130 results about "Vector (molecular biology)" patented technology

The invention belongs to the technical field of molecular biology, and particularly relates to a CeNF-YA3 gene, an expression vector and application of the CeNF-YA3 gene in vegetable fat regulation and control. The nucleotide sequence of the CeNF-YA3 gene is as shown in SEQ ID NO.1. After the CeNF-YA3 gene is over-expressed, the grease content of arabidopsis seeds and leaves can be remarkably increased; in addition, the invention also provides a series of vectors containing the CeNF-YA3 gene for subcellular localization, yeast hybridization and plant overexpression for the first time, and proves that the CeNF-YA3 protein has a transcriptional activation function, is localized in a cell nucleus and accords with the basic characteristics of transcription factors for the first time. Therefore, the technical scheme of the invention provides scientific guidance for the application of the CeNF-YA3 gene and the protein thereof.

The invention discloses a stropharia rugoso-annulata SRU6 gene promoter, a silent vector containing the promoter and application of silent stropharia rugoso-annulata endogenous genes, and relates to the field of molecular biology and gene engineering. According to the principle of shRNA mediated gene silencing, a silencing vector suitable for gene silencing of stropharia rugoso-annulata is constructed, and meanwhile, a stropharia rugoso-annulata efficient genetic transformation system is established; by utilizing the silent vector and the transformation system established by the invention, the expression quantity of the target gene can be reduced by about 80%, the purpose of gene function research is achieved, and the silent vector and the transformation system have very strong theoretical research and application values.

The invention belongs to the technical field of molecular biology and genetic engineering, and discloses a CRISPR / dCas9 vector for improving the yield of lactyl-N-tetrasaccharide of bacillus subtilis as well as a construction method and application of the CRISPR / dCas9 vector, and a CRISPR / dCas9 system is applied to production of lactyl-N-tetrasaccharide from bacillus subtilis 168 for the first time. According to the invention, pW-dcas9, pW-sgRNA and pW-sgRNA-zwf recombinant vectors are constructed, the transcriptional level of the zwf gene in a phosphopentose pathway is significantly reduced by 11.32 times by using a CRISPR / dCas9 system induced by xylose, and the metabolic flux of a competitive pathway is weakened. The yield of the lactyl-N-tetrasaccharide of the engineering strain obtained by the invention is greatly improved by 31% and reaches 7.25 g / l. Meanwhile, an effective method is provided for lowering metabolic flows of a cell wall competitive pathway and a glycolysis competitive pathway in a later period.

The invention discloses novel application of a GJB5 gene, a construction method of a GJB5 gene overexpression vector and a method for improving high temperature resistance of economic animals, and relates to the technical field of molecular biology. The preparation for overexpressing the GJB5 gene improves the heat resistance of the economic animal sertoli cells through overexpression of the GJB5 gene. Meanwhile, the invention also discloses a construction method of the GJB5 gene overexpression vector and a method for improving the high temperature resistance of economic animals. The effect of the GJB5 gene in heat stress is found for the first time, the gene is expected to be overexpressed in pigs or genetically modified into economic animals such as cattle and sheep in the future, so that the heat resistance of the pigs is improved, the semen quality of the pigs under the heat stress condition is further improved, and the GJB5 gene has important social benefits and economic benefits.

This application relates to the field of molecular biology, and more specifically to isolated transposases and their use. More specifically to nucleic acids and nucleic acid constructs encoding transposases, nucleic acid sets and nucleic acid set constructs, and compositions, recombinant vectors, recombinant host cells and kits comprising transposases. More specifically to methods for introducing exogenous nucleic acid fragments into the genome of host cells, methods for editing the genome of host cells, and methods for obtaining host cells containing exogenous nucleic acid fragments in their genomes. More specifically to the use of transposases, nucleic acids and nucleic acid constructs, nucleic acid sets and nucleic acid set constructs, compositions, recombinant vectors, or recombinant host cells for introducing exogenous nucleic acid fragment genes into the genome of host cells, or for preparing drugs or formulations for gene therapy, cell therapy, genome research, or stem cell induction and post-induction differentiation.

