123 results about "Egfp gene" patented technology

The invention discloses single guide ribonucleic acid (sgRNA) capable of effectively editing a pig ROSA26 gene, and application of the sgRNA. Based on the sgRNA capable of specifically identifying the pig ROSA26 gene in a pig genome, a cell line of enhanced green fluorescent protein (EGFP) site-specific integrated porcine fetal fibroblasts is successfully established by using a CRISPR/Cas9-mediated gene knock-in technique; the results show that the cell line can stably and efficiently express an EGFP gene; furthermore, in the preparation process of the cell line, exogenous promoter genes and positive and negative screening marker genes are not introduced into the cell line. Therefore, the sgRNA greatly improves the safety of transgenic pigs, and has important significance for eliminating the biological and food potential safety hazards of agricultural products of the transgenic pigs.

Four zebrafish gene promoters, which are skin specific, muscle specific, skeletal muscle specific and ubiquitously expressed respectively, were isolated and ligated to the 5' end of the EGFP gene. When the resulting chimeric gene constructs were introduced into zebrafish, the transgenic zebrafish emit green fluorescence under a blue light or ultraviolet light according to the specificity of the promoters used. Thus, new varieties of ornamental fish of different fluorescence patterns, e.g., skin fluorescence, muscle fluorescence, skeletal muscle-specific and/or ubiquitous fluorescence, are developed.

The invention discloses an establishing method of a bacterial artificial chromosome recombinant duck plague virus rescue system platform and application of the platform. A bacterial artificial chromosome recombinant duck plague virus is obtained by inserting a recombinant duck plague virus transfer vector pUC18/EGFP-TKAB-BAC11 in a TK domain, wherein the recombinant duck plague virus transfer vector contains a TK gene left-right homologous arm, a reporter gene EGFP and a bacterial artificial chromosome core function component. By means of the platform, the in-vitro biologics characteristics of a UL55 gene-deleted strain established through an inside-bacterium two-step RED recombination method and a back mutation strain and parent strain of the UL55 gene-deleted strain are quite close; the functions are not related to positioning of a UL26.5 gene in a cell. The method is beneficial to development of pathogenic mechanism and gene function research of DPV CHv and is beneficial to the duck plague virus prevention and the research and application of recombinant duck plague virus vaccines of other poultry infectious diseases based on the platform; in addition, due to the fact that the recombinant virus carries a TK deletion mark and an EGFP gene, a mark vaccine can be developed to clinically distinguish a wild virus and a recombinant vaccine virus.

A pseudo-virus packaging system of coronaviruses comprises a vesicular stomatitis virus (VSV) vector with a Fluc and EGFP double reporter gene replacing a GP gene and an assembly cell expressing coronavirus spike protein S. The double reporter gene is selected from luciferase and fluorescent protein, and the luciferase reporter gene is preferably a Fluc gene. The fluorescent protein reporter geneis preferably an EGFP gene, and the assembly cell is preferably 293T. The invention further relates to a one-step packaging method of the packaging system; a pseudo-virus with single-cycle infection,low background value and high titer can be quickly packaged through simple, convenient and rapid operation, and the packaging system has the characteristic of being rapid in detection compared with aslow virus mediated pseudo-virus system; the one-step packaging method can be used for researching COVID-19, SARS, MERS and other coronaviruses and other viruses, provides a powerful screening tool for evaluation of antiviral preparations and vaccine, and has wide application value.

