62 results about "Reverse genetics" patented technology

The invention discloses a method for quickly rescuing bovine coronavirus epidemic strains and application of the method. The bovine coronavirus SHZ isolate is subjected to efficient segmented cloning, the obtained segment and a Linker sequence are subjected to a cyclic polymerase extension reaction, and the obtained reaction product can be directly transfected to 293T cells for virus packaging without purification. Different from a traditional method, the method does not need to amplify full-length virus cDNA clone by means of bacteria and yeast, but directly obtains a sufficient amount of preliminary products through PCR, and avoids the problems of instability and low efficiency when partial sequences of a virus genome proliferate in a bacteria or yeast host. By adopting the reverse genetic system, the recombinant bovine coronavirus expressing the foreign protein is successfully rescued, a brand new technical platform is provided for dynamic visualization research of in-vivo and in-vitro replication of the virus, and an efficient and flexible tool is also provided for virology research, development of virus vector vaccines and screening of antiviral drugs.

The invention discloses a sugarcane ScPP2C49 gene and application thereof, and relates to the technical field of plant biology, the nucleotide sequence of the ScPP2C49 gene is as shown in SEQ ID No.1, and the amino acid sequence of protein coded by the ScPP2C49 gene is as shown in SEQ ID No.2. The gene is induced to express by drought and abscisic acid, and the encoded protein of the gene is positioned in a cell nucleus. Functional studies show that after overexpression of the ScPP2C49 gene in the plant, the drought resistance of the plant is negatively regulated through various mechanisms of inhibiting an abscisic acid signal channel, hindering stomatal closure under drought stress, weakening active oxygen scavenging ability, reducing photosynthetic efficiency, inhibiting root growth and the like. The invention also provides a recombinant overexpression vector containing the gene and a method for obtaining a transgenic plant with reduced drought resistance by using the vector through an agrobacterium-mediated method. According to the invention, a new gene resource is provided for deep analysis of a plant drought-resistant molecular mechanism, and an important target gene is provided for cultivation of high-stress-resistance crop varieties through a reverse genetics means (such as gene knockout).

The application belongs to the technical field of biotechnology, and particularly relates to a porcine epidemic diarrhea virus recombinant strain, a construction method and application. The S gene sequence of the existing epidemic porcine epidemic diarrhea virus PEDV CH / SX / 2016 strain is replaced by the S2015 gene sequence shown in SEQ ID NO. 1 to construct a PEDV CH / SX / 2016 strain infectious clone. A porcine epidemic diarrhea virus recombinant strain rCH / SX / 2016-S 2015 is successfully rescued by transfecting cells with a plasmid containing the infectious cDNA shown in SEQ ID NO. 4. The problem that the original strain cannot be successfully rescued by reverse genetics engineering is solved, and the recombinant virus strain rCH / SX / 2016-S 2015 has an activity comparable to that of the original strain, and can be used for preparing a PEDV diagnostic reagent and / or vaccine.

This invention discloses a method for directed evolution of antibiotic-resistant Escherichia coli strains based on cytidine deaminase, belonging to the field of microbial directed evolution and genetic engineering technology. This method uses E. coli as the host, introducing a recombinant plasmid expressing an optimized double-stranded cytidine deaminase mutant. Utilizing the low toxicity and high mutagenicity of this mutant, continuous passage evolution is carried out under gradient concentrations of aminoglycoside antibiotics (kanamycin and streptomycin). Combined with whole-genome sequencing, molecular docking, and reverse genetics verification, the A145T missense mutation in the wcaE gene is identified as the core functional site. This invention overcomes the shortcomings of traditional spontaneous and chemical mutagenesis, which suffer from low efficiency and significant strain damage. The mutation type is controllable, the evolutionary cycle is short, and the obtained engineered strains can tolerate up to 300 mg / L kanamycin while exhibiting streptomycin cross-resistance, and the genetic stability of the tolerance trait is strong. This method is simple, highly reproducible, and suitable for industrial breeding of stress-resistant E. coli, and can be widely applied in antibiotic fermentation, industrial microbial culture, and other scenarios.