The invention relates to a Sebastes schlegeli endogenous retrovirus envelope protein Penv, a lentiviral vector and application thereof, and belongs to the field of genetic breeding of molecular biology, and the amino acid sequence of the envelope protein Penv is as shown in SEQ ID NO.1. The invention further provides an in-vitro assembly system, a transformant and a kit containing membrane fusion protein particles and lentiviral particles of the envelope protein Penv and application of the envelope protein Penv. VSVG protein is replaced with Penv protein from sclerobone fish, so that the transduction efficiency of the lentiviral particles to sclerobone fish cells is effectively improved, and the transduction efficiency of the lentiviral particles to the sclerobone fish cells is improved. And efficient gene delivery is realized. Meanwhile, as the endogenous Env protein, the Env protein overcomes the immunological rejection of a host to the exogenous Env protein, and also has the potential of in-vivo application.

The invention belongs to the technical field of molecular biology, and particularly relates to a method for transferring an exogenous nucleic acid fragment into a plant by using a plant virus. The technical problem to be solved by the invention is to provide a new method which can be applied to transferring an exogenous fragment into a plant. According to the technical scheme, the method for transferring the exogenous nucleic acid fragment into the plant by using the plant virus comprises the following steps: fusing the exogenous nucleic acid fragment into a 3'non-coding RNA region of TVMVwt to obtain a fused fragment, constructing the fused fragment onto an infectious clone skeleton vector, and transforming the plant. According to the invention, an exogenous coding sequence is inserted into a 3'non-coding RNA region of TVMVwt to construct an infectious cloning vector, the infectious cloning vector is transferred into a plant, and expression of the exogenous coding sequence is realized.

The present application relates to the field of molecular biology, in particular to a recombinant human heparin N-deacetylase encoding gene, a recombinant vector, a recombinant strain and application thereof, the recombinant human heparin N-deacetylase constructed in the expression process shows excellent stability and activity, has the ability of specific catalysis of deacetylation of acetyl group in heparin precursor, and can effectively generate deacetylated heparin precursor. This property significantly improves the conversion efficiency and reduces the generation of by-products. The present application optimizes the codon of the recombinant human heparin N-deacetylase, and uses the promoter P AOX1 or P GAP to regulate the expression, so that the expression level of the recombinant human heparin N-deacetylase in Pichia pastoris is significantly improved, and the obtained GS115-AOX1-hNDase and GS115-PGAP-hNDase strains successfully realize the secretory expression of the target gene, providing a new strategy and method for the biosynthesis of heparin.

This application belongs to the field of plant molecular biology technology and specifically provides a PsbHLH35 gene and encoded protein, and their use in tree peony somatic embryogenesis. A PsbHLH35 gene, comprising the nucleotide sequence shown in SEQ ID NO. 1. A protein encoded by the PsbHLH35 gene, comprising the amino acid sequence shown in SEQ ID NO. 2. A method for using the PsbHLH35 gene in tree peony somatic embryogenesis, comprising the following steps: S1) cloning the PsbHLH35 gene into a vector to construct a PsbHLH35 gene overexpression vector; S2) transforming the PsbHLH35 gene overexpression vector into tree peony. The PsbHLH35 gene of this application has a significant promoting effect on tree peony somatic embryogenesis.

The present invention belongs to the field of molecular biology and genetics, and in particular relates to a method for editing a bovine gene based on Pro-iCHI. The present invention provides a method for editing a bovine gene based on Pro-iCHI. Protamine is transiently expressed in gene-edited b-haSCs, which are then injected to mature oocytes to obtain reconstructed embryos. Protamine can eliminate abnormal DNA methylation resulting from oocyte intracytoplasmic haSCs injection and enable the nucli to compress into sperm-like structures, and the obtained bovine Pro-iCHI embryos can successfully develop into blastocysts, with a blastocyst rate comparable to that of the embryos obtained by in vitro fertilization. Moreover, in the present invention, a protamine-encoding gene is inserted into a Saccharomyces cerevisiae protein expression vector for transient expression, which ensures that abnormal DNA methylation is erased, without integration into the genome resulting in the insertion of exogenous genes.

This invention discloses a novel site-specific recombinase-mediated multisite chromosome editing tool and its applications. The invention provides a complete product suite for integrating or continuously integrating the genome of a host cell, comprising: a circular vector 1 composed of the following elements: loxF1, nox, ori, resistance gene, lox71, P3, P1, P2; and / or a circular vector 2 composed of the following elements: lox66, nox, ori, resistance gene, loxF2, P3, P1, P2; and a circular vector 3 for expressing Nigri and Cre recombinases; and a site-specific knock-in tool for knocking in the lox66-nox fragment at the desired integration site in the host cell genome; P1 and P2 are adapters, used to insert the DNA fragment to be integrated, and P3 is a universal primer. This invention enables rapid and continuous genome integration using simple molecular biology operations.

