1532 results about "Homomeric" patented technology

The invention discloses a site for stably expressing protein in a CHO cell gene NW023276806.1 and application of the site, and belongs to the technical field of biological genes. The site belongs to a fixed position in a CHO cell genome, different protein genes are introduced based on a micro-homologous end connection mechanism through a CRISPR / Cas9 tool, and stable expression is carried out. By adopting a site-specific integration method, a target gene is integrated to a stable expression area in a site-specific manner, repeated high-expression monoclonal screening is effectively avoided, and an MMEJ mechanism is introduced to integrate a donor fragment, so that the research and development time for constructing a stable expression cell strain in biological pharmacy can be effectively shortened, and the cost is reduced.

Methods and compositions for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed. Specifically, provided herein comprises compositions comprising an RNA molecule comprising a reverse complement sequence comprising an exogenous sequence and homology arms, an RNA molecule comprising an endonuclease, and one or more guide RNAs for specific genomic integration of the exogenous sequence.

Disclosed herein are certain thiadiazolyl derivatives Formula (I):that inhibit DNA Polymerase Theta (Polθ) activity, in particular inhibit Polθ activity by inhibiting ATP dependent helicase domain activity of Polθ. Also, disclosed are pharmaceutical compositions comprising such compounds and methods of treating and / or preventing diseases treatable by inhibition of Polθ such as cancer, including homologous recombination (HR) deficient cancers.

A stable transformant, in which a gene of interest is incorporated into the genome, is simply and efficiently produced.A method for producing a transformant, comprising a step of introducing into a host, linear genome-introduced nucleic acid fragment(s) comprising a gene of interest and a helper plasmid for transformation having a pair of homologous recombination sequences for incorporation of the linear genome-introduced nucleic acid fragment(s), and then selecting a transformant, in which the gene of interest is incorporated into the predetermined position in the host genome and the gene of interest is expressed therein.

According to the invention, a structural domain reaction substrate sequence consisting of Exon1 (E1), P1 and Exon2 (E2) sequences of ribozyme is mutated, and E1, P1 and E2 sequences are mutated under the condition of maintaining the structural stability, so that the Azoarcus group I intron ribozyme still has enzyme activity and can maintain the capability of forming circular RNA (Ribose Nucleic Acid). The invention discloses a flexible vector construction method for preparing circular RNA (Ribonucleic Acid) without limitation of a substrate sequence, which comprises the following steps: determining a target to-be-cyclized site sequence NNUNNNN, and segmenting the target to-be-cyclized site sequence NNUNNNN into E1: NNU and E2: NNNN; with E1 and E2 sequences as references, designing IGS sequences to respectively form complementary pairing with E1 and E2; and 5'and 3 'homologous arms, IRES, CDS and other elements are respectively added. The method provided by the invention can be used for preparing the circular RNA for any target sequence, has no residual sequence, and has relatively high cyclization efficiency. The FlexCirc cyclization system designed on the basis of Azoarcus group I intron ribozyme can form the circular RNA, the cyclization substrate sequence has the characteristic of flexible design, and the cyclization efficiency can realize a relatively high cyclization proportion.

The invention belongs to the technical field of biological medicines, and particularly relates to a pleiotropic protein MEL3 and application thereof. The amino acid sequence of the pleiotropic protein MEL3 or the homologous protein of the pleiotropic protein MEL3 provided by the invention has the following general formula: X1GAX2LX3VLX4GLX5ALISWEKRKRX6, x1 is M or L; the X2 is one of V, SI and AI; x3 is one of K, T, KI and KR; x4 is one of KK, TT, SS and VK; x5 is one of P, K, A and T; and X6 is QQ or is free of amino acid. The pleiotropic protein MEL3 or the homologous protein thereof provided by the invention has broad-spectrum antibacterial activity, can achieve anti-inflammatory and antiviral effects at the same time, and provides a new thought for preparing anti-pathogenic microorganism or anti-inflammatory products.

The invention discloses a construction method and application of an NMD escape model of an Alport syndrome mouse. A non-human animal model carrying Col4a5 gene c.4432delG frame shift mutation is prepared on the basis of a CRISPR / Cas9 gene editing technology. The method comprises the following steps: co-injecting gRNA of a 49 exon of a targeted Col4a5 gene, homologous recombinant donor oligonucleotide containing c.4432delG mutation and Cas9 nuclease into a mouse fertilized egg, and carrying out embryo transplantation to obtain an F0-generation mutant mouse; a mutation site is verified by combining PCR (Polymerase Chain Reaction) with sequencing, and a stably inherited mutation line is established through two generations of breeding. Through verification, the model accords with pathological characteristics of the Alport syndrome, can stably simulate typical clinical manifestation and pathological characteristics of the human X-linked Alport syndrome, and can be used as an important tool for research of the Alport syndrome.

