107 results about "Enhanced green fluorescent protein" patented technology

The invention belongs to the field of biomedical science, and aims to provide a high metastatic potential hepatoma cell line capable of steady autophagy indication, and an establishment method and an application method thereof. Lentivirus expression vectors containing EGFP (enhanced green fluorescent protein)-LC3 (light chain 3) autophagy reporter genes are constructed and used to infect hepatoma cells with high metastatic potential, so that host cells can express the EGFP-LC3 steadily and efficiently and indicate autophagy changes of the hepatoma cells steadily. To apply the cell line, metastasis research models can be established in vitro and in vivo for the observation on autophagy changes during metastasis, and the cell autophagy changes can be analyzed rapidly and quantitatively by adopting an image analysis method based on Top-hat operators and morphological filter. The high metastatic potential hepatoma cell line has the characteristics that autophagy indication is conducted steadily, reliably and sensitively in a real-time manner, the metastasis occurs definitely, the autophagy is quantified rapidly and accurately, the operation is simple and convenient, and the like, and can greatly facilitate correlation study on autophagy and metastasis.

The invention relates to the field of genetic engineering, and particularly provides a self-deleting free carrier. A core function region of the free carrier comprises a human EF1alpha promoter sequence, a 1oxp sequence, an enhanced green fluorescent protein (EGFP) encoding sequence, an S/MAR sequence from a human beta interferon gene, a PolyA sequence of SV40, an expression cassette sequence of a mice Oct4 promoter drive Cre gene, and a 1oxp sequence from 5'-terminal to 3'-terminal. Preparation of gene targeting cells and animals is carried out by using the carrier disclosed by the invention, genes can be deleted, screened and marked and no gene insertion mutation is generated, and the effect on adjacent gene expression caused by a marker gene and potential biological safety hazard on an organism are removed.

The invention relates to a method for screening non-essential regions for replication of a goat pox virus and universal transfer vectors for same. The method comprises the steps of amplifying two-end gene segments of any two regions of a goat pox virus gene by using a PCR (Polymerase Chain Reaction) method; then, inserting an enhanced green fluorescent protein (EGFP) gene and a xanthine-guanine phosphoribosyl transferase (gpt) gene expression cassette into the segments; establishing two universal transfer vectors of the goat pox virus; and acquiring a recombinant virus expressing an exogenous gene stably from the transfer vectors, thereby determining the selected regions to be non-essential regions for replication of the goat pox virus, wherein each universal transfer vector contains one unique restriction enzyme cutting site Sal I and allows gene expression cassettes of other items to insert in. The recombinant virus obtained by means of the two universal transfer vectors provided by the invention not only has a growth performance similar to a parent virus, but also has better safety because a plurality of toxicity related genes in a genome are knocked out in an orientation way, and has the potential to be developed into an attenuated vaccine strain for gene engineering.

The invention discloses a method for synthetizing secretion lysozyme by middle silkgland cell of silkworm. The method comprises the following steps: building a pBSer1hLYZ (lysozyme)-A3EGFP (Enhanced Green Fluorescent Protein) plasmid for synthetizing secretion lysozyme by the silkworm; then, introducing the plasmid and an assistant plasmid capable of providing transposase into a silkworm germ cell according to the microinjection transgenosis silkworm technology; according to the transposition characteristics of a piggyBac transposon, introducing a green fluorescent protein gene and a lysozymegene into a silkworm gene group, to obtain stable heredity and expression so as to create the transgenosis silkworm capable of synthetizing secretion lysozyme by middle silkgland cell of silkworm by specificity; further, hybridizing the transgenosis silkworm with sericin silkworm; carrying out back crossing on the hybridized descendant with the sericin silkworm for 3-5 generations; and finally, carrying out selfing on the obtained product to carry out homozygosis on the lysozyme gene so as to obtain the new species of the transgenosis silkworm of the secretion lysozyme. According to the method, a basis for improving lysozyme production efficiency and lowering production cost is laid.