The invention relates to the technical field of crossing of molecular biology, nucleic acid detection and bioinformatics, and discloses an in-vivo detection method for synchronously detecting CRISPR off-target sites and viral vector integration sites. According to the in-vivo detection method for synchronously detecting the off-target site of the CRISPR and the integration site of the virus vector, the reverse terminal repeat (ITR) of the AAV vector is used as a tag sequence for the first time, and a universal PCR (Polymerase Chain Reaction) amplification primer is designed and a unique molecular identifier (UMI) technology is introduced to detect the off-target site of the CRISPR and the integration site of the virus vector. And meanwhile, an off-target event and an AAV carrier integration event in an in-vivo CRISPR editing process are captured and accurately recognized. Compared with the existing schemes such as GUIDE-seq, DISCOVER-Seq +, OliTag-seq, U-DiTaS and the like, the in-vivo detection method for synchronously detecting the CRISPR off-target site and the virus vector integration site, provided by the invention, has the advantage that remarkable breakthrough is realized in the aspects of in-vivo applicability, detection depth, operation economy and industrialization standardization.

The invention belongs to the technical field of molecular biology, and particularly relates to an anti-GLP-1R nano antibody as well as a preparation method and application thereof. The invention provides an anti-GLP-1R nano antibody, and also provides a construction method of the anti-GLP-1R nano antibody. The method comprises the following steps: constructing GLP-1R overexpression plasmids and overexpression cell strains, immunizing alpaca by using the plasmids and the cells to obtain PBMC cells, extracting total RNA, carrying out reverse transcription to obtain cDNA, amplifying antibody gene segments through PCR, connecting the segments to phage expression vectors, and electrically transforming the constructed vectors into SS320 competent cells to finally obtain the GLP-1R immune antibody library. The method comprises the following steps: carrying out cell panning for one round, carrying out Cell ELISA detection to obtain an antibody combined with GLP-1R, carrying out sequencing to obtain an antibody sequence, obtaining antibody protein through mammalian cells, and carrying out FACS detection by using CHOK1-GLP-1R cells to finally obtain the antibody combined with GLP-1R. The method is simple and convenient to operate, and the activity of the antibody obtained by using a mammalian cell expression system is closer to that of a natural antibody.

The present invention relates to the technical field of molecular biology, and in particular, to an anti-CD40 nanobody, and a preparation method therefor and use thereof. The anti-CD40 nanobody provided by the present invention has three unique complementarity determining regions, namely CDR1, CDR2, and CDR3. The present invention further provides a sequence encoding the nanobody or its VHH chain, a corresponding expression vector, and a host cell capable of expressing the nanobody. The present invention includes immunizing an alpaca by constructing a CD40 antigen to obtain a PBMC of the alpaca and constructing a vector; screening an anti-CD40 antibody through phage display; and finally obtaining a highly sensitive and specifically binding antibody through functional assays and sequencing result analysis. The nanobody provided by the present invention can specifically recognize and bind to CD40.

The present invention relates to the fields of molecular biology and genetic engineering, and relates to crayfish egg cell guide peptides, fusion proteins, nucleic acid molecules, recombinant expression vectors, host cells, methods for guiding exogenous proteins into crayfish eggs, and applications thereof. The amino acid sequence of the crayfish egg cell guide peptide is as shown in SEQ ID NO.1. The present invention uses green fluorescent protein (eGFP) as an example protein to prove that the polypeptide can successfully deliver exogenous proteins. This breakthrough can lay a technical foundation for the introduction of gene editing tools (such as the CRISPR-Cas9 system) into crayfish oocytes, and is expected to be widely used in the fields of crayfish breeding, functional gene research, and aquaculture gene improvement.

The present invention discloses circApbb1 molecules, siRNA targeting circApbb1 expression, adeno-associated virus vectors, preparation methods and applications, belonging to the field of molecular biology applications. A siRNA capable of knocking down circApbb1 expression was designed, and an adeno-associated virus vector HBAAV2 / 9‑cTNT‑mir30‑circApbb1Green of the siRNA was constructed. Detection showed that circApbb1 was significantly upregulated in the hearts of mice with atherosclerosis. The prepared siRNA adeno-associated virus vector can effectively reduce the expression of circApbb1 in the mouse heart, reduce the area of ​​atherosclerotic plaques, inhibit the progression of atherosclerosis and improve cardiac function, thus realizing the use of circApbb1 to treat atherosclerosis, providing an important supplement to the biological function of circApbb1, and providing a new technical means for the treatment of atherosclerosis.