The invention relates to the technical field of biomedicine, discloses mutant photo-induced ion channel ChR-2 protein, and further discloses a nucleic acid construct, an expression vector, a cell, related application and a computer model. The light-sensitive channel ChR2 protein mutant obtained by the invention has stronger light current, and the light-sensitive capability of the light-sensitive channel ChR2 protein mutant is at least improved by 100 times; besides, the invention also obtains a nucleotide sequence for coding the light-sensitive channel ChR2 protein mutant, constructs a recombinant expression vector, and obtains the light-sensitive channel ChR2 protein with higher expression quantity and stronger light sensitivity, and the light-sensitive channel ChR2 protein is very suitable for expression in cells of mammals (especially human); according to the invention, the rhodopsin in different channels is systematically studied by modifying the position G224 in the helix 6 of seven transmembrane helix motifs, which proves that the mutation of the position G224 in the helix 6 in WT ChR2 accelerates the photosensitivity of the channels, and the considered position is homologous in the helix 6 of the rhodopsin in different channels.

The invention belongs to the technical field of biological agriculture, and particularly relates to application of a receptor-like kinase gene MdSRF3 and an encoding protein thereof in regulation and control of apple disease resistance. The receptor-like kinase gene MdSRF3 for negative regulation and control of rot resistance is identified from apples, the CDS sequence of the receptor-like kinase gene MdSRF3 is as shown in SEQ ID NO: 1, and the amino acid sequence of coded protein is as shown in SEQ ID NO: 2. Researches show that over-expression of the gene significantly weakens apple disease resistance, and down-regulates expression of defense-related genes; the silence of the gene enhances the disease resistance of the apple. Different from the positive regulation and control functions of homologous proteins in arabidopsis thaliana, potatoes and rice, the negative regulation and control characteristics of the receptor-like kinase gene MdSRF3 provide a new perspective for research of a plant disease-resistant mechanism, provide a new gene resource for apple disease-resistant breeding, and have significant application values and industrialization prospects.

The invention discloses methanogen lyase homologous with a PeiR lyase sequence and application of the methanogen lyase, and aims to solve the problem that PeiR lyase protein of a methanogen cell wall peptide bond can participate in a biological process of hydrolyzing archaea cell walls, effectively kill methanogen and reduce methane production, and protein homologous with the PeiR lyase protein has potential methane reduction potential. According to the invention, a series of biological information software is utilized to identify protein sequences coded by methanogens virus from rumen microbiome sequencing data, and sequence homology between the proteins and PeiR protein is further analyzed to obtain a series of methanogens lyases. In order to realize the expression of the lyase, multiple segments of primers are designed to synthesize a target sequence, and the target sequence is successfully expressed in a prokaryotic expression system. Through the detection of crude enzyme liquid, the effectiveness of the lyase in the aspect of reducing the methane yield is verified in an in-vitro gas production test.

The invention belongs to the technical field of biotechnology and animal models, and discloses a double-person-derived mouse model for simulating a tumor immune microenvironment and a construction method and application thereof. The method comprises the following steps: firstly, pretreating NSG immunodeficient mice by adopting low-dose whole-body irradiation in combination with double-antibody targeted bone marrow depletion, and transplanting CD34 + hematopoietic stem cells from the same human donor to complete human immune system reconstruction; separating tumor primary cells, tumor-related fibroblasts and tumor vascular endothelial cells of the same donor, performing three-dimensional co-culture to obtain homologous human tumor organs, and performing in-situ inoculation to immune reconstruction mice to obtain a target model. The core defects of MHC mismatching, low immune reconstruction efficiency, poor tumor immune microenvironment simulation degree, low clinical consistency and the like of an existing model are overcome, and the method can be used for tumor immune treatment drug screening, microenvironment mechanism research and personalized tumor treatment scheme verification.

The present invention relates to modified Dda helicases which can be used to control the movement of analytes such as polynucleotides. The modified Dda helicases are used in analyte detection and characterisation. The present invention also relates to novel protein pores and their uses in analyte detection and characterisation. The invention particularly relates to an isolated pore complex formed by a CsgG-like pore and a modified CsgF peptide, or a homologue or mutant thereof, thereby incorporating an additional channel constriction or reader head in the nanopore.