The invention discloses a TK (Thymidine Kinase) gene removed recombinant VTT (Tian Tan strain) oncolytic VACA (Vaccinia Virus). A VTT oncolytic VACA is subjected to recombination transformation, a TKgene is removed, and an EGFP (Enhanced Green Fluorescent Protein) gene and a Luciferase gene are expressed. The invention further discloses a preparation method of the TK gene removed recombinant VTToncolytic VACA, which comprises the steps of: recombining a wild type VTT virus and a targeting plasmid aiming at the TK gene of the VTT virus in cells to obtain virus suspension; screening and purifying the virus suspension to obtain the attenuated VTT oncolytic VACA. According to the TK gene removed recombinant VTT oncolytic VACA, which is disclosed by the invention, the TK gene is used as an attenuation target, the EGFP gene and the Luciferase gene are expressed, virus virulence is reduced, and tumor selectivity of the virus is improved; the TK gene removed recombinant VTT oncolytic VACA has a function of monitoring distribution of virus particles in a body, provides a novel antitumor drug for the clinic, can be used for researching and developing a live vaccine and is applicable to various tumors such as the lung cancer, the liver cancer, the melanoma and the like.

The invention discloses an Armillaria selection marker. Hygromycin is adopted as the Armillaria selection marker. Antibiotics capable of inhibiting Armillaria growth are screened out by experiments; the eukaryotic expression vector pH2GW7-35S-EGFP is selected for agrobacterium transformation, the hygromycin is taken as the selection marker, and an EGFP gene is transferred into Armillaria with an agrobacterium-mediated method; GFP fluorescence is seen under a fluorescence microscope, which proves expression of pH2GW7-35S-EGFP in Armillaria, and the Armillaria can serve as a host cell for transgenic operation; establishment of a transgenic method based on Armillaria lays foundation for further research of Armillaria gene expression, and a genetic engineering research method for deep researchof biosynthesis and regulation of active components in Armillaria, the molecular mechanism of exchange between Armillaria and gastrodia elata nutrients and the like is provided.

The invention discloses a recombinant pseudorabies virus (PRV), a construction method and application thereof. The recombinant pseudorabies virus takes recombinant PRV/TK<->/gE<->/EGFP<+> as the female parent, an expression cassette containing the main immunogenic gene ORF2 of porcine circovirus type 2 is inserted to replace an EGFP gene expression cassette in a gE gene so as to obtain the recombinant pseudorabies virus named as rPRV-ORF2. The recombinant pseudorabies virus provided by the invention is used for recombinant pseudorabies virus vaccines and has safety and effectiveness.

The invention discloses a method for measuring sortase based on pichia pastoris surface display, comprising the following steps: A. infusing target gene QALPETGEE and linker-eGFP gene into QALPETGEE-linker-eGFP; B. constructing an expression carrier for constructing QALPETGEE-linker--eGFP; C. converting the expression carrier pKFS-QALPETGEE-linker-eGFP into pichia pastoris; D. expressing yeast surface protein: inoculating and culturing positive transformant by YPD and BGMY liquid culture medium, and inducing expression in BMMY liquid culture medium; E. measuring the yeast surface protein; F. expressing sortase; and G. testing activatedly the sortase. The invention uses the yeast surface display technology, fluorospectrophotometer and flow cytometry are adopted for measuring, and the used materials are lower in price, thus greatly reducing the measuring cost of sortase and facilitating operation.

The present invention belongs to the technical field of animal infectious disease prevention and control and specifically relates to a method for efficiently and rapidly constructing a porcine intestinal coronavirus recombinant virus. A BAC plasmid containing full-length cDNA of PEDV AJ1102 strain is cleaved by a CRISPR/Cas9 system, then a recombinant BAC plasmid of EGFP gene substituted with PEDVAJ1102 strain ORF3 gene is obtained by a homologous recombination method, and after transfecting cells, the recombinant virus rAJ1102-delta ORF3-EGFP is rescued. Since different porcine intestinal coronaviruses have similar genomic structure and replication strategies, the method is also suitable for construction of other porcine intestinal coronavirus recombinant viruses. Compared with traditional methods, the method has prominent advantages of wide target selection, high specificity, simple operation, high efficiency, short experimental period, etc., can obtain the recombinant virus withinone week, and is the method for efficiently and rapidly constructing the porcine intestinal coronavirus recombinant virus.