The application discloses a PEDV chimeric virus attenuation identification method based on reverse genetic operation and application of screening potential attenuated vaccine, and the PEDV chimeric virus CHM2013-SP BJ , CHM2013- BJ , CHM2013-SP BJ -ORF3 CHM , CHM2013-SP BJ -E CHM , CHM2013-SP BJ -M CHM , CHM2013-SP BJ -N CHM and CHM2013-(S+ORF3) BJ are constructed and rescued, loss-of-function test and gain-of-function test are carried out, and the attenuated chimeric virus CHM2013-SP BJ -ORF3 CHM and CHM2013-SP BJ -M CHM are obtained, and it is disclosed that the S gene of the PEDV variant BJ2011C promotes virus pathogenicity in cooperation with the ORF3, E and M genes, and the ORF3 gene is the most critical.

The high pathogenicity of the rift valley fever virus limits the research on the pathogenic mechanism and prevention, control and treatment products of the rift valley fever virus, the offspring replication-deficient virus particles are successfully saved through a reverse genetic manipulation technology, and the virus particles do not have the capacity of multiple rounds of infection on common cells; stable replication and passage can only be realized in a cell line for providing exogenous envelope glycoprotein; in addition, the virus particle carries an exogenous indication protein and can indicate cell infection. The Rift Valley fever virus-like particle provided by the invention can be used as a Rift Valley fever biosafety secondary laboratory research platform, the establishment of the virus particle greatly simplifies tedious procedures in the Rift Valley fever virus research process, and paves a way for screening of Rift Valley fever virus neutralizing antibodies, research and development of candidate vaccines and research of pathogenic mechanisms of the Rift Valley fever viruses.

This invention provides a chalcone isopentenyltransferase gene. GiPT16 This invention relates to the GiPT16 protein, amplification primer set, and applications, belonging to the field of biogenetics technology. It is based on *Glycyrrhiza inflata* (GiPT16 protein, amplification primer set, and applications). Glycyrrhiza inflata Using whole-genome sequencing data and a reverse genetics strategy, the key aromatic isopentenyltransferase GiPT16, involved in the isopentenylation modification of chalcone active ingredients, was successfully identified and functionally characterized. This enzyme was confirmed to specifically catalyze the biosynthesis of psoralen and glycyrrhizin C using DMAPP as a donor. It is the first chalcone isopentenyltransferase characterized in *Glycyrrhiza inflata*, filling a gap in the study of key enzymes in the chalcone isopentenylation metabolic pathway of this species and providing important evidence for further elucidating the molecular mechanisms of quality formation in *Glycyrrhiza inflata* medicinal materials.

The invention belongs to the field of veterinary virology, and discloses a bovine ephemeral fever virus strain and reverse genetic application thereof, and the reverse genetic steps are as follows: taking pCI plasmid as a skeleton to prepare plasmid for expressing a complete genome of bovine ephemeral fever virus; the method comprises the following steps: respectively preparing auxiliary plasmids for expressing an N protein gene, a P protein gene, an L protein gene and a G protein gene of the bovine ephemeral fever virus by taking pCAGGS plasmids as a skeleton, co-transfecting BHK-21 cells by using the five plasmids, and culturing the cells to obtain the rescued bovine ephemeral fever virus. The rescued bovine ephemeral fever virus has no obvious difference from the reproductive capacity of the parent virus, and the genome is still stable after 10 generations of continuous passage in BHK-21 cells. The invention provides methodological support for genetic modification of the bovine ephemeral fever virus, and has an application prospect.

The present application relates to viral sensitizers. More specifically, the present application relates to compounds of Formula (I), as well as methods of using such compounds as viral sensitizers, specifically for increasing permissiveness of a cell to the production of virus from reverse genetics systems, replicating viruses and increasing transduction.