The invention discloses a recombinant porcine pseudorabies virus strain (belonging to Herpesviridae) and a preparation method thereof. The recombinant porcine pseudorabies virus strain has the accession number of CCTCC NO: V201345. Based on porcine pseudorabies virus vectors, green fluorescent protein labels are used so that green fluorescent protein expression gG-less universal transfer vector PG is constructed, and the recombinant porcine pseudorabies virus strain contains a pseudorabies virus self-late gene gG promoter, a CMV promoter and SV40Poly(A), has upstream flanks of 0.8kb and downstream flanks of 1.7kb and completely satisfies homologous recombination demands. Enhanced green fluorescent protein (EGFP) can be used as a gene expression indicator and comprises porcine IL-18 gene inserted into multiple cloning sites of the EGFP so that connection construction of different expression cassettes by a common vector is realized and two expression cassettes are individually expressed. The exogenous gene expression quantity is closely related to the connection direction of the two expression cassettes.

The invention aims at providing a duck plague virus gene deletion transfer vector and an application of the duck plague virus gene deletion transfer vector. The transfer vector is subjected to homologous recombination with duck plague virus to obtain duck plague virus gene deletion engineering strains which can be prepared into a high-safety and high-prevention-effect duck plague virus gene deletion vaccine. The duck plague virus gene deletion vaccine does not influence the virus immunogenicity while lowering down the virus pathogenicity; therefore, the prepared duck plague virus gene deletion vaccines are high in safety, able to remain the original virus immunogenicity, suitable for prevention and control for the current duck plague diseases in domestic, and beneficial for the cleaning treatment of duck plague in a duck farm; in addition, the recombined duck plague virus strains do not contain any exogenous genes; and compared with the similar duck plague virus gene vaccines reported in the prior art, the duck plague virus gene deletion vaccine has the advantage that no EGFP (Enhanced Green Fluorescent Protein), lacZ and other marker gene are contained, so that the bio-safety is improved.

The invention discloses a multimeric protein having the effect of brain targeting, and a preparation method and usage thereof. The method comprises the steps: firstly, expressing cholera toxin B subunit and a fusion protein EGFP-CTA2-TAT of three proteins: an enhanced green fluorescent protein, a cholera toxin A subunit and cell-penetrating peptide in escherichia coli by incompatible double plasmid systems to obtain CTB gene by PCR amplification; cloning the gene segment into a carrier pET-28a to obtain a recombinant plasmid pET-28a-CTB; using wild type CTA2, EGFP and TAT amino acid sequences as templates, inserting 3 enzyme cutting sites and linkers between EGFP and CTA2, and optimizing to obtain codons suitable for expression in escherichia coli; cloning the gene segment into a carrier PET-22b (+) to obtain recombinant plasmid PET-22b-EGFP-CTA2-TAT; using different resistances of PET-28a-CTB and PET-22b-EGFP-CTA2-TAT, and co-transforming the different resistances of the PET-28a-CTB and the PET-22b-EGFP-CTA2-TAT into escherichia coli BL21, to obtain engineering bacteria after screening under double resistance selection pressure of penbritin and kanamycin. CTB5/EGFP-CTA2-TAT chimeric proteins can be obtained after inducible expression of the engineering bacteria by IPTG. The invention further discloses the preparation method and usage of the protein.

The present invention is directed to a transformation system for making transgenic organisms that includes a vector containing a modified piggyBac transposon into which is inserted an enhanced green fluorescent protein gene linked to a polyubiquitin promoter sequence and a nuclear localizing sequence; and a helper transposase vector that includes an hsp70 promoter sequence upstream of the putative piggyBac promoter that increases the transformation frequency of this system.

