1221 results about "Gene clone" patented technology

Introducing blocks of foreign genes in a single operon would avoid complications such as position effect and gene silencing inherent in putting one gene at a time into random locations in the nuclear genome. Cloning several genes into a single T-DNA does not avoid the compounded variable expression problem encountered in nuclear transgenic plants. This disclosure shows that a bacterial operon can be expressed in a single integration event as opposed to multiple events requiring several years to accomplish. Expression of multiple genes via a single transformation event opens the possibility of expressing foreign pathways or pharmaceutical proteins involving multiple genes. Expressing the Cry2aA2 operon, including a putative chaperonin to aid in protein folding, in the chloroplast via a single transformation event leads to production of crystalized insecticidal proteins. Expressing the Mer operon via a single transformation event leads to a phytoremediation system.

The invention provides a kit for separating genome DNA by using magnetic balls and application thereof. The kit comprises magnetic balls, a magnetic frame, a genome DNA extraction reagent (lysing solution, binding solution, rinsing solution A, rinsing solution B and eluent) and specifications; the main steps of extracting the gene DNA comprise cell lysis, nucleic acid absorption, impurity removing and nucleic acid elution. The application of the kit for extracting the genome DNA does not need to use large-toxicity organic solvents of phenol, chloroform and the like, has good safety, and simple, fast and time-saving operation, and simultaneously can extract a plurality of samples. The kit can extract the genome DNA from materials of animals, plants, bacteria, fungus, blood, virus, animal source feed stuff, samples in the forensic medicine and the like, the DNA has high yield and purity, and the obtained genome DNA can be used for experiments such as PCR amplification, gene cloning, construction of genomic library, sequence measurement, molecular hybridization, molecular marking and the like. The kit can be stored at the temperature of 4 DEG C, also can be placed at room temperature, and is convenient to transport.

The invention discloses directional knockout on sheep MSTN gene by applying the CRISPR / Cas9 technology, and a method for verifying the impact effect of directional knockout on skeletal muscle satellite cell differentiation. The concrete contents are as follows: firstly, target gene cloning; secondly, gRBA design and synthesis; thirdly, CRISPR / Cas9 gene knockout vector construction; fourthly, CRISPR / Cas9 gene knockout vector exogenous activity detection; fifthly, CRISPR / Cas9 gene knockout vector endogenous activity detection; sixthly, CRISPR / Cas9 gene knockout vector knockout effect detection. The method has the following advantages: firstly, the experimental period is shorter; secondly, the method is simple and practicable, has high repeatability, and can be performed in common laboratories; thirdly, active vector constructed through the method is small in toxic and side effects, and can be applied to the preparation and production of transgenic animals; fourthly, the method is small in non-specific shear, and obviously improves the gene targeting knockout efficiency; fifthly, the method is high in applicability.

The invention discloses a recombined chimeric antigen receptor with a photoinduction element. The chimeric antigen receptor comprises an antigen combining area, a transmembrane structure area, a co-stimulatory signal transduction area and T cell signal transduction area functional structure domains, wherein the photoinduction element is inserted at the N terminal or C terminal of the recombined chimeric antigen receptor or between the functional structure domains. According to the chimeric antigen receptor, photoinduction element LOV2 genes are cloned into plasmids Lenti-EF1alpha-CD19CAR with a molecular cloning method to establish a co-expression vector of the recombined chimeric antigen receptor with the photoinduction element, 293T cells are used for packaging to obtain a lentiviral vector with photoinduction CAR molecules. After the recombined chimeric antigen receptor is expressed in T cells through the lentiviral vector, the damage toxicity of T cells is reversely controlled by LOV2, and an importable approach is provided for improving the safety of CAR-T in treating cancer clinically.

The present invention relates to the technical field of plant gene engineering and concretely relates to the separation clone of paddy wide compatibility gene S5, a function validation and an application thereof for improving the paddy. The segment of the gene contains paddy wide compatibility gene coding aspartic proteinase. Subspecific Indica-Japonica paddy has strong hybrid advantage than varietal paddy, but the sterility of the subspecific Indica-Japonica hybrid restricts using the hybrid advantage thereof. The wide compatibility gene cloned by the present invention can conquer the sterility of the subspecific Indica-Japonica hybrid of the paddy and accordingly uses the strong hybrid advantage of the subspecific Indica-Japonica to further improve the output of the paddy.