The invention belongs to the technical field of virus and gene engineering, and particularly relates to an expression vector of a respiratory syncytial virus (RSV) ON1 genotype consensus sequence strain and an editing strain, a reverse genetic manipulation system and an application of the reverse genetic manipulation system of the respiratory syncytial virus ON1 genotype consensus sequence strain and the editing strain of the RSV ON1 genotype consensus sequence strain and the editing strain of the respiratory syncytial virus ON1 genotype consensus sequence strain. According to the invention, an RSV-ON1 genotype consensus sequence strain is firstly constructed and saved, on the basis, 72 nucleotides which are repeatedly inserted into a G gene of the consensus sequence strain are removed through a gene editing technology, and two strains with different virus G gene sequences are obtained. According to the invention, through construction of RSV infectious clone edited by RSV-ON1 genotype G gene and rescue of the two strains, a fast, simple and accurate RSV reverse genetic system is constructed, and the RSV reverse genetic system can be applied to research on RSV in-vitro virus replication mechanism and pathogenesis and neutralizing antibody immune escape. A etiological technical platform is provided for research, development and evaluation of RSV vaccines, antibodies, drugs and the like.

The invention discloses a recombinant foot-and-mouth disease virus carrying a HiBiT luciferase reporter gene as well as a construction method and application of the recombinant foot-and-mouth disease virus. According to the invention, based on a reverse genetics technology, on the basis of full-length cDNA infectious clone of an FMDV O / BY / CHA / 2010 strain, a recombinant foot-and-mouth disease virus carrying a HiBiT luciferase reporter gene is constructed, and the recombinant foot-and-mouth disease virus is successfully rescued in a BSR / T7 cell. Experimental results show that the recombinant foot-and-mouth disease virus has no significant difference from parent viruses in the aspects of replication ability and pathogenicity, and good biological characteristics are maintained. Meanwhile, a cell line capable of stably expressing the LgBiT protein is successfully constructed, real-time detection of light-emitting signals in the virus infection process is achieved through specific binding of the LgBiT and the HiBiT protein, and an effective technical means is provided for virus research and rapid detection.

The present application relates to viral sensitizers. More specifically, the present application relates to inhibitors of PDKP1, as well as methods of using such compounds as viral sensitizers, specifically for increasing permissiveness of a cell to the production of virus from reverse genetics systems, replicating viruses and increasing transduction.

The invention provides an astragalus membranaceus calycosin-7-methyltransferase protein AmOMT1, a coding gene, a primer group and application of the astragalus membranaceus calycosin-7-methyltransferase protein AmOMT1, and belongs to the technical field of biology. The amino acid sequence of the astragalus membranaceus calycosin-7-methyltransferase protein AmOMT1 is as shown in SEQ ID No. 1 (sequence identifier number 1). According to the present invention, based on the radix astragali seu hedysari genome and the second-generation transcriptome data, the transmethylase AmOMT1 having 4 'and 7-site methylation functions on 3', 4 ', 7-trihydroxy isoflavone simultaneously is found and identified by using the reverse genetics method, and the experiment results show that the AmOMT1 can catalyze the methylation of the 3', 4 ', 7-trihydroxy isoflavone to generate calycosin and 3', 4 '-dihydroxy-7-isoflavone, and the transmethylase AmOMT1 can catalyze the methylation of the 3', 4 ', 7-trihydroxy isoflavone can catalyze the methylation of the 3', 4 ', 7-trihydroxy isoflavone to generate the 3', 4 '-dihydroxy-7-isoflavone.

This invention belongs to the field of genetic engineering technology and relates to the application of wheat receptor kinase TaSPR12 in improving wheat disease resistance. This invention utilizes reverse genetics methods to analyze and screen wheat receptor kinase genes. TaSPR12 This gene is induced to express by the stripe rust pathogen. The wheat receptor kinase gene was knocked out using CRISPR-Cas9 gene editing technology. TaSPR12 The created loss-of-function mutants exhibited enhanced resistance to the main prevalent races of wheat stripe rust. This invention identified the wheat receptor kinase gene. TaSPR12 It plays a negative regulatory role in wheat resistance to stripe rust and can be used for genetic improvement of wheat resistance to rust.

