90 results about "A-site" patented technology

The invention relates to the technical field of gene engineering and enzyme engineering, in particular to an esterase mutant for degrading ethyl carbamate and application of the esterase mutant, the esterase mutant has an amino acid sequence as shown in SEQ ID NO.1 which is subjected to site mutation, the site mutation is any one or more site combined mutation of V129S, V161L or I229M, and the amino acid sequence of the esterase mutant is shown in SEQ ID NO.1. The V129S site mutation is that valine at the 129 site is mutated into serine, the V161L site mutation is that valine at the 161 site is mutated into leucine, and the I229M site mutation is that isoleucine at the 229 site is mutated into methionine. Mutation is carried out through a site-directed mutagenesis method, so that the amino acid sequence of the esterase mutant V129S / I229M is changed, the autoplasmic structure and function of eggs are changed, the esterase mutant V129S / I229M with the ethyl carbamate hydrolysis rate increased to 6.55 times in an acid environment is finally obtained, and the esterase mutant V129S / I229M has high industrial application value.

There is provided a fluorinase variant thereof having at least 70% sequence identity to a sequence MSDLGX6TDDSVAQCKGLMLSICPX24VX26IX28DX30CHX33MTPX37DVVEGARYIVDLPR X52FPEGTVFATTTYPATGTX70X71RSVAX76RX78KX80AALGGARGQX90AGSGX95GX97E RAEGX103YIYIAPNNGLLTX116VIEEHGYX124EAYEVSX131TX133VIPX137X138PEPTFYSR EMVAIPSAHLAAGFPLX163X164VGRX168LX170DX172EIVRFEX179X180KX182X183X184VX186G X188X189LX191GX193X194X195X196X197DHPFGNX204WTNX208HRTDLEKAGIX219YX221TX223X 224KX226VX228DGVLX233FX235LPLX239PTFADAX246X247X248GX250PVX253YX255NSRGYLX2 62X263ARNAAX269LAYPYNLX277AGX280SVX283VTX286A (SEQ ID NO: 1), wherein X is a natural amino acid, and wherein the variant comprises one or more mutations at a site selected from the group consisting of an ion-egress site, a S-Adenosyl-L-Methionine (SAM) binding site, an ion-binding site (IBS), and a conserved site. Also disclosed are polynucleotides encoding the variants, vectors comprising the polynucleotides encoding the variants, and host cells comprising the vectors thereof. Also disclosed are Methods for producing the variants, methods of catalyzing the fluorination of a compound, uses of the fluorinase variants, and methods of treatment using the fluorinase variants thereof.

The invention belongs to the technical field of genes, and discloses a method for efficiently expressing foreign protein based on an NC048599.1 site in a CHO cell genome. A site for stably expressing a protein in a CHO cell genome is located in a range of 69195000 to 69199000 of a CHO cell gene NC048599.1 with a sequence as shown in SEQ ID NO: 1 (Sequence Identifier Number 1), and the sequence of the CHO cell gene NC048599.1 is shown in SEQ ID NO: 1. Different protein genes are introduced at fixed positions in a CHO cell genome, and stable expression is carried out.

Provided herein are fusion proteins and related methods and systems for increasing the efficiency of genome editing using site-specific nuclease enzymes. These fusion proteins, systems, and methods may selectively increase a desired editing outcome (e.g., insertion of a donor polynucleotide sequence). Also provided are various useful compositions for producing and using these fusion proteins and practicing these methods.

A homologous recombination method of excising a specific DNA sequence from a DNA molecule. The method includes providing the DNA molecule including a pair of target sequences that are a target of a site-specific recombination enzyme and a pair of homologous sequences that are DNA sequences sharing homology with each other. The pair of target sequences are oriented in different directions from each other and arranged on opposite sides of the specific DNA sequence. The pair of homologous sequences are arranged on opposite sides of the specific DNA sequence at positions further outside of the pair of target sequences. The method further includes inducing homologous recombination between the pair of homologous sequences by action of the site-specific recombination enzyme on the DNA molecule so that the specific DNA sequence and the pair of target sequences are excised from the DNA molecule.