The invention discloses a ribosome group or selective ribosome group detection and analysis method based on an immune principle, and relates to the field of molecular biology and genomics. Comprising the following steps: sample preparation and cross-linking: treating cells by using a translation inhibitor to suspend ribosome translation, and then cross-linking RNA-protein in the cells by using a membrane permeable cross-linking agent; then cracking the cells, digesting RNA of a cracking product by using ribonuclease, and retaining ribosome and mRNA fragments protected by the ribosome; immune enrichment: carrying out specific immune enrichment on the digested sample by using an antibody combined with a carrier, and eluting to obtain a ribosome-mRNA compound; then decrosslinking and removing protein, extracting mRNA fragments and carrying out phosphorylation treatment so as to add joints, build a library and carry out sequencing; and finally, library construction, sequencing and bioinformatics analysis are carried out. The method has the advantages of high-specificity enrichment, accurate positioning of translation sites, wide application range and accurate sequence information.

The invention relates to the technical field of gene engineering, in particular to a DNA (Deoxyribonucleic Acid) oriented demethylation double lentivirus plasmid system as well as a construction method and application thereof. The double lentivirus plasmid system comprises a dCas9-GCN4s sequence and an Anti-GCN4-scFv-TET1 sequence, and the dCas9-GCN4s sequence and the Anti-GCN4-scFv-TET1 sequence are The sequence of the dCas9-GCN4s is as shown in SEQ ID (sequence identifier number) No.36; the sequence of the Anti-GCN4-scFv-GFP-TET1CD is as shown in SEQ ID No. 37, and the sequence of the Anti-GCN4-scFv-GFP-TET1CD is as shown in SEQ A guidance system and a demethylation system are respectively inserted into a lentiviral vector by means of molecular biology, and are associated by an antigen-antibody technology, and the demethylation effect is amplified, so that an obvious demethylation effect is finally achieved on a target position of a target cell genome.

This application relates to the fields of gene therapy and molecular biology. More specifically, the present invention relates to an isolated altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid comprising one or more amino acid substitutions as compared to the VP1 protein of wild-type AAV5 capsid, which increase transduction efficiency, as well as to a capsid and a vector based thereon.

This invention relates to the fields of molecular biology and plant genetic engineering, and proposes a gene expression enhancement technology based on reprogramming 3'UTR functional elements. The technology comprises the following steps: S1, reconstructing translational regulatory elements within the 3'UTR region of a target gene to construct a dual-luciferase reporter vector; S2, using the dual-luciferase reporter vector plasmid as plasmid DNA, transforming plant protoplasts using the PEG method, and subsequently extracting the target protein. This invention repositions translational regulatory elements (Omega, 6MΔT, and N50‑high) within the 3'UTR region of a target gene, significantly enhancing protein expression levels of exogenous genes in plants. This effect is independent of transcriptional regulation, revealing an innovative approach to functional reprogramming of 3'UTR elements and providing a novel solution to addressing the bottleneck of translation efficiency in existing technologies.

The invention relates to a construction method of an OriCiro cell-free cloning system based on an RCR technology, and belongs to the technical field of molecular biology. The construction method comprises the following steps: expression and purification of high-activity Tn5 transposase, transposon-mediated oriC insertion, RCR amplification, traceless excision of oriC transposon and repair cyclization. The construction method provided by the invention provides a rapid, reliable and mutation-burden-free new genome construction and rescue path for the existing research, can solve the difficult problem of construction of large-size and toxic DNA sequence vectors for gene therapy, shortens the research and development and production periods, and improves the production efficiency. Therefore, a powerful tool is provided for the fields of preparation of mRNA vaccines or therapy plasmids, construction of gene therapy vectors, supply of pharmaceutical-grade plasmid DNA, research of a virus molecular mechanism and the like.