The invention discloses a recombinant oncolytic influenza virus for delivering an anti-gpA33 * CD3 bispecific antibody and application of the recombinant oncolytic influenza virus. Belongs to the technical field of biological medicine. According to the recombinant oncolytic influenza virus provided by the invention, a heavy chain variable region and a light chain variable region of an anti-gpA33 antibody and an anti-CD3 antibody are inserted into an influenza virus genome, the recombinant oncolytic influenza virus is rescued through a reverse genetics technology, and the virus can stably express an anti-gpA33 * CD3 bispecific antibody in colorectal cancer cells; one end of the antibody can be specifically combined with CD3 molecules on the surfaces of the T cells to efficiently activate the T cells, the other end of the antibody can be specifically combined with biomarker molecules gpA33 on the surfaces of the colorectal cancer cells, and under the bridging action of the antibody, the activated T cells are collected to the surfaces of the colorectal cancer cells to induce the T cells to directly generate the effect of targeted killing of the colorectal cancer cells, so that the colorectal cancer cells are efficiently killed. Therefore, the purpose of treating tumors is achieved.

The invention belongs to the technical field of plant genetic engineering and crop breeding, and particularly relates to application of wheat lectin receptor-like kinase TaSIT2 and a coding gene TaSIT2 thereof in improving stripe rust resistance of crops. A wheat lectin receptor-like kinase gene TaSIT2 is analyzed and screened by using a reverse genetics method, a TaSIT2 gene editing wheat plant is obtained by using an agrobacterium-mediated genetic transformation method through a gene editing technology, the resistance of the transgenic plant to wheat stripe rust physiological races is remarkably enhanced, and the wheat stripe rust physiological races can be obtained. It is shown that knockout of the TaSIT2 gene enhances the resistance of wheat to stripe rust, it is determined that the wheat lectin receptor-like kinase TaSIT2 gene plays a negative regulation role in wheat stripe rust resistance, and a new technical thought and gene resources are provided for wheat stripe rust resistance variety cultivation from the perspective of molecular biology. And more paths are provided for cultivating lasting broad-spectrum disease-resistant varieties.

The invention belongs to the field of veterinary biological products, and particularly relates to an O-type foot-and-mouth disease strain containing a VP1 N133K mutation site and a construction method of the O-type foot-and-mouth disease strain. The invention provides an attenuated O-type foot and mouth disease virus mutant strain. Asparagine at the 133rd site of VP1 protein is mutated into lysine. The amino acid sequence of the mutated VP1 protein is as shown in SEQ ID NO: 2. The invention also provides a construction method of the attenuated O-type foot-and-mouth disease virus mutant strain, wherein N133K mutation is introduced by adopting a reverse genetics technology. The FMDV VP1 N133K mutant strain constructed by the method disclosed by the invention is beneficial to research on an FMDV attenuating mechanism. In addition, by reversely introducing VP1 N133K mutation, conservative epitopes can be screened or multivalent vaccines can be designed, and the cross protection capability is improved.

The present application relates to viral sensitizers. More specifically, the present application relates to compounds of Formula (I), as well as methods of using such compounds as viral sensitizers, specifically for increasing permissiveness of a cell to the production of virus from reverse genetics systems, replicating viruses, and increasing transduction. Formula (I).

The application provides a astragalus membranaceus calyx heterophylla isoflavone methyltransferase protein AmOMT1, a coding gene, a primer set and application thereof, and belongs to the technical field of biotechnology.The amino acid sequence of the astragalus membranaceus calyx heterophylla isoflavone methyltransferase protein AmOMT1 is shown as SEQ ID No.1.Based on astragalus membranaceus genomic and second-generation transcriptomic data, a methyltransferase AmOMT1 with 4' and 7 position methylation functions for 3', 4', 7-trihydroxyisoflavone is found and identified by a reverse genetics method.Experiments show that AmOMT1 can catalyze the methylation of 3', 4', 7-trihydroxyisoflavone to generate calyx heterophylla isoflavone and 3', 4'-dihydroxy-7-methoxyisoflavone.

The present application relates to viral sensitizers. More specifically, the present application relates to compounds, as well as methods of using such compounds as viral sensitizers, specifically for increasing permissiveness of a cell to the production of virus from reverse genetics systems, replicating viruses, and increasing transduction.