The invention discloses an expression vector resisting PCV2 and a transgenic pig and construction methods thereof, belonging to the field of gene engineering. The expression vector is constructed by respectively connecting an H1 promoter and a pPB-sh2 sequence to a pUC57-H1 plasmid and then connecting a fusion fragment containing a loxP-Neo/EGFP gene expression cassette to pPB-H1-sh2. The vector is introduced into the genome of a pig, and somatic cell nuclear transfer technology is employed to produce the transgenic pig. The expression vector provided by the invention has a piggyback transposon, greatly improves transformation efficiency, changes a common plasmid series recombination transgene integration model into a single-site single-copy integration model and better simulates the internal environment of a biological gene. The constructed transgenic pig degrades PCV2mRNA through siRNA synthesized by itself, thereby fundamentally inhibiting PCV2 infection.

The invention relates to a construction method and applications of bacillus subtilis with high yield of glucosamine. The construction method comprises the following steps: transforming a GFA1 gene coming from a brewer's yeast, a glms riboswitch gene and an EGFP enhanced fluorescent protein coding gene into bacillus subtilis, and carrying out construction to obtain bacillus subtilis with high yield of glucosamine. According to the invention, for the first time, the glms riboswitch gene and the EGFP gene are connected into PHT01, then a GLMS-EGFP sensor is constructed, the change of fluorescence intensity is shown by responding to the change of the amount of glucosamine 6-phosphate in bacillus subtilis cells, and through a flow cytometry, the synthesis condition of glucosamine can be known in real time.

The invention relates to a method for constructing a bionic three-dimensional vessel network of human parenchymal viscera, which is characterized by comprising the following steps: acquiring a vessel network image of the liver, heart or kidney of a normal human, and converting a three-dimensional vessel model into a two-dimensional vessel tomograph; placing P(CL-EC) in the middle of fibrin glue and pressing the P(CL-EC) into a diaphragm; punching the degradable diaphragm with a computer laser high-precision perforating machine; co-culturing the degradable diaphragm and bone marrow mesenchymal stem cells of a wild C57BL/6J mouse, wherein the degradable diaphragm with the middle serial number is co-cultured with the bone marrow mesenchymal stem cells of an SDF-1/VEGF transfected wild C57BL/6J mouse; after the cells covers the diaphragm substantially, stacking materials according to the serial numbers, and injecting endothelial progenitor cells of the EGFP gene transfected C57BL/6J mouse into a preformed hole of a vessel. The method has the advantage of capability of bionically constructing the vessel network of each human parenchymal viscus without the limitation of size.

The invention discloses an adeno-associated virus capable of being used for bimodal imaging and application of the adeno-associated virus. A CAG promoter, an AQP1 gene, a 2A connecting sequence, an EGFP gene, a WPRE after-transcription adjustment element and a polyA sequence are sequentially inserted in the middle of ITR of an AAV vector plasmid. After animal living nerve cells are infected by the prepared recombinant adeno-associated virus, expression of AQP1 and EGFP proteins can be mediated in the nerve cells, so that an expression region has green fluorescence signals, diffusion weighted MRI (Magnetic Resonance Imaging) signals can be changed, and the characteristic of bimodal imaging is achieved. The expression level of the exogenous gene transduced by the virus vector disclosed by the invention can be observed in situ in a living body tissue through noninvasive magnetic resonance imaging, can also be observed in an in-vitro tissue through fluorescence imaging, and can be used for magnetic resonance imaging contrast, neuron marking, neural circuit tracing and the like.

The invention discloses a method for improving homologous recombination efficiency and recombination virus screening of pseudorabies virus. According to the method, a pseudorabies virus AH strain transfer vector containing an EGFP gene complete expression box is subjected to enzyme digestion linearization, and then subjected to homologous recombination with the pseudorabies virus AH strain; and the obtained recombination virus screening method comprises the steps of separating fluorescent single host cells with pathological changes, and then performing further screening by improved plaque experiment. After recombinant plasmid enzyme is subjected to digestion linearization, the enzyme is subjected to cell transfection; next, virus homologous recombination is performed, so that the homologous recombination efficiency is improved; by virtue of the method in combination with single cell absorption and plaque purification, the screening and purifying time of the recombination viruses is greatly shortened; and therefore, the method is of great significance to improvement of the virus homologous recombination efficiency, has high application prospect in single cell separation, particularly recombination virus screening, and can be widely applied to the recombination virus screening.