The invention belongs to the technical field of cell research, and particularly relates to a method for constructing a laryngeal cancer cell line for stably expressing the green fluorescent protein. The method comprises the following steps: (1) designing and synthesizing a primer; (2) digesting the gene sequence of pLenti-puro carrier plasmids with BamHI and XhoI according to the gene sequence andpLenti-puro sequence of the green fluorescent protein, and connecting digested products to obtain a target carrier pLenti-puro-GFP; (3) verifying the carrier pLenti-puro-GFP with endotoxin extraction, digesting the pLenti-puro-GFP plasmids with BamHI and XhoI, and detecting the gene sequence by agarose gel electrophoresis, and sequencing the carrier pLenti-puro-GFP plasmids to detect the sequencesituation of the GFP-LC3; and (4) co-transfecting a 293T cell with the carrier pLenti-puro-GFP plasmids and a lentivirus package carrier, collecting virus supernatant, and transfecting a laryngeal cancer cell Hep-2 with the virus supernatant, infecting for 24 hours, then adding a culture medium containing puromycin, screening the cell, and observing under high content until a stable strain is obtained.

The invention discloses a preparation method and an application for a dual-fluorescence reporting system for detecting a micro-RNA (ribonucleic acid) function, wherein the preparation method comprises the following preparation steps of: A, obtaining a mCherry gene sequence via a PCR (polymerase chain reaction) amplification, inserting a rho EGFP-C1 carrier to replace the EGFP (enhanced green fluorescent protein) sequence of the mCherry gene sequence, and constructing a carrier rho mCherry-C1; B, using a RNAi-Ready rho SIREN-RetroQ plasmid as a template, obtaining a human U6 promoter gene via a PCR amplification, and inserting the carrier rho mCherry-C1 to obtain a plasmid rho hU6-mCherry-C1; and C, obtaining a gene sequence carried with a CMV (cytomegalovirus) promoter and an EGFP expression dialog sequence in an interval of 358 to 1944 on a plasmid rho Adtrack-CMV, and inserting the sequence in the plasmid rho hU6-mCherry-C1 to obtain a plasmid rho MGhU6. The plasmid rho MGhU6 is a dual-fluorescence reporting system simultaneously containing a mCherry reporting gene, an internal reference EGFP, a micro-RNA and the target sequence insertion site thereof. The reporting system can be used for detecting the inhibition function of the micro-RNA to the target sequence expression. The detection method is easy, as well as simple and convenient in operation; and a function detection for the micro-RNA can be realized by transfecting one plasmid only. The system also can be used for visually detecting the inhibition function of a micro-RNA in a single living cell to the target thereof by the aid of fluorescence microscopic imaging.

The invention discloses a Cre fusion protein functional test cell and a modified Cre fusion protein. The Cre fusion protein functional test cell takes an HEK (human embryonic kidney) 293 cell as a host cell, a pAcmy (African cassava mosaic virus)-stop-EGFP (enhanced green fluorescent protein) carrier and phi C31 integrase are used for co-transfection of the host cell. Through the action of the phi C31 integrase, the carrier which carries an LoxP (locus of X-over P1) element having a Cre recombinase detection function is integrated to a pseudo attP (attachment P) site of an HEK 293 cell line in a single copying manner, so a stable and reliable cell model is provided for researching the Cre fusion protein membrane penetrating efficiency and biological activity. The Cre fusion protein HNTC (hemagglutinin-neuraminidase cytotoxic T) which is modified by TAT (transactivator) and NLS (nuclear localization signal) polypeptides has high membrane penetrating efficiency and good biological activity, can be effectively used as a tool for genetic manipulation at a mammalian cell chromosome level.

The invention relates to an expression vector containing a green fluorescent protein gene. The green fluorescent protein gene is a gene sfGFP, wherein the nucleotide sequence of the gene sfGFP is shown in SEQ ID NO: 1. The invention further relates to a construction method of the expression vector containing the green fluorescent protein gene and application of the expression vector containing the green fluorescent protein gene in positive clone screening. According to the expression vector containing the green fluorescent protein gene and the construction method and application thereof, the characteristic that the sfGFP can complete super-folding and illuminate under various conditions is utilized, the advantages of luminous power and luminous intensity of the sfGFP are obviously stronger than those of ordinary proteins, such as GFP (Green Fluorescent Protein), EGFP (Enhanced Green Fluorescent Protein) and the like, and are 1.6 times the EGFP, and obvious green fluorescent light can be observed by unaided eyes, so that the screening of positive clones during protein expressed cloning can be facilitated, and the fast and accurate screening for the positive clones is facilitated.