The invention provides an application of WNT7B in construction of a myopia animal model. Homologous genes wnt7ba and wnt7bb of the WNT7B in the zebra fish are knocked out or knocked down by applying a CRISPR / Cas9 gene editing technology, an animal model of which the eye axis length is remarkably increased, the eyeball movement frequency is remarkably reduced and the light response is reduced is obtained, and the result shows that the WNT7B is related to the high myopia. The model provides a favorable tool for pathogenesis and drug screening of high myopia, and has a good application prospect.

The invention relates to the field of agricultural products, and particularly discloses a polypeptide for improving plant disease resistance, and the polypeptide is LAND2 small peptide and homologous peptide thereof. The LAND2 small peptide disclosed by the invention is screened and found from an arabidopsis thaliana proteome, and homologous peptides of the LAND2 small peptide are respectively derived from chilies and cucumbers. Tests prove that when the LAND2 small peptide is externally applied to arabidopsis thaliana, the local and systematic resistance of plants to botrytis cinerea can be enhanced, and the LAND2 small peptide has broad-spectrum immunocompetence in chilies and cucumbers. The polypeptide is practically applied to agricultural production, can enhance the disease resistance of plants and improve the yield and quality of the plants, and has practical popularization and application values.

The present invention belongs to the technical field of genetic engineering, and particularly relates to the use of an exogenous gene integration site, a homologous recombination vector, and a method for constructing a transgenic pig. The safe site for exogenous gene integration provided by the present invention is the region between the genes LOC100155859 tubulin alpha-1B chain-like and LOC100517052 histone H3.3-like on porcine chromosome 11. The present invention further fully verifies this safe site in transgenic animals and cells. During the passage process, the exogenous gene can be stably and highly expressed, and has no side effects on cells and tissues. Therefore, the safe site provided by the present invention has good application prospects in genetic engineering.

The invention discloses recombinant streptomyces mobaraensis as well as a construction method and application thereof, and belongs to the technical field of bioengineering. According to the invention, smY2019 is used as an original strain, and a CRISPR / cas9 technology is utilized to construct five chassis cells of a GIs gene cluster; on the basis of smY2019-deltaG5, two kinds of chassis cells in which endogenous CRISPR / Cas system genes are knocked out are constructed. And on the basis of the smY2019-deltaG5-C2, further constructing a chassis cell smY2019-deltaG5-C3 of which a non-homologous end connection (NHEJ) repair pathway gene is knocked out. The TGase secretory enzyme activity of the chassis cell smY2019-deltaG5-C3 is improved by 37.3% compared with that of the original strain smY2019, and meanwhile, the transformation efficiency, the growth speed, the protein synthesis capability and the energy metabolism level of the chassis cell are obviously improved compared with that of the original strain smY2019.

The application discloses a kind of carrier, kit and application for long-acting gene knockdown of animal parasitic nematode, belong to the field of animal parasitic disease prevention and control.The lentivirus vector in the application integrates specific coding sequence to the genome of the infected nematode, stably and continuously expresses short hairpin RNA (shRNA) in the nematode, efficiently produces primary single-stranded small RNA (siRNA), these siRNAs target homologous target gene mRNA to cause its degradation, realize stable and continuous gene silencing effect.The application breaks through the bottleneck of traditional animal parasitic nematode RNA interference (RNAi) technology with low efficiency and instability, significantly improves the timeliness of conventional RNAi, can be used to establish long-acting RNAi technology system of animal parasitic nematode, has application value in the research of host in vivo process such as nematode infection, parasitism and pathogenicity and animal parasitic nematode disease prevention and control.

The invention discloses an E3 ubiquitin ligase gene ulA, the nucleotide sequence of which is shown as SEQ ID NO: 1. The gene is knocked out from fusarium oxysporum by using a homologous recombination method to obtain a mutant strain ulA; compared with a wild strain, the mutant hulA has the advantages that the spore yield is reduced, the spore germination is delayed, the response capability to various abiotic stresses is reduced, the pathogenicity to panax notoginseng plants is reduced, and the sensitivity to six bactericides is increased, and experimental results show that the gene plays an important role in pathogenicity, drug resistance and the like of fusarium oxysporum. The gene can be used as a target gene for developing an anti-fusarium oxysporum preparation.

The invention belongs to the technical field of molecular biology breeding, and particularly relates to application of a GhDCP5L gene in creation of high-temperature response cotton germplasm, and an amino acid sequence of a protein coded by the GhDCP5L or a homologous gene thereof is as shown in SEQ ID NO.2 or SEQ ID NO.4. The invention provides a novel thermal response gene, a gene resource library of high-temperature response of cotton is enriched, the GhDCP5L gene and a homologous gene thereof are applied to plant genetic engineering breeding, and new germplasm of high-temperature-resistant or sensitive high-temperature-resistant cotton can be accurately created by regulating and controlling the expression of the gene, so that the high-temperature-resistant or sensitive high-temperature-resistant cotton is obtained. The method provides technical support for coping with stable yield of cotton under the background of global warming climate, can be used for preparing male sterile line cotton germplasm by performing high-temperature treatment on the high-temperature-sensitive material, has good application value in the aspects of cotton hybrid seed production, promotion of hybrid cotton development and the like, and is beneficial to improving the economic benefit of the cotton industry.