The present invention relates to a noninfectious virus recombinant gene SARS-CoV-EGFP for screening medicine for resisting SARS virus and making basic research of virology. In 5' end of total genome of SARS coronate virus the promotor sequence T7 Pro. is introduced, the coding sequence of external structural domain related to viral pathogenicity and toxicity in S protein can be sustituted with EGFD gene of code enhanced green fluorescin, and in the 3' end of total genome HDV antisense genome ribozyme sequence HDV R2 and T7 transcription terminator sequence T7 Ter are successively introduced to create recombinant gene SARS-CoV-EGFP, then it is inserted into carrier to construct recombinant plasmid pUC19/SARS-CoV-EGFP, after cell transfection, through replication, transcription, traslation, processing and packaging to obtain recombinant virus capable of making transfected cell give out green fluorescence under the fluorescent microscope.

The application discloses a GAMYB gene based cultivation method for a transgenic rice sterile line. The GAMYB gene based cultivation method comprises the steps as follows: A, acquisition of a rice gene cassette; B, acquisition of a maize lethal gene ZMAA1; C, amplification of a maize pollen specific promoter Pg47; D, acquisition of an EGFP gene cassette; and E, connection of each gene expression element. The step E of connection of each gene expression element comprises: a first step of introducing a complete rice GAMYB gene into a pCAMBIA1300 vector, and then connecting the maize lethal geneZMAA1, the maize pollen specific promoter pPg47 and the EGFP gene expression element onto the pCAMBIA1300 vector. According to the invention, a F1-generation heterozygote follows the Mendel segregation law in the self fruitfulness process, and generated progenies not only contain heterozygotes which can keep a triple linked gene, but also contain the sterile line without fertility.

The invention discloses a duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein (EGFP) genes and a constructing method and application of the duck plague virus recombinant vaccine strain rDEVTK-EGFP. The microorganism preservation number of the vaccine strain rDEVTK-EGFP is CGMCC No. 9456. According to the duck plague virus recombinant vaccine strain rDEVTK-EGFP, the constructing method and the application, a recombinant clone technology is adopted; a gene segment sCMV-EGFP containing enhanced green fluorescent proteins (EGFP) and sCMV promoter sequences is inserted into a duck plague virus TK gene region; recombinant EGFP duck plague viruses with an sCMV-EGFP expression cassette inserted into the corresponding position of a TK gene is constructed; the EGFP genes are stably expressed by the recombinant viruses. The invention further relates to a method for constructing the recombinant duck plague virus vaccine strain for stably expressing other poultry pathogeny exogenous genes and the application of the recombinant duck plague virus vaccine strain to preparation of vaccines for preventing duck plagues and other poultry infectious diseases.

The invention provides to a method for constructing a recombinant rhabdovirus expression vector for EGFP (enhanced green fluorescent protein) gene, relating to a method for constructing the recombinant rhabdovirus expression vector for the EGFP gene. The invention aims to solve the problems of low transduction efficiency, large distance in expression efficiency from requirement of industrial production, short expression time and the like of the traditional recombinant rhabdovirus expression vector. The method comprises the following steps: firstly, amplifying EF1 alpha gene through PCR (polymerase chain reaction); secondly, constructing a plasmid pT-EF1 alpha; thirdly, constructing a plasmid pWK; fourthly, amplifying L-ITR (L-inverted terminal repeat) gene through PCR, and constructing pWK-L-ITR; fifthly, amplifying R-ITR gene through PCR, and constructing pWK-ITR; sixthly, amplifying the EGFP gene through PCR, and constructing the recombinant rhabdovirus expression vector pWK-ITR-EGFP for the EGFP gene. In the invention, through the addition of elements of VSV (vesicular stomatitis virus)-GED, WPRE (Woodchuck Posttranscriptional Regulatory Element) and ITR, the expression efficiency of exogenous genes is enhanced, the transduction efficiency of virus is improved, and the expression time is prolonged.