The invention relates to the technical field of viruses, in particular to a construction method and application of a porcine parvovirus full-length infectious clone carrying a stable genetic marker. The method comprises the following steps: cloning a complete genome of a porcine parvovirus SXND18 strain to a plasmid pBluescript II SK (+) through an In-fusion seamless cloning technology, so as to obtain a recombinant plasmid pSXND18; meanwhile, a new EcoRI restriction enzyme cutting site is introduced into a genome by utilizing an overlapping PCR (Polymerase Chain Reaction) technology and is used as a genetic marker for identifying a wild virus and a rescue virus; and mixing the recombinant plasmid with a transfection reagent, and transfecting ST cells to obtain the rescue virus rSXND18. The porcine parvovirus reverse genetic operating system established by the invention can be used for research in the directions of porcine parvovirus virulence analysis, cross-species transmission mechanism and the like, and also lays a foundation for research on novel vaccines of the porcine parvovirus.

The invention relates to a porcine Getavirus full-length infectious clone as well as a construction method and application thereof, and belongs to the technical field of molecular biology. The invention provides a porcine Getavirus full-length infectious clone. The porcine Getavirus full-length infectious clone comprises a segment C, a segment G1, a segment G2, a segment G3, a segment G4 and a segment G5. The invention provides two GETV HN2024 strain full-length infectious clones carrying stable genetic markers, rescue viruses can be successfully prepared through the full-length infectious clones, and an established swine Getavirus reverse genetic operating system can be used for research in the directions of swine Getavirus virulence analysis, cross-species transmission mechanism and the like. And meanwhile, a foundation is laid for researching a novel vaccine of the swine Getavirus.

The application belongs to the technical field of biology, and particularly relates to a 3D protein mutant O foot-and-mouth disease recombinant virus strain and construction and application thereof. The application mutates methionine at the 403th position of the 3D protein of the O foot-and-mouth disease virus into alanine, and successfully constructs and rescues a 3D protein mutant O foot-and-mouth disease recombinant virus strain rFMDV-M403A by using reverse genetic technology. The amino acid of the recombinant virus strain can be stably mutated in the process of subculture, and the growth speed of the mutant strain is slower than that of the wild strain, and the virus titer is significantly reduced. In individual experiments, the infection of the mutant strain to suckling mice shows that the mutant strain can improve the survival rate of the suckling mice, and significantly reduce the viral load in the liver and the tissue lesions. The application provides an attenuated foot-and-mouth disease recombinant virus strain, which has important significance for in-depth understanding of the pathogenic mechanism and prevention and control strategy of the foot-and-mouth disease virus, and provides an important theoretical basis for further development of the foot-and-mouth disease genetic engineering vaccine.

The application discloses a TaTCP15 gene for regulating nitrogen utilization of wheat as well as a biological material and application thereof, and identifies an upstream regulatory factor of a key gene TaNRT2.1 for regulating nitrogen utilization of wheat through a research strategy combining forward and reverse genetics, and screens and clones a TaTCP15 gene for regulating nitrogen utilization of wheat. It is shown by function prediction and identification results that the TaTCP15 gene is expressed in roots, stems and young panicles at high abundance, and after overexpression of the TaTCP15 gene in Arabidopsis and wheat, the plant height is significantly dwarfed, the ear length is shortened, the yield per plant is reduced, the thousand-grain weight is reduced, the grain length and width are reduced, the grains are round, the tillering is reduced, the heading stage is lengthened, the growth period is lengthened, and the leaf is greener. The application accumulates new data and information for in-depth research on a molecular regulation mechanism of nitrogen utilization of wheat.

The application provides a phloridzin glycosyltransferase gene in dendrobium officinale DoUGT885 and application thereof, and belongs to the technical field of gene cloning. Based on the dendrobium officinale genome data, a key enzyme for synthesizing phloridzin, i.e. a phloridzin glycosyltransferase gene, is successfully explored and identified by a reverse genetics method DoUGT885 , and it is confirmed that DoUGT885 the glycosyltransferase can specifically catalyze the glycosylation of phloridzin to generate phloridzin, which proves that the glycosyltransferase can specifically catalyze the glycosylation of phloridzin to generate phloridzin, and solves the problem of the scarcity of high-efficiency and specific glycosyltransferase elements in the biosynthesis of phloridzin. The discovery of the enzyme breaks the source limitation of the existing phloridzin synthesis enzyme, provides a new high-quality enzyme element selection for the biosynthesis system, not only enriches the glycosyltransferase resource library, but also reduces the dependence of phloridzin biosynthesis on traditional enzyme elements, and lays a key foundation for constructing an efficient and stable phloridzin biosynthesis pathway.