The invention relates to the field of biology, and discloses a double-antibody connection probe for detecting lactylated PGK1, a preparation method and application, the double-antibody connection probe comprises an antibody A, an antibody B and complementary oligonucleotides respectively coupled with the antibody A and the antibody B; the antibody A is a polypeptide which is subjected to K139-lactic acid modification and is used for recognizing a K139la site of the PGK1; the antibody B is a polypeptide which is used for identifying more than or equal to 50 amino acids away from the K139 site in the PGK1 and has the length of 12-15 amino acids; the double-antibody connection probe obtained by the invention realizes the ultra-sensitive detection of the targeted protein.

The present invention relates to a T7 RNA polymerase mutant and its application. Specifically, the present invention discloses a T7 RNA polymerase mutant, which has at least one amino acid mutation at a site corresponding to the wild-type T7 RNA polymerase selected from the group consisting of: position 44, position 45, position 46, position 172, position 389, position 425, position 441, position 632, position 643, position 754, and position 789; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO:1, and the mutation results in a significant reduction in the by-product dsRNA produced during the process of the enzyme synthesizing mRNA, and at the same time reduces the production of RNA aggregates.

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

In a homologous recombination method in which a specific DNA sequence is deleted from a DNA molecule, the DNA molecule is used as the DNA molecule. A DNA molecule is prepared in which a pair of target sequences, which are targets for a site-specific recombinase, are disposed so as to sandwich a specific DNA sequence therebetween and so as to have different orientations from each other, and a pair of homologous sequences, which are DNA sequences having homology, are disposed on the outside of the pair of target sequences disposed so as to sandwich the specific DNA sequence. The site-specific recombinase is caused to act on a DNA molecule, and a specific DNA sequence and a pair of target sequences are deleted from the DNA molecule by homologous recombination between a pair of homologous sequences.

There is provided a homologous recombination method of excising a specific DNA sequence from a DNA molecule, comprising: providing the DNA molecule that has a pair of target sequences as a target of a site-specific recombination enzyme are placed in different directions from each other across the specific DNA sequence and that a pair of homologous sequences as DNA sequences sharing homology with each other are placed on further outside of the pair of target sequences placed across the specific DNA sequence; and making the site-specific recombination enzyme act on the DNA molecule and thereby causing homologous recombination between the pair of homologous sequences, so as to excise the specific DNA sequence along with the pair of target sequences from the DNA molecule.

The invention discloses a site for stably expressing a protein in an HEK293T cell gene NG027973.1 and an application of the site for stably expressing the protein in the HEK293T cell gene NG027973.1. According to the present invention, the obtained stable expression site is located at the upstream 69 bp and the downstream 134bp of the 135153 base in the HEK293T cell gene NG027973.1, such that the 135084-135287 base can be integrated, and the stable expression can be performed; according to the invention, a target gene is integrated to a stable expression region in a site-specific integration manner. The site information provided by the invention is clear, site-specific integration of foreign protein genes can be realized at the site, the protein can be stably expressed, the screening process and time in the cell construction process can be greatly shortened, and the research and development cost can be reduced.

There is provided a method of altering a eukaryotic cell comprising transfecting the eukaryotic cell with a nucleic acid encoding an RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site-specific manner wherein the cell expresses the RNA and the enzyme, and modifying the eukaryotic cell with the nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site-specific manner. The RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site-specific manner.

Methods and compositions are provided for driving expression of a coding sequence that has been integrated within the genome of a cell. A donor polynucleotide is integrated within the genome so that a non-functional promoter is located downstream of a genomic target sequence. This genomic target sequence can be bound by a site-specific promoter activation tool, wherein the site-specific promoter activation tool drives robust expression of a coding sequence that is operably linked to the non-functional promoter.