The invention discloses a method for obtaining transgenic bovine fetal fibroblast by using Cas9 cutting nuclease-mediated Ipr1 fixed point insertion. The bovine fetal fibroblast is cotransfected by adonor vector and a CRISPR / Cas0 expression vector for a target locus through an electroporation method, an MSR1 promoter in the donor vector can realize specific expression in full-time phagocytic cells, and after puromycin drug screening, the targeted positive clone cell is obtained through PCR authentication. The transgenic positive clone cell is used as a donor cell so as to obtain a transgenicclone embryo, the transgenic clone embryo is further transplanted into the uterus of a rutting acceptor calf, and finally a living Ipr1 fixed point insertion transgenic cloned calf is obtained.

The invention discloses a monoclonal antibody 64360-52D1 capable of specifically combining with SARS-Cov-2 virus structural nucleocapsid protein (N protein), a preparation method thereof, and 6 complementarity-determining regions (CDR) of heavy chain and light chain variable regions; and more specifically, the monoclonal antibody is secreted by a hybridoma strain 64360#52D1, and can specifically recognize the SARS-Cov-2 virus N protein rather than SARS virus N protein. Thus, the monoclonal antibody can be used for identifying the two coronaviruses with high similarity. The invention further provides an enzyme-linked immunosorbent assay (ELISA) and an immune colloidal gold test strip detection method for specifically detecting the SARS-Cov-2 virus N protein by preparing the 64360#52D1 antibody. The antigen of the antibody is SARS-Cov-2 virus N protein subjected to heat treatment and expressed in mammalian cells; the finally obtained antibody belongs to an IgG1 subtype; and a sequence for encoding the variable region of the antibody is obtained in a gene cloning mode.

The cystic fibrosis gene and its gene product are described for both the normal and mutant forms. The genetic and protein information is used in developing DNA diagnosis, protein diagnosis, carrier and patient screening, drug and gene therapy, cloning of the gene and manufacture of the protein, and development of cystic fibrosis affected animals.

The invention discloses soluble programmed dead protein-1-IgV and a preparation process. The protein sequence of is showed as followed: PPTFFPAL LVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQ DSRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAE LRVTERRAEV PTAHPSP. The invention obtains gene of Hpd-1-IgV through applying artificial synthesis according to the structure of human PD-1, target gene is cloned into pQE-30 pronucleus expression carrier to transform colibacillus and is expressed in colibacillus with high efficiency, bacterioclasis shows that target protein is existed in inclusion body form, and purified soluble programmed dead protein-1-IgV is finally obtained through inclusion body scouring, affinity chromatography, gel column chromatography and dialysis renaturation. The soluble programmed dead protein-1-IgV can respectively combine with mPD-L1 / Fc and hPD-L1 / Fc in extra-corporal shPD-1IgV and on cell level and molecular level, and has certain tumor killing effect in internal shPD-1IgV. Therefore, shPD-1IgV has excellent internal and external biological activity, provides experiment basis for curing tumor, establishes experiment foundation, and has potential application prospect.

The invention relates to a host cell and a method for the host cell applied in efficient secretion expression of a recombinant protein, which belongs to the filed of microbiological engineering and fermentation engineering. The invention provides a bacillus licheniformis host cell CBB3008 which is preserved in the China Center for Type Culture Collection with the preserving number of CCTCC NO: M 208236. The invention transforms an expression plasmid of an industrial enzyme which is obtained by a gene cloning technology into the host cell to synthesize an industrial enzyme preparation in the host cell efficiently, and then secretes the synthesized enzyme protein into a culture medium efficiently through a protein secretion system of the host cell, thus the invention can guide efficient secretion production of the industrial enzyme preparation. Therefore, the invention is helpful to reduce the fermentative production cost of the industrial enzyme preparation, simplify the fermentative production process and reduce the environmental pressure of the fermentation industry. A method for host cell screening and genetic improvement of the invention can also be used for other types of host cells, in particular for the breeding of the host cells of bacillus subtilis, bacillus megaterium, bacillus pumilus, bacillus starch solution and the like.

The invention provides a novel species of α-1,3-fucosyltransferase expressed by a gene cloned from animal cells, a cDNA coding for the α-1,3-fucosyltransferase, a method of detecting, or inhibiting the production of, the α-1,3-fucosyltransferase which involves the use of the cDNA, a recombinant vector containing the DNA as an insert, a cell harboring the recombinant vector, and a method of producing same. The α-1,3-fucosyltransferase of the invention is useful in the production of carbohydrate chains having useful physiological activity, for example sialyl Lewis x, and modifications thereof.