The invention relates to a genetic modification method capable of enhancing disinsection efficiency of baculovirus, belonging to the field of gene engineering. The method comprises the following steps: amplifying EGFP gene by using a pEGFP-N1 vector as a template, carrying out double enzyme digestion, and connecting into a plasmid pFastBacDUAL to obtain a recombinant plasmid pDUAL-EGFP; designing and synthesizing primers according to the Clbi138 gene sequence and open reading frame thereof; carrying out PCR (polymerase chain reaction) amplification by using greenish brown hawk moth karyotype polyhedrosis virus genome DNA (deoxyribonucleic acid) as a template, recovering the target segment, carrying out enzyme digestion, and connecting into the recombinant plasmid pDUAL-EGFP subjected to double enzyme digestion to obtain a recombinant plasmid pDUAL-EGFP-Clbi138; and transforming a Escherichia coli strain containing silkworm karyotype polyhedrosis virus, culturing, purifying, inoculating into an LB (Langmuir-Blodgett) liquid culture medium, carrying out shake culture, and extracting DNA of the recombinant BmNPV. The experiment proves for the first time that the disinsection efficiency of the recombinant BmNPV is obviously enhanced, the median lethal concentration is reduced by 11 times as compared with the control group, the median lethal time is shortened by 42.9% as compared with the control group, and the liquefaction in the sick polypide is more severe, thereby achieving the effect of enhancing control effects on pests. The recombinant BmNPV has obvious economic and ecological benefits, and has wide application prospects.

The invention discloses a double-fluorescence screening method for fungal gene knockout, and relates to the technical field of fungal gene knockout. The method comprises the steps that a 2.9-kb URA3-2micro2_origin fragment in a pYES2 carrier DNA template is amplified, the fragment is inserted into a pCAMBIA1300 carrier, and a pUM carrier is obtained; then, a tef1 promoter, an eGFP gene, a PgpdA promoter, a ble resistance gene, an RFP gene, a TtrpC terminator, an upstream homologous arm of a target gene x and a downstream homologous arm of the target gene x are amplified respectively; finally,a knockout carrier pKO-x of the target gene x is established; the pKO-x is transformed into agrobacterium competent cells, fungi is transformed through an agrobacterium mediated method, antibiotic screening is carried out to obtain a resistance transformant, then fluorescence microscope detection is carried out on the resistance transformant, the resistance transformant is a target transformant ifonly red fluorescence is emitted, the resistance transformant is an ectopic integration transformant if both green fluorescence and red fluorescence exist, and the transformant is a wild type if no fluorescence is emitted. The method can be used for effectively distinguishing the target transformant from the ectopic integration transformant.

The invention relates to a visual tracking method for rapid expression of an NS1 protein by a bombyx mori bioreactor. The method comprises the steps: a donor plasmid carrying an egfp expression cassette and an nsl gene expression cassette is constructed, an scherichia coli DH10B competent cell is converted by the donor plasmid, the egfp expression cassette and the nsl gene expression cassette which are between Tn7R-Tn7L of sequences of the donor plasmid can be specifically transposed onto a Bm-Bacmid vector, an integrated recombinant eukaryotic expression vector for expressing an egfp gene and an nsl gene is constructed, a bombyx mori is infected by recombinant buddedvirus BV particles produced after insect cells are transfected with the integrated recombinant eukaryotic expression vector, and real-time monitoring of a situation of virus multiplication in a bombyx mori body is carried out through visual green fluorescent signals. The method can perform large-scale and real-time monitoring of expression dynamic situation of an exogenous gene in the bombyx mori body, and lays the foundation for revealing characteristics of the NS1 protein and other functional proteins.