The application provides a construction method of a recombinant canine distemper virus strain expressing mink parvovirus VP2 protein and relates to the technical field of genetic engineering. The construction method uses a mink canine distemper virus attenuated vaccine CDV3 strain as a basis, clones mink parvovirus VP2 genes into a canine distemper virus genome cDNA, and obtains a recombinant canine distemper virus rCDV3-mVP2 strain capable of simultaneously expressing CDV proteins and MEV VP2 proteins through virus reverse genetic technology, so as to provide a candidate vaccine strain for the research and development of a mink canine distemper and parvovirus enteritis double recombinant live vaccine.

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of wheat receptor-like cytoplasm kinase TaRLCK118 or a coding gene thereof in the aspect of improving stripe rust resistance of crops. A reverse genetics method verifies that the TaRLCK118 gene plays a positive regulation role in wheat stripe rust resistance, so that the expression quantity of wheat receptor cytoplasm kinase TaRLCK118 can be improved by improving the expression of the TaRLCK118 gene in a receptor crop, and the resistance of the crop to stripe rust is improved; and gene resources and biological materials are provided for cultivation of stripe rust resistant wheat varieties.

The invention belongs to the technical field of biology, and particularly relates to a PEDV (porcine epidemic diarrhea virus) reverse genetic operating system as well as a preparation method and application thereof. The construction method of the PEDV reverse genetic operating system comprises the following steps: constructing pBAC-M and pBAC-T; the method comprises the following steps: dividing a PEDV whole genome into 8 fragments, and amplifying and connecting the 8 fragments to a pcDNA3.1 vector by taking whole genome cDNA of a PEDV epidemic strain as a template; converting a connection product into escherichia coli, and culturing, so as to obtain pcDNA3.1-F1-F8; the method comprises the following steps: by taking pcDNA3.1-F1-F8 as a template, amplifying F1-F8 fragments, connecting the F1-F8 fragments to form a PEDV whole genome sequence, and inserting pBAC-M and pBAC-T to obtain pBAC-M-PEDV and pBAC-T-PEDV. The PEDV reverse genetic operating system provided by the invention provides support for research and development of PEDV vaccines.

The application discloses a non-neurotoxic VSV vector recombinant oncolytic virus carrying a Sindbis virus G protein, and is a self-replicating recombinant oncolytic virus VSV-SING which is obtained by replacing the G protein coding gene of a wild type VSV with the G protein coding gene of a Sindbis virus through reverse genetic manipulation technology, and has non-neurotoxicity and high oncolytic activity.

The invention belongs to the technical field of virus construction, and particularly relates to a bovine parainfluenza virus type 3 V protein deletion strain and application thereof. The construction process of the strain comprises the following steps: by taking pBPIV3-SX full-length infectious clone plasmid as a template, respectively amplifying two gene segments before and after a base editing site of a V gene, and fusing the obtained amplified segments to obtain a segment containing V gene deletion; and replacing a fragment containing V gene deletion to BssHII and PacI restriction enzyme cutting sites of the pBPIV3-SX plasmid, and transfecting to carry out virus rescue, thereby obtaining the recombinant plasmid pBPIV3-SX. According to the invention, on the basis of a reverse genetics operation technology, a V protein deleted infectious clone plasmid is constructed, and a recombinant deleted virus is successfully rescued; meanwhile, the V protein is obtained through an escherichia coli expression system, a mouse is immunized to obtain a polyclonal antibody, a theoretical basis is provided for functional research of the BPIV3 non-structural V protein and development of genetic engineering vaccines, and the BPIV3 non-structural V protein has important scientific value and application value.