The invention relates to the field of tumor molecular biology, and particularly discloses a LentiCRISPRv2 / Cas9-sgRNAs gene editing system based on a cancer promoting gene Cebpg as well as a preparation method and application of the LentiCRISPRv2 / Cas9-sgRNAs gene editing system. Aiming at the current situation that CEBPG high expression in gastric cancer is related to tumor progression, a LentiCRISPRv2-Cas9 plasmid is connected with sgRNA of a specific targeting CEBPG gene, and an efficient gene editing system is constructed. The preparation method comprises the following steps: designing an sgRNA sequence of a targeted CEBPG coding region, carrying out single-chain sgRNA annealing, carrying out vector enzyme digestion and connection, preparing a LentiCRISPRv2 / Cas9-sgRNAs vector, and carrying out lentivirus packaging to realize gene knockout. The system can significantly inhibit CEBPG expression, slow down gastric cancer cell proliferation and improve an immunosuppressive microenvironment, and is especially suitable for CEBPG high-expression gastric cancer treatment.

The invention relates to the technical field of molecular biology, in particular to an antibacterial peptide SAV-546 as well as a construction method and application thereof, and the construction method of the antibacterial peptide SAV-546 comprises the following steps: obtaining a target gene segment through gene cloning; constructing an expression recombinant vector by using the target gene segment to obtain a positive recombinant plasmid; and carrying out prokaryotic induced expression on the positive recombinant plasmid to obtain the antibacterial peptide SAV-546. The antibacterial peptide SAV-546 disclosed by the embodiment of the invention is not easy to induce bacteria to generate drug resistance, the construction method of the antibacterial peptide SAV-546 is simple, convenient and efficient, the cost is low, the antibacterial peptide SAV-546 can be widely applied to preparation of antibacterial drugs, medical dressings and food preservatives, and a new scheme is provided for solving the problem of clinical drug-resistant bacterial infection.

The present invention relates to the fields of biotechnology and molecular biology, in particular to a universal inducible promoter, vector and host cell based thereon, as well as to a method for producing said host cell. The proposed inventions make it possible to analyze the activity of a target protein, for example, a receptor ligand, for example, a cytokine.

The present application relates to the fields of genetics, gene therapy, and molecular biology. More specifically, the present invention relates to a nucleic acid having promoter activity (variants), an expression cassette and a vector based thereon, a host cell for producing a target product or expression vector.

The present invention provides a method for constructing a oligospora spore that spontaneously produces a predatory organ, belonging to the field of molecular biology technology. The method comprises: sequentially connecting a promoter, a target gene, a GFP sequence, a hygromycin selection marker, and a terminator, and constructing the entire construct in a pCE-Zero vector; then using primer 1 for PCR amplification to obtain a fluorescent conversion fragment; preparing oligospora spore protoplasts, and transforming the fluorescent conversion fragment into the oligospora spore protoplasts; and screening for positive transformants. The oligospora spores obtained by this method can spontaneously produce a predatory organ, greatly advance the response to nematodes, and have a better control effect on nematodes.

The present application relates to the fields of genetics, gene therapy, and molecular biology. More specifically, the present invention relates to a codon-optimized nucleic acid that encodes the FVIII-BDD (B-domain deleted coagulation factor VIII) protein, to an expression cassette and a vector based thereon, to a host cell for producing FVIII-BDD, as well as to various uses of the above vector.

The invention relates to the technical field of molecular biology, in particular to a construction method of a J774A.1 cell line with a baz2b gene knocked out. The invention provides a construction method of a J774A.1 cell line with a baz2b gene knocked out. The construction method comprises the following steps: constructing a J774A.1 cell line for expressing Cas9 protein; the recombinant vector contains the sgRNA; a recombinant vector, pMD2. G and psPAX2 are co-transfected into cells, and then the cells, a J774A.1 cell line for expressing Cas9 protein and puromycin antibiotic are screened to obtain a J774A.1-KO baz2b cell line with inactivated baz2b function, and the J774A.1 cell line with the baz2b gene knocked out is obtained. The cell line can regulate the formation of a macrophage extracellular trapping net.

The invention relates to the field of genetic engineering and molecular biology, in particular to a nucleic acid construct, and a vector, a host cell, a viroid particle, a vaccine and a pharmaceutical composition containing the nucleic acid construct. Furthermore, the invention also provides application of the nucleic acid construct vector, the host cell, the viroid particle, the vaccine and the pharmaceutical composition in prevention and / or treatment of virus (e.g., SARS-CoV-2) infection or diseases and / or symptoms caused by virus infection.

The invention relates to the fields of genetic engineering and molecular biology, in particular to a method for optimizing a nucleotide sequence of an expression protein in a host cell and a method for optimizing a nucleotide sequence of an expression protein in a Vero cell. The invention also relates to nucleic acid molecules, vectors, host cells and viroid particles comprising the optimized nucleotide sequences obtained by said method.