The invention discloses a CA16 infectious clone with a green fluorescent protein gene as well as a construction method and an application of the CA16 infectious clone. The method comprises the steps of firstly, inserting full-length cDNA (complementary deoxyribonucleic acid) of a CA16/GD09/24 virus strain by taking a low copy number plasmid pACYC177 as a carrier; then, inserting an eGFP (enhanced green fluorescent protein) reporter gene and adding a 2A protease cleavage site (AITTL) between 5'UTR and VP4 by taking the full-length infectious clone as a skeleton so as to obtain a full-length infectious clone of the CA16 with the eGFP reporter gene. The CA16 infectious clone with the green fluorescent protein gene, constructed by the method, disclosed by the invention, can be used for saving an eGFP-CA16 reporter virus of which the growth tendency is similar to that of a recombinant CA16 virus and a parent CA16, and can also be used for screening antiviral drugs, thereby providing a convenient platform for deepening virus replication and pathogenesis researches and laying a solid foundation for screening and developing novel vaccines in future.

The invention relates to an rHVT (recombinant Herpesvirus of Turkey)-H9HA (H9 hemagglutinin) for expressing H9 subtype AIV (avian influenza virus) HA protein and a construction method thereof. The collection number of a vaccine strain rHVT-H9HA is CGMCC No: 10907. A GFP (green fluorescent protein) expression cassette is separated from a carrier pEGFP-C1 (plasmid enhanced green fluorescent protein) and is inserted into an HVT genome, and a recombinant virus rHVT-GFP is obtained. Through homologous recombination, a GFP gene of the rHVT-GFP is replaced with an HA gene of H9N2 subtype AIV epidemic strain A/Chicken/Jiangsu/WJ57/2012, a recombinant virus without fluorescence is selected, and the rHVT-H9HA for stably expressing the H9 subtype AIV HA gene is obtained. The recombinant virus strain is low in cost, good in safety, long in immunity period and suitable for large-scale production of vaccine and can be used for making the vaccine.

The invention relates to a high-throughput screening system for breeding high-nucleic-acid yeast. The high-throughput screening system comprises reporter plasmid Yep-Hyg B-yeGFP and a host cell containing the Yep-Hyg B-yeGFP, wherein the reporter plasmid Yep-Hyg B-yeGFP is annular and sequentially comprises a YEplac195 plasmid skeleton, a yeast enhanced green fluorescent protein gene yeGFP expression box and a hygromycin B resistance gene expression box from 3' to 5'; the original strain of the host cell is a saccharomyces cerevisiae industrial strain whose nucleic acid content is confirmed tobe higher than a normal value through screening. The invention further discloses application of the screening system in high-nucleic-acid yeast engineering bacterium breeding. The screening system has the advantages that the defect that traditional high-nucleic-acid yeast breeding cannot directly reflect RNA content can be overcome, the instability of the plasmid in the yeast can be utilized to easily eliminate the plasmid through continuous passage, and the system is significant to the application of the high-nucleic-acid yeast in food industry.

The invention discloses fusion protein for screening and evaluating an anti-enterovirus 71 (EV 71) medicine and application of the fusion protein. According to the fusion protein, firefly luciferase (Fluc) is used as a reporter gene, two micromolecule polypeptides which can be bonded tightly pepA and pepB are bonded with the N end and the C end of the firefly luciferase respectively, the middles of the firefly luciferase are connected by an EV713C protease effect substrate, and a fusion gene is inserted into an eukaryotic expression vector and is expressed to generate the fusion protein. Simultaneously, enhanced green fluorescent protein (EGFP) which is inserted reversely can indicate the transfection efficiency of the EGFP in cells which are transfected in vitro by observing the condition of green fluorescence. By utilizing an indication vector provided by the fusion protein, the reproduction condition of EV 71 can be indicated simply, quickly, flexibly and quantitatively, and the indication vector also can be applied to the screening and evaluation of the anti-EV 71 medicine, has high actual application value and has a broad application prospect in the field of medical science.