The invention discloses a luciferase reporter gene dog interferon alpha biological activity detection method and application thereof. The method comprises the following steps: performing PCR amplification, and obtaining pM*1 gene segments of M*1 proteins; inserting the pM*1 gene segments into a 5' end of a pGL3-basic carrier luc gene, then performing PCR amplification, and obtaining pM*1-luc fusedgene segments; using the pM*1-luc fused gene segments to substitute pCMV and EGFP gene segments in a pEGFP-N1 carrier, building pM*1-luc plasmids, transfecting cells, and selecting a stable transfected cell line; performing cloning culture on the selected stable transfected cell line; and preparing a standard curve by adopting a gradient diluted dog interferon alpha standard product, co-incubating a dog interferon alpha sample to be detected in the cloning cultured cell, detecting a fluorescence intensity, and evaluating titer of the dog interferon alpha sample to be detected according to thestandard curve.

The invention discloses a method for breeding transgenic buffalos by using somatic cell nuclear transfer technology, which comprises: obtaining buffalo fetal fibroblasts (BFF) by separation from buffalo fetuses and culture, implanting a transgenic vector (pCE-EGFP-IRES-neo), which carries a neomycin resistant gene and an enhanced green fluorescent protein (EGFP) gene, into the BFF by an electroporation process, and selecting BFF into which the EGFP gene is transferred by using G418; transplanting the BFF into which the EGFP gene is transferred into denucleated oocytes of the buffalo to construct cloned embryos, treating the cloned embryos for 5 minutes with ionomycin at a concentration of 5 mu mol/L, culturing cloned embryos in 6-dimethyl-aminopurine at a concentration of 2mmol/L for 3 hours, performing chemical activation treatment, co-culturing the cloned embryos in granulose cell monolayer liquid drops for 6 to 7 days, and obtaining the transgenic cloned blastaea; selecting transgenic cloned blastaea in which the EGFP is expressed under a reversed fluorescence microscope, and transplanting the transgenic cloned blastaea into receptor buffalos; and obtaining the transgenic buffalos after full-term pregnancy. After the irradiation by an ultraviolet lamp, the EGFP marker genes are obviously expressed in the heads and limbs of the transgenic buffalo calves, and this proves the method disclosed by the invention can successfully breed transgenic cloned buffalos.

The invention discloses a lentiviral vector, wherein the original hUbc promoter in FUGW is replaced by a U6 promoter or a strong promoter EF1alpha, the original EGFP gene is replaced by a strong fluorescent protein copGFP or a Tomato gene, a eukaryon resistance label gene, multiple clone sites and tag protein Flag are linked to the same cistron of the gene, and splicing protein T2A and P2A are adopted to link a plurality of genes on the same cistron. Compared with the conventional lentivirus vector, the lentiviral vector of the present invention has the following advantages that: a plurality of proteins can be concurrently and efficiently expressed with the same cistron, fluorescence intensity is high, induced function recovery is performed on the interfered gene, the vector is suitable for carrying large fragment genes, and the like. In addition, the lentivirus vector has characteristics of wide application, high efficiency, benefits for applications of the lentiviral vector in clinical and science researches, good practical value and good application prospects.

The invention discloses a lentiviral vector of fusion green fluorescent protein with over expression of RAB7A and EGFP genes, a building method and application thereof. The vector takes a lentiviral vector LV11 as a skeleton vector, wherein an EGFP gene segments and RAB7A gene segments are inserted behind a CMV promoter of the vector LV11 in a direction from 5' to 3'. By virtue of the lentiviral vector of fusion green fluorescent protein with over expression of RAB7A and EGFP genes, the problem that over-expression vector is effective and intuitive in the study of RAB7A genes can be solved. The vector takes CMV as the promoter, so that the high-efficiency expression of eukaryotic gene can be ensured; EGFP is used as a marker, the autophagy and endocytosis degree of cells and the effect of RAB7A genes in cell hypoxia process can be judged by intuitively observing the intensity of the expressed fluorescence; a good tool is provided for studying the functions of the RAB7A genes; the basis is provided for the subsequent correlation studies of RAB7A.