97 results about "Reverse genetics" patented technology

The present application relates to a virus of reverse genetic operation construction of NADC34-like PRRSV2. Specifically, the full gene sequence of NADC34-like PRRSV2 BJ1805-2 isolated strain is segmented and connected to the vector by using PCR amplification, enzyme digestion and other methods with pACY177 plasmid as the carrier, and the BJ1805-2 full-length infectious clone recombinant plasmid (rBJ1805-2) is obtained. The infectious clone virus rBJ1805-2 is rescued in vitro, and the first NADC34-like PRRSV2 strain infectious clone platform in China is successfully built. The present application also relates to a modification method for constructing NADC34-like PRRSV2 strain which can adapt to Marc-145 cell passage culture. The infectious clone virus constructed by the present application has good in vitro and in vivo proliferation efficiency, and can cause specific viremia after inoculation in pigs; it has good safety, does not cause fever after inoculation in pigs, and will not cause piglet death. The modified strain can be used as a candidate strain for developing the first NADC34-like PRRSV2 specific vaccine in China, and is conducive to the prevention and control of PRRSV epidemic in China.

The invention discloses an effect protein E56, a coding gene and application of the effect protein E56 to improvement of southern rust resistance of corn, and belongs to the technical field of agricultural biology. The amino acid sequence of the effector protein E56 is as shown in SEQ ID NO. 1. A reverse genetics method is utilized to analyze the Puccinia polystachys effect protein E56 gene, and the E56 gene is up-regulated in expression in the Puccinia polystachys infection process. The E56 gene is silenced by adopting a host-mediated gene silencing technology, and the toxic function of the E56 gene in the infection process of the southern rust disease is determined. A specific fragment of the E56 gene is cloned to an RNAi interference vector, a corn immature embryo is transformed by using an agrobacterium tumefaciens-mediated transgenic technology, and an obtained transgenic corn plant shows resistance to the corn southern rust disease. The E56-RNAi transgenic plant shows resistance to puccinia polypoda, so that the effect protein E56 can be used for creating a new strain for resisting the southern rust disease.

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 an application of a TaAP2-4 gene in regulating and controlling starch content of wheat grains, which is used for enhancing the expression quantity of the TaAP2-4 gene to improve the starch content of the wheat grains and weakening the expression quantity of the TaAP2-4 gene to reduce the starch content of the wheat grains. The TaAP2-4 gene is located on a chromosome 4 of a wheat genome, and three copies, namely TaAP2-4-A, TaAP2-4-B and TaAP2-4-D, are formed on the chromosome 4. According to the invention, a Crispr / Cas9 gene editing vector for knocking out the TaAP2-4 gene is constructed by adopting a reverse genetics method, the TaAP2-4 gene is successfully knocked out in a wheat material by adopting an agrobacterium-mediated method, and the starch content of a knocked-out mutant is reduced, so that the condition that the TaAP2-4 gene plays a positive regulation role in a wheat grain starch synthesis process is known. In practical application, directional knockout mutation of the genome level TaAP2-4 gene can be achieved through a gene transformation process, the gene knockout mutation method can be used for verifying functions of the TaAP2-4 gene, and materials are provided for mining other starch synthesis related genes.

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 discloses an SFTSV pseudovirus as well as a preparation method and application thereof. Based on the characteristics of high pathogenicity and transmissibility of the SFTSV, the virus infection animal experiment needs to be operated in a laboratory at the biosafety third level or above, so that the research and development of vaccines and therapeutic drugs are limited to a great extent. A novel SFTSV pseudovirus is obtained by deleting a nucleoprotein coding gene in an S gene of the SFTSV virus based on a reverse genetics technology, and the pseudovirus lacks a complete virus genome and replication ability and can be researched in a low-biosecurity-level laboratory; the pseudovirus system carries luciferase or a green fluorescent protein reporter gene, the virus load is quantified through the expression of the reporter gene, and the pseudovirus system can be used for evaluating the inhibition effect of a small-molecule inhibitor or a natural product on virus invasion; the pseudovirus can be used for simulating a natural infection process and evaluating a neutralizing antibody level induced by a vaccine. The invention provides a new development platform for discovery of anti-SFTSV drugs.

The invention discloses an H9N2 influenza virus M1 protein mutant and application thereof, and belongs to the technical field of biology. The H9N2 influenza virus M1 protein mutant is a protein obtained by mutating histidine H at the 222 position of an amino acid sequence of H9N2 influenza virus M1 protein into glutamine Q and keeping other amino acid residues unchanged, and the amino acid sequence of the H9N2 influenza virus M1 protein mutant is as shown in SEQ ID NO. 1. After a mouse is infected with the M1-H2222Q point mutation recombinant virus obtained by a reverse genetics technology, the weight loss of the mouse is obvious, the replication ability of the virus in the lung of the mouse is enhanced, and the pathological change is obvious. The M1 protein mutant disclosed by the invention can be used for rapidly obtaining the mouse adaptive H9N2 influenza virus.

The invention discloses construction and application of a recombinant PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) strain for expressing porcine circovirus Cap protein. The recombinant PRRSV capable of expressing the Cap protein of the porcine circovirus type 2 is successfully constructed and saved by adopting a reverse genetic manipulation technology. The modified virus is inoculated to Marc-145 cells for subculture, and the result shows that the modified virus can trigger cytopathy and stably express Cap protein. The obtained recombinant strain shows a stable passage capability in a Marc-145 cell, and has an extremely high virus load. The strain can be used as a vaccine candidate strain, provides multiple protection for PRRSV and porcine circovirus of different pedigree, and provides a brand new solution for healthy breeding of pigs.

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 invention discloses a neurotoxicity-free VSV vector recombinant oncolytic virus carrying Marburg virus deficient G protein, which is characterized in that a G protein coding gene of wild VSV is replaced by a coding gene of Marburg virus deficient G protein through a reverse genetic manipulation technology, so as to save and obtain a recombinant oncolytic virus VSV-MARG-delta MLD capable of being autonomously replicated. The invention has the advantages of no neurotoxicity and efficient oncolytic activity.

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 application discloses a chimeric Kunjin virus of tamabi virus prM-E and a preparation method and application thereof. On the basis of a reverse genetic manipulation system of the Kunjin virus KUNV, the KUNV prM / E gene is replaced by the DTMUV prM / E gene to construct a chimeric DTMUV virus infectious clone plasmid with the KUNV as a skeleton, which is named as pUC19-KUNV-DTMUV prM / E. Then, the chimeric tamabi virus prM-E Kunjin virus is obtained through virus rescue, and is named as rKUNV-CHv-prM / E (CHv). Analysis on the virus characteristics shows that the CHv can effectively proliferate in BHK-21 and DEF cells, and the duck embryo virulence is weaker than that of the KUNV and the DTMUV. The animal experiment results show that the CHv only detects obvious virus levels in the brain, and does not produce viremia; the CHv after immunization can produce similar IgG antibody levels to those after immunization of the KUNV and the DTMUV. Therefore, the recombinant virus of the chimeric DTMUV prM / E with the KUNV as a skeleton prepared by the application can induce the duckling to produce sufficient immune protective neutralizing antibody levels, and can be applied to preparation of a duck tamabi virus vaccine.

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.