, in vitro transcription into virusRNA and then transfection are not needed, and the shortcomings of being not stable in RNA in vitro and easy in degradation are overcome. The obtained HP-PRRSV infectious clone platform can be appliedto research on an in vitro virus reproduction mechanism and an infective mechanism. The constructed HP-PRRSV natural weak strain infectious clone virus can be used for researching a safer and more efficient novel PRRSV genetic engineering vaccine, and prevention and control of Chinese PRRSV is facilitated.

The invention relates to a preparation method and an application of a monoclonal antibody. The preparation method comprises the following steps: 1, obtaining a B cell, enriching, and purifying; 2, binding a labeled antigen to a specific B cell; 3, obtaining an antigen specific B cell; 4, amplifying the variable region gene of an antibody; 5, constructing an expression vector; and 6, expressing a recombinant antibody and purifying: transfecting a 293T cell in the logarithmic phase by using purified expression vector DNA, centrifuging to remove cell fragments, and carrying out protein G column purification on the obtained centrifuge solution to obtain a recombinant monoclonal antibody solution. The preparation method substantially rises the concentration of the recombinant monoclonal antibody, reduces the cost, and is suitable for industrial large-scale production application. The obtained monoclonal antibody can specifically neutralize hepatitis C E1 antigens, and can be used for preparing drugs for preventing and treating hepatitis C.

The invention discloses a preparation method of a cell strain with sterol carrier protein (SCP) stable transformation of prodenia litura. The preparation method sequentially includes the steps of 1), cloning cDNA (complementary deoxyribonucleic acid) of SCP (single-cell protein)-2 gene from prodenia litura; 2), cloning the cDNA of the SCP-2 encoded protein into pcDNA5 / FRT plasmid to form recombined pcDNA5 / FRT-SCP2 plasmid; 3), applying the pcDNA5 / FRT-SCP2 plasmid to transform colon bacillus and obtaining transformed colon bacillus via cultivation; 4), extracting recombined plasmid DNA from the transformed colon bacillus and performing purification; 5), transfecting Chinese hamster ovary cells with purified and recombined pcDNA5 / FRT-SCP2 plasmid DNA; 6), selecting transfected CHO cells in a culture medium containing antibiotic hygromycinB and performing continuous subculture to obtain the cell strain. The invention aims to provide the cell strain with SCP stable transformation of the prodenia liture, the preparation method of the cell strain and an application of the cell strain. The cell strain has the advantages of high screening accuracy, simpleness in operation and accuracy in result.

The present invention provides a method of culturing PAMAM-D mediated sperm vector of transgenic mammalian, including following steps: foreign gene enzyme is cut into a linear configuration of end cohesive, determining its dosage range aimed at different sperms; screening PAMAM-D in the composition algebra which is more than G5 and ranges of exogenous charge ratio 2-50:1, transfecting sperm by the exogenous DNA has high effect, and also keeping the optimum combination condition of the exogenous DNA usage of sperm activity and PAMAM-D generation and dose, so as to prepare PAMAM-D / exogenous DNA compound, adequately washing collected sperm under the monitor of the supernatant fluid IF-1 after washed; transfecting sperm outside of compound by incubates method, the female animal is conceived by individually selecting single sperm of transfection exogenous DNA, or sperm of transfection exogenous DNA formed by injecting in testicle, in vitro fertilization embryo transfer, in vivo artificial insemination and natural mating, cultivating transgenic animal.

The invention relates to the technical field of biopolymer materials, in particular to a preparation method and application of a cationic polymer gene vector with a fluorinated end group. The invention provides a cationic polymer with a fluorinated end group. The structure of the cationic polymer is shown in the specification. According to the method, a fluorinated group is introduced through a fluorinated initiator by a one-step method to initiate synthesis of various terminal group fluorinated linear polyethyleneimine F-PEI with different molecular weights, the gene transfection efficiency is improved by introduction of the fluorine group, and high-efficiency DNA transfection is realized. The gene vector can achieve an efficient transfection effect in the cell transfection process, the toxicity generated to cells in the transfection process is small, gene molecules can be effectively and safely conveyed into the cells, and the gene transfection vector has the advantages of being efficient, low in toxicity, low in price, easy to synthesize and the like. The method has the advantages of simple reaction, easily available raw materials, low preparation cost, easily controlled molecular weight, high yield and simple post-treatment, and is suitable for large-scale production. # imgabs0 #