The invention provides a site-specific mutagenesis vector and a use method thereof, and relates to the field of gene engineering in the biotechnological pharmaceutical industry, the vector contains BbsI restriction enzyme cutting sites except for a polyclone region, and site-specific mutagenesis comprises the steps of carrying out base mutation on the BbsI restriction enzyme cutting sites, removing the BbsI sites and keeping amino acid unchanged. According to the invention, a Golden gate method is adopted to construct the multi-copy vector, and a plurality of copies are directly assembled on a system in one step, so that the time is saved, and the tedious steps of multiple times of enzyme digestion and connection are omitted. According to the invention, a multi-copy vector is constructed in vitro, the copy number of the antibacterial peptide is increased, simultaneous expression of each unit is realized, and the expression quantity is improved. Meanwhile, the antibacterial peptide genes are connected in series by adopting a serial connection technology of the antibacterial peptide genes, so that the expression quantity of the antibacterial peptide is improved on the DNA level.

A DNA editing method for inserting a desired nucleotide sequence into a target site of a target DNA in a cell or removing the target site includes an introduction step of introducing a vector and a site-specific nuclease system into the cell and bringing the vector and the site-specific nuclease system into contact with the target DNA, the vector comprises a first promoter P1 and structure (1): 5 '-M1-Hv1-D-Hv2-M2-3', M1 represents a 5 '-side fragment of a nucleotide sequence encoding a selection marker gene and is operably linked to P1, Hv1 represents a nucleotide sequence homologous to a first nucleotide sequence Ht1 on the 5' side of a target site of the target DNA and D-Hv2-M2-3 'represents a nucleotide sequence homologous to a second nucleotide sequence Ht2 on the 5' side of the target site of the target DNA and D-Hv2-M2-3 'is operably linked to P1. D represents the desired nucleotide sequence but may not exist, Hv2 represents a nucleotide sequence homologous to a second nucleotide sequence Ht2 on the 3'side of the target site of the target DNA, and M2 represents the remaining 3 '-side fragment of the nucleotide sequence encoding the selection marker gene; the DNA editing method comprises a cutting process, an editing process and a selecting process, and the cutting process is carried out through the site-specific nuclease system. And a step in which a fragment represented by structure (2): 5 '-Hv1-D-Hv2-3' and a fragment represented by structure (3): 5 '-M2-P1-M1-3' are generated from the vector, and the target site or the surroundings thereof is cleaved in the target DNA, in which the target DNA is bound to structure (2) depending on the homology of Ht1 and Hv1 and bound to structure (2) depending on the homology of Ht2 and Hv2, and the fragment represented by structure (3): 5 '-Hv1-D-Hv2-3' is cleaved in the target DNA depending on the homology of Ht2 and Hv2. And a selection step for obtaining a selection vector containing a selection marker gene that binds to the 3'terminal of M1 and the 5 'terminal of M2 in the fragment represented by structure (3) and is operably linked to P1, and that can function, whereby the desired nucleotide sequence D is inserted into the target site or the target site is removed, and a selection step for obtaining a selection vector that contains a selection marker gene that binds to the 3' terminal of M1 and the 5 'terminal of M2 in the fragment represented by structure (3).

The invention relates to the technical field of bioengineering and provides a method for improving the specificity and affinity of an aptamer. The method includes: S1, screening a target of an aptamer from a compound information database by virtual computing; S2, verifying the screening result in Step S1 through experiments; S3, performing virtual saturation mutation on a site of the aptamer, and screening out a mutation site of the aptamer; S4, performing base substitution to the mutation site of the aptamer; and S5, detecting the binding parameter of the aptamer after base substitution with the target screened in Step S1, and selecting an aptamer with improved specificity and affinity after base substitution. An efficient molecular design-guided method is developed by computer rational calculation, to improve the specificity and binding affinity of the aptamer by directional modification. The present invention is of great significance for the practical application of aptamers.