The invention belongs to the field of antibody development, and discloses a screening method and application of a human-mouse protein high homologous target antibody based on a fully humanized antibody mouse. Aiming at the problem of weak antibody response caused by immune tolerance of human-mouse high homologous targets (protein homology is greater than or equal to 95%), the following scheme is provided: in embryonic stem cells (ES cells) of HUGO-Mabfully humanized antibody transgenic mice, a mouse target gene (such as ACVR2A) is knocked out through a Turbo Knockout technology, and homozygous knockout ES clones are screened; carrying out microinjection on the clones to the whitened B6 mouse blastocyst, and transplanting a pregnant mouse to obtain a Founder mouse; the Founder mouse is subjected to target antigen immunization for more than or equal to 4 times (the Freund's complete adjuvant is used for the first time), and the titer of the serum antibody is detected. According to the invention, 100% homozygous knockout chimeric efficiency is realized in the Founder stage, the mouse construction period is shortened from traditional 8-10 months to 3-4 months, and the diversity and affinity of the antibody are significantly improved (titer reaches 1: 729,000). The obtained antibody can be used for preparing medicines for treating tumors or autoimmune diseases.

The invention discloses a VpCas9 protein double-site mutant and an application of the VpCas9 protein double-site mutant in gene editing. The invention firstly provides a homologue VpCas9 protein of Cas9, the amino acid sequence of the homologue VpCas9 protein is shown as SEQ ID No.5, and compared with SpCas9, the homologue VpCas9 protein has a better editing effect in the aspects of gene editing efficiency, PAM compatibility or specificity and the like; in order to further improve the gene editing effect of the VpCas9 protein in genome editing, the VpCas9 protein is subjected to double-site mutation, the VpCas9 protein double-site mutant is obtained, and compared with SpCas9 protein or VpCas9 protein, the VpCas9 protein double-site mutant has a better gene editing effect in genome editing, and the gene editing effect of the VpCas9 protein is improved. The method has an important application prospect in genome editing of plants, animals or microorganisms.

The invention discloses methanogen lyase homologous with high-dimensional characteristics of PeiW lyase and application of the methanogen lyase, and aims to solve the problem that PeiW lyase protein of methanogen cell wall peptide bonds can participate in the biological process of hydrolyzing archaea cell walls, effectively kill methanogen and reduce methane production, and protein homologous with the protein has potential methane reduction potential. According to the method, a series of bioinformatics software is utilized, a protein sequence coded by methanogens virus is identified from a large amount of rumen microorganism virus group sequencing data, the high-dimensional homology of a deep learning model between the protein sequence and PeiW is analyzed, and two methanogens lyases similar to the high-dimensional characteristics obtained by the PeiW model are obtained. In order to realize the expression of the lyase, multiple segments of primers are designed to synthesize a target sequence, and the target sequence is successfully expressed in a prokaryotic expression system. Through detection of a crude enzyme solution, the effectiveness of the lyase in the aspect of reducing the yield of methane is verified in an in-vitro gas production test.

Methods and compositions for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed. Specifically, provided herein comprises compositions comprising an RNA molecule comprising a reverse complement sequence comprising an exogenous sequence and homology arms, an RNA molecule comprising an endonuclease, and one or more guide RNAs for specific genomic integration of the exogenous sequence.

The invention discloses methanogen lyase with the same protein family annotation as PeiR lyase and application of the methanogen lyase, and the methanogen lyase is obtained through homologous screening of Pfam protein family database annotation based on known PeiR lyase characteristics aiming at methanogen cell wall peptide bonds. The PeiR lyase protein can participate in the biological process of hydrolyzing archaea cell walls, methanogens are effectively killed, methane production is reduced, and the protein homologous with the protein has potential methane reduction potential. According to the invention, by integrating metagenome sequencing data of rumen microorganisms, screening methanogenic bacterium virus protein by using series bioinformatics software, and combining Pfam functional domain annotation to carry out homologous relationship analysis, a series of lyase is finally identified and is successfully expressed in a prokaryotic expression system, and meanwhile, an in-vitro gas production experiment result also shows that the methanogenic bacterium virus protein can be successfully expressed in a prokaryotic expression system. The crude enzyme liquid can significantly reduce the methane generation amount.