The invention provides large stokes displacement fluorescent protein CyOFP and application of the fluorescent protein to microimaging and highly-sensitive living creature light-emitting imaging. The fluorescent protein is obtained through mNeptune site directed mutagenesis, the 1-230 positions of the amino acid sequence of the fluorescent protein sequentially correspond to 4-234 positions shown by the SEQ ID No: 2, or the fluorescent protein can be further obtained by conducting gene synthesizing on the fluorescent protein DNA segment and expression. The invention further provides a novel BRET system and fusion protein Antares obtained through system optimization. The CyOFP can be stimulated by blue light, and a quite high quantum yield is achived; the CyOFP and the enhanced green fluorescent protein (EGFP) are jointly stimulated by monochromatic light at the same time in a two-photon mode, and the resolution ratio of two-photon imaging is raised; a novel BRET system formed together with fluorescent protease NanoLuc greatly improves the depth and sensitivity of living creature imaging.

The invention provides a transgenic carrier with function of automatic control and elimination of selection marker and application thereof in zea mays marker-free transgenic breeding, and belongs to the technical field of breeding of zea mays molecules. The carrier uses a pEGAD carrier as a skeleton carrier, and a fusion gene of EGFP (enhanced green fluorescent protein) and IPT (isopentenyl transferase) genes is used for replacing a bar gene and is used as the selection marker; an expression framework of the selection marker is integrated into a Cre/lox site specific recombination system. Theapplication comprises the following steps of transferring the carrier into a zea mays embryonic callus, obtaining the zea mays embryonic callus with the green fluorescent expression effect; after heatshock, culturing the zea mays embryonic callus without the green fluorescent expression effect by differentiating and rooting, so as to obtain a transgenic plant. The carrier has the advantages thatthe selection efficiency is improved, the removal of the transgene selection marker is realized, and the theoretical and application basis is provided for the large-range development of marker-free zea mays transgene.

The present invention discloses a recombinant canine adenovirus-2 transfer vector, a method for constructing the recombinant canine adenovirus-2 transfer vector and an applications thereof. The recombinant canine adenovirus-2 transfer vector loses the 1412bp fragment of the E3 region, a large fragment of exogenous gene can be inserted, and moreover, a hCMV IE promoter, a multiple cloning site, an enhanced green fluorescent protein gene and a SV40 early transcribed Poly A signal sequence can be inserted in the position of the lost fragment of the E3 region. The method successfully constructs the lost recombinant CAV-2 transfer vector of the E3 region, obtains a purified canine adenovirus-2 strain containing EGFP reporter gene and optimizes the cloning and purification method thereof, an evaluation indicates that the biological property of the recombinant virus is stable, and therefore the present invention provides a technical platform for the further development of CAV-2 live vector vaccine and related fundamental researches.

The invention discloses a degumming method applicable to green fluorescent protein-containing bave. The degumming method comprises the following steps: drying the bave to be constant-weight, soaking the bave in a pretreating solution in a bath ratio of 1: 50, degumming for 30-120 minutes under the conditions that the concentration of alkaline protease is 0.5-3g/L and the temperature is 40-60 DEG C, washing by water, and drying to obtain the degummed bave, wherein the pretreating solution is a solution with pH of 10 and containing 1g/L sodium carbonate and 0.5g/L penetrant JFC. The degumming method is suitable for degumming the green fluorescent protein-containing bave; by the degumming method, the degumming rate is high and the fluorescent characteristic of the bave can be retained to the maximum extent; after the bave is degummed through the degumming method, the bave has relatively small mechanical performance loss, soft hand feel and good mercerization; through the degumming method, a requirement on degumming of transgenic green bave can be met.