The application discloses a pig HBB gene site-directed modification system and application. The application provides a system for site-directed modification of a pig HBB gene, the first carrier, the second carrier and the third carrier contained in the system can express gene editing proteins and sgRNA, three target sites of the HBB gene are effectively cleaved, a site-directed modification fragment of a donor DNA is used to replace a to-be-site-directed modification fragment of the target site, and accurate deletion of bases from 176 to 179 of a CDS sequence of the HBB gene is realized. 41‑42(‑CTTT) The model accurately simulates the most common beta thalassemia mutation genotype beta of Chinese population, provides a precise model for analyzing the pathogenesis of beta thalassemia of Chinese population and developing a new treatment method.

The present invention provides a chimeric polyketide synthase (PKS) or polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) and uses thereof, wherein the PKS or PKS-NRPS comprises two or more recombinant units (RUs) linked to each other, wherein each RU comprises one or two or more contiguous domains derived from a parent PKS or PKS-NRPS, and the junction of at least one RU is defined by a recombination site selected from a site at the LPTY (A / P) FQ (H / R) xRYWL motif located in the AT post- linker (RYWL motif site) and a site at the RL motif of ACP helix 0 (RL motif site); wherein the two or more RUs are not naturally linked to each other.

A protein-glycan conjugate having multiple payloads. By means of a site-specific glycan conjugation technique, different types of payloads are conjugated to specific positions of a protein-glycan chain, so as to obtain a conjugate having multiple payloads. The conjugate exhibits good biological effects.

The invention discloses a rice OsRDR5 gene, an encoding protein, a fixed-point editing system, application and an application method. The nucleotide sequence of the OsRDR5 gene is shown as SEQ ID NO.1; the amino acid sequence of the encoding protein of the rice OsRDR5 gene is as shown in SEQ ID NO. 2. The gene has a Coa3cc structural domain and belongs to a cytochrome c oxidase assembly factor 3 gene family. A knockout mutant of the OsRDR5 gene shows a relatively high deep root ratio, and meanwhile, the gravitational property of a rice root system is changed. The rice OsRDR5 gene disclosed by the invention is used for regulating and controlling the drought resistance of rice, and a knockout mutant of the rice OsRDR5 gene has a relatively high deep root ratio and can be applied to breeding of a new variety of water-saving and drought-resistant rice, so that the drought resistance of the rice is improved.

Disclosed herein is a method for training a framework comprising one or more models for site-specific ribonucleic acid (RNA) modification detection on an RNA sequence of interest, comprising receiving a training dataset of sequences, each sequence being either an unmodified RNA sequence or a modified RNA sequence and, for each sequence, annotations indicating whether the respective sequence contains a modification at one or more sites; extracting a sequence context read j from each sequence, and extracting, for each site of interest i in read j, i being a site at which it is desired to detect a modification, a site-specific annotation from the annotations, the site-specific annotation indicating whether a specific modification m occurs at site i; training at least one said model to determine a probability of modification m at site i, using a plurality of k-mers, extracted from each read j, including one or more k-mers from before site i, at site i, and after site i; updating the at least one model based on a loss between the probability and the site-specific annotation; and outputting the framework, after a predefined number of epochs for training has been completed.

The application discloses a rice OsRDR5 gene and a coding protein, a site-directed editing system, application and an application method, a nucleotide sequence of the OsRDR5 gene is shown as SEQ ID NO. 1; and an amino acid sequence of the coding protein of the rice OsRDR5 gene is shown as SEQ ID NO. 2. The gene has a Coa3_cc domain and belongs to a cytochrome c oxidase assembly factor 3 gene family. Knockout mutants of the OsRDR5 gene show a higher deep root ratio, and simultaneously change the gravitropism of rice root systems. The rice OsRDR5 gene of the application regulates the drought avoidance of rice, the knockout mutants have a higher deep root ratio, and can be applied in breeding of new water-saving and drought-resistant rice varieties, so that the drought resistance of rice is improved.

A non-naturally occurring peptide linker, comprising a site-specific conjugation motif of GGX1X2Q, which allows for site-specific conjugation mediated by a transglutaminase; wherein each of X1 and X2 independently represents any naturally-occurring amino acid or one of X1 and X2 is absent; and wherein the non-naturally occurring peptide has a length of 9-20 amino acids. Also provided herein are fusion polypeptides such as tandem single-domain multi-specific antibodies containing such peptide linkers.

