3963 results about "Techniques of genetic engineering" patented technology

A hyperthermostable protease having the amino acid sequence represented by the SEQ ID NO:1 of the Sequence Listing or a sequence derived therefrom by deletion, substitution, insertion or addition of one to several amino acid residues, a gene encoding the hyperthermostable protease, and a process for preparing the protease, aiming at providing by genetic engineering techniques a hyperthermophile protease which is advantageous for industrial use.

The invention relates to a gene deletion attenuated African swine fever virus as a vaccine and the vaccine, and a construction method thereof. An African swine fever Chinese epidemic strain Pig / CN / HLJ / 2018 is adopted, a virulence gene of the African swine fever virus is deleted by a genetic engineering technology, and the gene deletion virus of MGF360-505R deletion and joint deletion of CD2V and MGF360-505R is obtained. Experiments show that the two virus strains can provide 100% immune protection against the African swine fever Chinese epidemic virulent strains, can be used as vaccines for safe and effective prevention and control of African swine fever in China, and have great social value.

Various human monoclonal antibodies that bind to human TGF-β type II receptor to inhibit the signal transduction of human TGF-β signal into cells are produced by immunizing human antibody-producing transgenic mice generated by genetic engineering techniques with the soluble human TGF-β type II receptor. Further, these human monoclonal antibodies were demonstrated to be effective for preventing and treating various diseases induced by human TGF-β in various organs (for example, tissue fibrosis).

Production of recombinant human serum albumin with rice albuminous cell as biological reactor is carried out by taking rice albuminous cell protein as storage site for recombinant protein, taking rice albuminous specific expression starter and signal peptide, entering inductive recombinant human serum albumin into inner-film system of rice serum albumin cell and storing it in rice albuminous protein. It is cheap, has more yield and higher expression.

The invention discloses a recombinant human collagen and a production method thereof and belongs to the technical field of genetic engineering. The structure of the recombinant human collagen is a single-chain structure; the basic repeating unit is gergapgfrgpagpngipgekgpagergap which is a human collagen III peptide fragment; the terminal sequence is GPPGPCCGGG which is a human collagen II peptide fragment. The production method of the recombinant human collagen comprises the following steps of: constructing escherichia coli genetically engineered bacterium; fermenting and cultivating the escherichia coli genetically engineered bacterium; inducing and expressing the recombinant human collagent; and purifying the recombinant human collagen. According to the recombinant human collagen and the production method thereof disclosed by the invention, the escherichia coli expression system is adopted for being amplified in a large-scale manner, the production cost is low and the yield is high.

The invention relates to the technical field of genetic engineering, in particular to a method for specifically knocking out a human PCSK9 gene by virtue of CRISPR-Cas9 and sgRNA for specifically targeting the PCSK9 gene; through high-throughput cloud computing and designing, a group of sgRNA for specifically knocking out the specifically targeted PCSK9 gene in the human PCSK9 gene by virtue of the CRISPR-Cas9 is synthesized; meanwhile, each sgRNA is linked to a linear pCG-U6-SgRNA plasmid, so that a carrier is obtained, and a pair of forward and reverse sgRNA oligonucleotide carriers, together with the pCG-NLS-FLAG-Cas9 plasmid, are used for successfully transfecting cells, so that the PCSK9 gene is knocked out; the method has the advantage that the large-scale production of the sgRNA can be achieved just by synthesizing a few of polynucleotide segments; and the preparation method is simple and easy to implement, good in sgRNA targeting properties and high in knocking-out efficiency on the CRISPR-Cas9 system.

Immunization of human antibody-producing transgenic mice, which have been created using genetic engineering techniques, with AILIM molecule as an antigen resulted in various human monoclonal antibodies capable of binding to AILIM and capable of controlling a variety of biological reactions (for example, cell proliferation, cytokine production, immune cytolysis, cell death, induction of ADCC, etc.) associated with AILIM-mediated costimulatory signal (secondary signal) transduction. Furthermore, it has been revealed that the human monoclonal antibody is effective to treat and prevent various diseases associated with AILIM-mediated costimulatory signal transduction, being capable of inhibiting the onset and / or advancement of the diseases.

The invention relates to the preparation of a functional area of a sea purse blood vessel growth inhibition factor 1 and the use of the functional area of the sea purse blood vessel growth inhibition factor 1 in medicaments for preventing and curing tumors. In the invention, a gene engineering technique is used to realize the cloning, expression and recombination of the sea purse blood vessel growth inhibition factor 1; the recombinant functional fragment of the sea purse blood vessel growth inhibition factor 1 has the bioactivities for resisting the growth of blood vessels, tumor growth, tumor transplantation, acute toxicity tests and stability tests; and the functional area of the sea purse blood vessel growth inhibition factor 1 can be mixed with or dissolved in pharmaceutically acceptable carriers to prepare the medicaments for curing various tumors. The functional area of the sea purse blood vessel growth inhibition factor 1 has high action specificity, has the characteristics of easy expression, low degradation rate and the like of gene engineering medicaments and has the effects of inhibiting blood vessel growth, tumor growth and tumor transplantation; the provided gene engineering technique can realize the industrial production of the functional area of the sea purse blood vessel growth inhibition factor 1; and the functional area of the sea purse blood vessel growth inhibition factor 1 prepared by the gene engineering technique can be used in the preparation of medicaments for inhibiting blood vessel growth and preventing and curing tumors.