The invention discloses a cat omega interferon mutant as well as a preparation method and an application thereof, and belongs to the field of preparation and application of cat omega interferons. A cat omega 2 or omega 11 interferon mutant is obtained by comparing gene sequences and amino acid sequences of 13 subtypes of cat omega interferon and performing amino acid site-directed mutation on cat omega 2 or omega 11 interferon. The invention further discloses the method for preparing the cat omega interferon mutant. The method comprises the following steps: cloning a gene for coding the cat omega 2 or omega 11 interferon mutant into a baculovirus transfer vector, recombining with a baculovirus for infecting an insect host, expressing an exogenous gene, and obtaining a cat omega interferon protein expression product. The method is simple in process, the cat omega interferon capable of being safe to use can be efficiently and stably obtained, and the antiviral activity of the cat omega interferon mutant is remarkably improved. The cat omega 2 or omega 11 interferon mutant can be used for preparing drugs or reagents for preventing or treating cat viral diseases.

This invention relates to a new clone mycose synthesized zyme gene and its expression in the correlated host which zyme gene clone belong to thermobifida turning the malt sager to mycose under normal biochemical reaction, its zyme substrate conversion efficiency increases along with the decreasing of the reaction temperature, taking the malt sager or amylum as the raw material and turn it to alpha 1-mycose alpha-1 by cloned.

The invention discloses a gene sequence of Incision alginate lyase Alg2B from Flavobacterium strain (Flavobacterium sp. S20). The invention also provides a method for preparing novel alginate lyase, which is characterized in that by using a gene engineering technical method, the gene of the alginate lyase is cloned to an escherichia coli expression vector, the escherichia coli recombinant strain capable of realizing heterogenous expression of enzyme can be obtained, the recombinant strain is used for heterogenous expression of the alginate lyase Alg2B, and thereby monosaccharide and oligosaccharides produced by sodium alginate can be degraded. The provided alginate lyase Alg2B can be widely used in the fields of agriculture, food, feed addictives, medicine and alga genetic engineering.

The invention discloses a recombinant alginate lyase and a construction method and an application thereof, which belong to the technical field of biology. The alginate lyase Aly-Cob is derived from anenvironment sample (a mixture of sea-tangle stacking place and factory production waste material). By using a gene engineering technology, a gene of the alginate lyase is cloned to an expression vector, and a recombinant bacterial strain for heterogenous expression of the alginate lyase is obtained. The alginate lyase Aly-Cob produced by the bacterial strain through fermentation has the functionfor degrading sodium alginate to prepare alginate oligosaccharide. The provided alginate lyase Aly-Cob can be widely used for the fields of agriculture, food, feed addition, medicine and alga processing.

The invention relates to the biomedical field, and in particular relates to a method for efficiently expressing recombinant liraglutide. The method for efficiently expressing recombinant liraglutide is used for performing gene cloning and transformed expression. The recombinant liraglutide is constructed, and liraglutide protein is prepared through an escherichia coli expression technology. Compared with the two methods, the method disclosed by the invention is simple and convenient to operate, high in yield and low in cost.

The invention belongs to the technical field of plant gene engineering, disclosing a separated and cloned major gene GS5 capable of controlling the width and the weight of a rice grain and the DNA sequence of the allelic gene of the major gene GS5. The DNA sequence is shown as SEQ ID NO.1 (Zhenshan 97B) and SEQ ID NO.3 (H94) and contains 10 exons. The amino acid sequence of the major gene and the amino acid sequence of the allelic gene are shown as SEQ ID NO.2 and SEQ ID NO.4. By using two large-grain rice varieties and two small-grain rice varieties for comparative sequencing, in an approximately 6.1kb range, 22 common base differences exist between the large-grain variety and the small-grain variety, wherein 18 mutations are in a promoter area, 4 mutations are in a code area and 5 amino acids are caused to be changed. By using a transgenic technology, GS5 transgenic rice plants are obtained and express that the width and the weight of the rice grain are obviously improved when being compared with the control width and the control weight of the rice grain. The character changes are quite coincident with the two genotype expressions of a Zhenshan 97 near-isogenic line and a GS5 near-isogenic line. The invention additionally discloses a method of near-isogenic line breeding, gene cloning and gene transfer and application thereof.

The invention discloses a method for acquiring a rhodotorula glutinis phenylalanine deaminase gene. The method comprises the following steps of: extracting RNA (Ribose Nucleic Acid); carrying out inverse transcription; carrying out PCR (Polymerase Chain Reaction) amplification; establishing a cloning vector pUCm-T-pal; and measuring a sequence to obtain a full-length phenylalanine deaminase gene sequence. The invention belongs to the technical field of gene clone. The method disclosed by the invention has the advantages of easiness for operation and higher efficiency and success rate.