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

A site-specifically-modified engineered tRNA and the use thereof. Said modification refers to replacement, with one or more modified nucleotides, of one or more native unmodified nucleotides selected from sites 4, 9, 13, 14, 17, 18, 27, 28, 32, 34, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58 and 72 of a tRNA molecule. Compared with unmodified tRNAs, the site-specifically-modified tRNA has higher structural stability and / or higher nuclease degradation resistance, the aminoacylation efficiency thereof is not remarkably reduced, and immunogenicity thereof is not remarkably increased. The modified tRNA can be used for eliminating premature translation terminations caused by nonsense mutations, and thus has great application potentials in treating nonsense mutation-related genetic diseases, tumors, etc. The modification of the engineered tRNA also provides valuable insights for drug development of tRNAs and thus can guide the modification design of native tRNAs.

This invention discloses a method based on SOD The primers and methods for gene screening of heat-resistant scallop varieties were developed using scallop cDNA and DNA as templates. PCR amplification was performed using upstream and downstream primers (SEQ ID No. 3 and SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6) to obtain samples containing scallop cDNA and DNA. SOD Gene sequence fragments from the exon and promoter regions were used as molecular markers. The genotypes were CT at the c.229 C>T site, CA at the c.284 C>A site, and CA at the c.348 C>A site in the exon region, and CA at the g.1124 A>C site and AT at the g.1359 T>A site in the promoter region. This method screened for high-temperature resistant scallop varieties, demonstrating advantages such as genetic stability, accuracy, and efficiency.

It has been found that by using a site-specific nuclease system, it is possible to cause repair using both non-homologous end joining and homologous recombination between genomic DNA and donor DNA, and to produce cells and organisms into which a long donor sequence has been knocked in with high efficiency and accuracy. The site-specific nuclease system includes a combination of a molecule that simultaneously targets a homologous arm sequence on one side of the donor DNA and the genomic sequence corresponding to the homologous arm sequence for cleavage, and a molecule that targets a genomic region near the cleavage site of the molecule for cleavage.

The present invention provides a formate dehydrogenase mutant with improved electron transfer rate and its application. The formate dehydrogenase mutant uses the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity with SEQ ID No.1 as the parent, and has a site-directed saturation mutation corresponding to tyrosine at position 262 of SEQ ID NO.1. By performing site-directed saturation mutation on formate dehydrogenase from Thiobacillus, the present invention finally obtains an Ts FDH mutant with improved electron transfer rate and stable catalytic activity.

Provided is a simple and efficient method of plant genome editing that does not involve the incorporation of a gene cassette for the expression system of a site-specific DNA modification protein. The method of the present invention comprises: (i) a step for mixing plant cells or a tissue containing plant cells together with a site-specific DNA modification protein and a needle-shaped inorganic compound in a liquid medium and making a disturbance to perforate the cells with the needle-shaped inorganic compound, thereby introducing the site-specific DNA modification protein into the cells; and (ii) a step for culturing the plant cells or the tissue containing the plant cells, into which the site-specific DNA modification protein has been introduced in step (i), thereby causing a DNA mutation specific to a target site in the genome of the plant cells.

Provided are directed to compositions, methods, and systems for epigenetic editing of antigen presentation mechanism (APM) -related genes in a cancer cell. In some embodiments, provided are directed to methods of modulating methylation at a methylation site in an APM-related gene in a cancer cell. In some embodiments, the site-specific demethylation systems comprise a fusion protein or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises a site-specific DNA binding domain and an effector domain having demethylation activity. In some embodiments, provided are directed to such methods, wherein recruitment of the site-specific demethylation system to a target site results in demethylation at the methylation site. Also provided herein are nucleic acids encoding the polypeptides described herein, systems comprising additional components useful for editing, such as gRNA, methods of treating cancer, and kits directed to practicing the methods described herein.