The invention relates to a molecular dynamics simulation method based on interaction of nucleic acid and protein, which comprises the following steps: carrying out molecular modeling on a target nucleic acid sequence to obtain a nucleic acid three-dimensional structure model; performing structure prediction on the target protein sequence according to the homologous template and a deep learning method to obtain a protein three-dimensional structure model; performing interaction site screening on the nucleic acid three-dimensional structure model and the protein three-dimensional structure model by using a molecular docking algorithm to obtain a compound initial conformation of nucleic acid and protein; based on multi-stage energy minimization and ensemble balance conditions, performing dynamic simulation on the initial conformation of the compound to obtain conformation trajectory data; and performing multi-dimensional analysis on the conformation trajectory data to obtain a quantitative index of the interaction between the nucleic acid and the protein. According to the method, the modeling precision is improved, manual intervention is reduced, and the simulation efficiency is improved.

The invention discloses an engineering bacterium capable of efficiently degrading NP as well as a construction method and application of the engineering bacterium, and belongs to the technical field of wastewater treatment. The engineering bacterium is pseudomonas monteilii EBTRC-PM with an overexpressed ubiH gene, and the engineering bacterium is a pseudomonas monteilii EBTRC-PM The construction method comprises the following steps: designing a primer according to the genomic DNA of the pseudomonas monteilii EBTRC-PM, carrying out PCR to obtain a ubiH gene segment, carrying out homologous connection on the ubiH gene segment and a plasmid, replacing a T7 promoter of a reconstructed plasmid with a strong promoter P13, and finally transferring into the pseudomonas monteilii EBTRC-PM to obtain the engineering bacterium capable of efficiently inhibiting the toxicity in the NPEO degradation process, so that the NP degradation capability is greatly improved, and the engineering bacterium has the advantages that the construction is simple, and the construction cost is low. Good application prospects are realized in the field of wastewater treatment.

The invention discloses methanogen lyase homologous with high-dimensional characteristics of PeiR lyase and application of the methanogen lyase and aims to solve the problems that PeiR lyase protein of methanogen cell wall peptide bonds can participate in the biological process of hydrolyzing archaea cell walls, methanogen is effectively killed, methane production is reduced, and protein homologous with the protein has potential methane reduction potential. According to the invention, a series of biological information software is utilized to identify protein sequences coded by methanogen viruses from a large amount of rumen microbiome sequencing data, and high-dimensional homology between the proteins and PeiR proteins based on a deep learning model TM-Vec is further analyzed, so that a series of methanogen lyases are obtained. In order to realize the expression of the lyase, multiple segments of primers are designed to synthesize a target sequence, and the target sequence is successfully expressed in a prokaryotic expression system. Through the detection of crude enzyme liquid, the effectiveness of the lyase in the aspect of reducing the methane yield is verified in an in-vitro gas production test.

The invention discloses a method for obtaining and culturing ED pathway regulated and controlled engineering bacteria for fermentation production of vitamin B12, a mutant strain is based on ensifer adhesion, an ED pathway key gene edd gene in the ensifer adhesion of a vitamin B12 production strain is overexpressed by a homologous recombination single exchange method, an edd gene overexpressed mutant strain is constructed, and the edd gene overexpressed mutant strain is used for producing the vitamin B12. The purpose of disturbing the ED pathway to realize the metabolic migration of central carbon is achieved, and the method is applied to the production of vitamin B12. A shake flask fermentation experiment proves that overexpression of the edd gene can remarkably promote synthesis of a VB12 product, and the edd gene has good commercial and scientific research values.

The invention discloses a biotin ligase mutant and application thereof, and belongs to the technical field of gene engineering. According to the invention, a series of biotin ligase mutants are prepared by docking biotin ligase and substrate molecules thereof and carrying out multiple mutations on screened 10 amino acids with binding force acting sites. On the basis, a biotin ligase mutant is connected to escherichia coli through a homologous recombination technology, a series of escherichia coli recombinant bacteria with high biotin yield are obtained, and the biotin yield reaches 0.873 mg / L to the maximum after 24-hour fermentation.

The present application provides methods, compositions, and systems for decoding a pseudouridine-modified (psi-modified) codon. In some aspects, the present application provides methods for improving the translation of a target RNA in a host cell, the target RNA comprising a psi-modified codon wherein the method comprises: introducing a near homologous transport RNA (nctRNA) of the psi-modified codon or a nucleic acid encoding the nctRNA into the host cell; and wherein the psi-modified codon is decoded to an amino acid, thereby improving translation of the target RNA. In some cases, the codon of the psi modification is a premature termination codon (PTC).