The invention belongs to the technical field of preparation of molecular markers for livestock, and particularly relates to a preparation method of a sheep lambing number trait related molecular marker serving as MAS (marker-assisted selection) as well as an application of the molecular marker. The molecular marker is obtained through LH (luteinizing hormone) beta gene cloning, and the nucleotide sequence is represented in SEQ ID NO.1. One C713-T713 base is substituted at 713 bp in the SEQ ID NO.1, so that BSrB I-RFLP (restricted fragment length polymorphisms) is caused. The invention further discloses a primer used in a DNA (deoxyribonucleic acid) sequence of an amplified LH beta gene part and a polymorphism detection method, and provides one novel molecular marker for MAS of the sheep lambing number trait.

The invention discloses a preparation method for a recombinant coxsackie virus A16 like particle, which comprises the following steps: (1) cloning a P1 gene and a 3CD gene of a coxsackie virus A16 to a target plasmid to obtain a recombinant expression vector; (2) transforming a target yeast cell by using the recombinant expression vector obtained in the step (1) to obtain a recombinant yeast cellfor expressing the P1 gene and the 3CD gene; and (3) cracking the recombinant yeast cell obtained in the step (2), and separating to obtain the recombinant coxsackie virus A16 like particle. The recombinant coxsackie virus A16 like particle can be prepared in a yeast expression system by the method provided by the invention. Compared with a wild-type P1 gene and a wild-type 3CD gene, the yield ofthe coxsackie virus A16 like particle in the yeast expression system is greatly increased through the optimization of codons of the P1 gene and the 3CD gene, and the recombinant coxsackie virus A16 like particle can be further used for producing candidate preventive vaccines and pharmaceutical compositions for infant hand-foot-and-mouth diseases.

The invention provides a dust mite allergen and application thereof. The dust mite allergen provided by the invention belongs to a congener of cathepsin D, can be used for diagnosis, treatment and prevention of allergic diseases and especially dust mite allergic diseases, and is especially used for diagnosis, treatment and prevention of allergic diseases caused by the 34th component of the dust mite allergen. The dust mite allergen recombinant protein is prepared by gene cloning and protein expression, and has the advantages of high protein purity, high specificity and high yield. When being used for preparing reagents for diagnosing, preventing or treating allergic diseases caused by the dust mite allergen, the dust mite allergen provided by the invention has the characteristics of high specificity and low cost. Especially, the dust mite allergen can be efficiently used for diagnosing whether the patient is allergic to the 34th component of the dust mite allergen.

The invention discloses a preparation method and application of a classical swine fever virus recombinant subunit vaccine with the amino acid sequence shown as SEQ ID No.1. The preparation method of the classical swine fever virus recombinant subunit vaccine typically includes the following steps: classical swine fever E2 truncated protein (TE2) coding gene is cloned into baculovirus vector pFastBacTM1, and is then transfected into Sf9 insect cells to obtain recombinant baculovirus capable of expressing protein TE2. The high five insect cells in logarithmic growth phase are infected by the recombinant baculovirus, so that a large amount of the protein TE2 can be expressed in a cell culture supernatant. Finally, the cell culture supernatant is recovered and purified to obtain a large amount of the recombinant protein TE2 with the purity more than 90%. According to the method, the target protein can be harvested from the cell culture supernatant, the time of protein purification is reduced, consumption of a large amount of time can be avoided, and the vaccine production process can be simplified. Under the premise of simplification of the vaccine production process, the recombinant protein TE2 has the advantages of strong immunogenicity and high safety, and the animal experiments prove that the recombinant protein can effectively stimulate the body to produce a highly effective humoral immune response.

The invention discloses a method for producing a porcine parvovirus antigen and its product. The method comprises the following steps: porcine parvovirus capsid protein VP2 gene or optimized VP2 gene is cloned in a baculovirus carrier so as to obtain a transfer expression carrier; the constructed transfer expression carrier and baculovirus DNA are carried out cotransfection to obtain recombined baculovirus; the recombined baculovirus is used to infect insect host and cell; the infected insect host is cultured to express corresponding porcine parvovirus capsid protein; and the expressed antigen is ingathered and purified so as to obtain the porcine parvovirus antigen. The method adopts a baculovirus expression system to make safe and efficient porcine parvovirus antigen capsid particles in a domestic silkworm bioreactor; the prepared purified antigen by the method has high safety, and can be directly produced to vaccines for animal immunity. The method for producing porcine parvovirus antigen has the advantages of high expression efficiency, high immunization activity of the expressed antigen, low production cost, large scale production realization and the like.