A method for observing the infection process of citrus canker pathogens in real time comprises the following steps: (1) guiding an enhanced green fluorescent protein gene (egfp) on an extensive host vector pMP2463 into a wild-type citrus canker pathogen to obtain an enhanced green fluorescent protein (EGFP); (2) inoculating the obtained EGFP-marked citrus canker pathogens onto the surface of a citrus leaf in a spraying way, and inoculating the obtained EGFP-marked citrus canker pathogens into mesophyll clearances of the citrus leaf through injection; and (3) sampling by tearing leaf epidermisor by a frozen section method, and observing the number and the distribution of the citrus canker pathogens in mesophyll clearances and on the surface of the citrus leaf under a laser confocal microscope. Through the adoption of the method, the attachment, breeding and spatial distribution of the EGFP-marked citrus canker pathogens in the process of infecting citrus can be observed in real time so as to obtain interactive spatial and temporal relations of citrus canker pathogens and the citrus; and the method is easy and quick to operate.

The invention relates to a method for producing transgenic buffalo embryos by applying intracytoplasmic sperm injection (ICSI) mediation. The method comprises the following steps of: 1, incubating buffalo sperms and transgenic deoxyribonucleic acid (DNA) together; 2, performing intracytoplasmic microinjection on the buffalo sperms; and 3, cultivating and identifying the transgenic buffalo embryos. By the method of the invention, the transgenic buffalo embryos are obtained successfully. In 102 injection spawns of the transgenic buffalo embryos, 61.8 percent of the injection spawns are subjected to cleavage, 17 blastospheres are obtained in embryos expressed by blastomere serving as enhanced green fluorescent protein (EGFP), 11 blastospheres express the EGFP, and the transgenic positive rate of the blastospheres is up to 64.7 percent; and after one of the transgenic embryos is cultured in vitro for a long time, the expression of the EGFP can be still seen during 2 to 7 days, so the method makes the important progress for producing transgenic buffalos further.

The invention discloses an attachment expression vector of cells of human and other mammals, an expression system, a preparation method and application and belongs to the technical fields of genetic engineering and gene therapy. A segment beta-interferon MAR (matrix attachment region) sequence (or an nucleotide sequence which is 95 percent homologous with the sequence or more), which is shown as SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, is inserted into the attachment expression vector; compared with an expression vector containing a whole-length MAR sequence or a 387bp MAR sequence, the vector can efficiently, lastingly and stably express exogenous target genes; especially, the expression of the vector containing an E-segment MAR sequence is optimum and the expression vector can be used for the gene therapy. In the vector, a multiple cloning site where the segment MAR sequence is inserted into the vector is located at the downstream of eGFP (enhanced Green Fluorescent Protein); on one hand, transgene silencing can be overcome; on the other hand, an expression level of a target protein can be improved and the effectiveness of subsequent monoclonal cell strain screening are improved.

The invention relates to a cereal cyst nematode Ha34609 protein, an encoding gene and application of the encoding gene. The amino acid sequence of the cereal cyst nematode Ha34609 protein is shown as SEQ ID NO: 1. The nucleotide sequence of the encoding gene is shown as SEQ ID NO: 2. After a silent Ha34609 gene is treated by adopting dsRNA (double-stranded ribonucleic acid), compared with eGFP (enhanced green fluorescent protein) dsRNA, the length and the width of a white female worm are remarkably reduced (t-test, confidence interval: 95%), which indicates that the gene plays an important role in parasitic and pathogenic processes of cereal cyst nematodes and can serve as a target gene of plant nematode resistance engineering. The cereal cyst nematode Ha34609 protein, the encoding gene and the application of the encoding gene have significant values in a study on a nosogenesis of the cyst nematodes and preparation of nematode resistance plants.