The invention discloses a method for generating pterostilbene by utilizing grape resveratrol-oxygen-methyltransferase to catalyze resveratrol, grape ROMT (resveratrol-oxygen-methyl transferase) genes are cloned by utilizing 5' and 3' RACE (rapid amplification of cDNA ends) full-length gene cloning technology according to EST (expressed sequence tag) sequence of the ROMT genes of Chinese wild grapes in East China, and the full length of an open reading frame of the genes is 1074bp; and the cloned grape STS (steroid sulfatase) and ROMT genes can be simultaneously transferred into model plant tobacco, tobacco plant analysis is utilized for analyzing the biological synthesis path of generating the pterostilbene by utilizing the grape ROMT to catalyze the resveratrol, a grape ROMT gene sequence is obtained for orientation, and the ROMT genes can catalyze the resveratrol to generate the pterostilbene in the transgenic tobacco, thereby providing the sequence of the ROMT genes and the method for synthesizing the pterostilbene by utilizing plants and providing a basis for getting high-quality, disease-resistant and health care crops.

A simple and highly efficient method for cloning cDNAs including CD27 (SEQ ID NO:28) from mammalian expression libraries based on transient expression in mammalian host cells has been discovered. Novel expression vectors allowing highly efficient construction of mammalian cDNA libraries are disclosed. The cloning method of the invention which has been used to clone genes for cell surface antigens of human lymphocytes, has general application in gene cloning. Cell surface antigens cloned according to the present invention have been purified, and the nucleotide and amino acid sequences determined. These antigens have diagnostic and therapeutic utility in immune-mediated infections in mammals, including humans.

The invention belongs to the field of molecular biological gene engineering, and particularly relates to a double-control double-regulation prokaryotic expression vector system and a construction method and application thereof. Two expression vectors finish expression of a target gene in the vector system, wherein one expression vector is a main expression vector, and the target gene is cloned on the expression vector; the other expression vector is an auxiliary expression vector, and the auxiliary expression vector controls a switch for regulating the promoter of the main expression vector; the main expression vector contains an SP6 promoter and a lactose regulating gene; the auxiliary expression vector contains an arab promoter, an araC regulating gene and an SP6RNA polymerase gene; and the target gene expressed by the main expression vector and the auxiliary expression vector are controlled and / or regulated by an inducer.

The invention belongs to the field of virus genetic operation, and in particular to a Newcastle disease virus (NDV) heat resistant live vaccine vector system and application thereof. The Newcastle disease virus (NDV) heat resistant live vaccine vector system comprises a) a transcription plasmid, b) three auxiliary plasmids and c) host cells. The transcription plasmid is obtained by cloning genomic full-length cDNA of a NDV heat resistant vaccine strain to pBR322 vector; and the three auxiliary plasmids are obtained by cloning nucleoprotein, phosphoprotein and large polymerase protein gene of the NDV heat resistant vaccine strain to pcDNA3.1 vector. The artificial recombinant Newcastle disease virus has the characteristic of heat resistance, and the Newcastle disease virus (NDV) heat resistant live vaccine vector system is established for the first time. The artificial recombinant Newcastle disease virus has great application prospect in the aspects of research and development of multiple (multivalent) heat resistant genetic engineering live vaccines of the NDV, avian influenza and other major diseases of poultry, research on virus heat-resistant mechanism, and the like.

The invention provides a preparation method and application of a recombinant antimicrobial peptide (AMP) and belongs to the technical field of biological genetic engineering. The method comprises: amplification of an antibacterial peptide gene, gene cloning, transformation of beer yeast and efficient expression and purification of recombinant protein. The recombinant AMP provided by the invention is AMP-36 of the porcine Cathelicidins family, and the protein of the recombinant AMP contains short peptide shown by the sequence ID No.2. By utilizing the method provided by the invention, the prepared recombinant AMP can be used for producing a feed additive, a veterinary medicine and a preservative and preparing Gram-negative bacteria, Gram-positive bacteria and medicaments for diseases caused by fungi or virus.

The gene clone and expression of Japonese lampreyí»s oral gland model proteins RGD with antineoplastic action are disclosed. Three cDNA sequences with said model proteins RGD and their clones, the protein sequence and its expression in engineered bacteria, and the action of proteins RGD in closing the signal transfer channel to suppress the vascular neogenesis and tumor cell reproduction for antineoplastic purpose are also disclosed.