The invention belongs to the technical field of gene engineering, and concretely relates to a CRISPR / Cas9 single transcription unit directionally modified backbone vector and an application thereof. Technical problems to be solved in the invention comprise low species versatility and difficult realization of Cas9 protein expression and gRNA transcription cooperation of present CRISPR / Cas9 genome editing systems. A technical scheme in the invention is characterized in that a CRISPR / Cas9 single transcription unit backbone vector is constructed, and transcription of Cas9 and a guide RNA core unit are regulated by a promoter. The invention also provides a method for constructing a target site specifically modified Cas9-gRNA recombinant vector by adopting the CRISPR / Cas9 single transcription unit backbone vector. The efficient CRISPR / Cas9 single transcription unit backbone vector can effectively realize Pol II promoter driving-based Cas9 and gRNA unit cooperative transcription, and also can realize simple, fast and efficient genome directional heredity modification of various eukaryotes.

The invention discloses a CRISPR Cas9 system for Bacillus subtilis genome edition and an establishment method thereof, belonging to the technical field of gene engineering. A knock-out plasmid PHY300dsrf is established; and the plasmid comprises sgRNA and cas9 genes of the specific targeted target gene, a homologous restoration arm, and replication initial points and tolerance screening markers of Escherichia coli and Bacillus subtilis. The sgRNA of the specific targeted srfA-C gene can guide the cas9 protein to cut double strands at the specific site of the srfA-C gene, and can be used for accurate homologous recombination on the genome under the guide of the homologous restoration arm, thereby introducing the XhoI enzyme digestion site at the rupture. When the tank feed fermentation culture is carried out on the Bacillus subtilis of which the srfA-C gene is successfully knocked out, the foam is obviously reduced, which proves that the CRISPR / Cas9 gene editing system can effectively perform gene edition on the Bacillus subtilis genome.

The invention belongs to the technical field of gene engineering, and discloses sgRNA of targeting IGF-IR gene. The nucleotide sequence of the sgRNA of targeting IGF-IR gene is represented by SEQ ID NO:2. According to applications, a Crispr / cas9-sgRNA lentiviral vector system is adopted, knockout or modification of human hepatoma cell line IGF-IR gene on cellular level is realized successfully, hepatoma carcinoma cell IGF-IR gene transcriptional level is reduced obviously, hepatoma carcinoma cell proliferation, invasion, and migration capacities are reduced, a novel method is provided for treatment of hepatic carcinoma, and clinical application prospect is promising.

Anti-human Mpl antibodies were prepared, and from these three types of antibodies with strong binding activity were selected. An expression system for single-chain antibodies derived from these selected antibodies was constructed using genetic engineering techniques. The anti-human Mpl antibodies and anti-human Mpl single-chain antibodies were assessed for TPO-like agonist activity using BaF3-human Mpl that proliferates TPO-dependently. It was found that while the anti-human Mpl antibodies did not exhibit agonistic activity, the anti-human Mpl single-chain antibodies showed agonistic activity. This shows that when screening for modified antibodies with agonistic activity, it is beneficial to determine agonistic activity after modifying antibodies with antigen-binding activity.

The invention relates to the technical field of genetic engineering, and particularly relates to a guide RNA (gRNA) sequence based on a CRISPR / Cas9 system and combinations thereof, as well as gRNA for a specific targeted knockout hepatitis B virus cccDNA P gene. In the invention, 30 gRNAs are designed according to design principles of CRISPR / Cas9, have a sequence table as shown in SEQ ID NO.1-30, and are constructed on a PX458 vector, to screen out four more efficient gRNAs. The CRISPR / Cas9 system guided by the four gRNAs and combinations thereof is utilized in a human hepatocellular carcinoma cell line (HepG2.2.15) to effectively knock out the human hepatitis B virus cccDNA P gene. The gRNA of the specific targeted hepatitis B virus cccDNA prepared in the invention can accurately target the hepatitis B virus cccDNA and realize gene knockout. The preparation method is simple in operation; the gRNA has a good targeting property; the CRISPR / Cas9 system is high in knockout efficiency.

The invention relates to a DNA transmethylase defective CHO (Chinese hamster ovary) cell line and a preparation method and application thereof and belongs to the technical field of gene engineering. DNA Transmethylase Dnmt3a gene of CHO cells is knocked off by means of CRISPR / Cas9 gene editing technique, and screening and identifying are performed to obtain DNA transmethylase Dnmt3a defective CHOcells; the cells are transfected with a eukaryotic expression vector, stably expressed recombinant CHO cell strains are screened, and accordingly a novel CHO cell expression system based on DNA transmethylase deficiency is established. The recombinant gene CHO cell expression system is established via host CHO cell genetic modifications, expression level of recombinant proteins can be significantly increased, the problem of recombinant protein expression instability is solved, and recombinant protein expression stability is improved.

The invention relates to the technical field of genetic engineering and particularly relates to a method for constructing a human neuroblastoma cell line of which a CAPNS1 gene is knocked out based ona CRISPR / Cas technology. The invention provides the method for knocking out the CAPNS1 gene of human neuroblastoma cell. After plasmid transfection into SK-N-SH cells, a monoclonal antibody is prepared. After Cruiser<Tm> Enzyme digestion, the clone is positive clone. The monoclonal sequencing result shows that the CAPNS1 gene-free SK-N-SH cell line is successfully constructed. In the CAPNS1<- / ->group, the CAPNS1 protein and Calpain1 and Calpain2 protein levels are significantly reduced. The result shows that the CAPNS1 gene-free SK-N-SH cell line is successfully constructed.

The invention belongs to the field of genetic engineering technology, and specifically discloses a Cas9-mediated tomato gene editing vector and application thereof. A Cas9 gene is introduced into a tomato M82 by constructing a CRISPR / Cas9 tomato plant expression vector, and gene editing is carried out on the DFD gene of the tomato. The invention provides a method for constructing a storage-endurance transgenic tomato, the new storage-endurance tomato product is obtained, so that the storage endurance of the tomato fruit is improved, and besides, the growth and other qualities of plant are not influenced, and the method can compensate for shortcomings of former research programs.

The invention belongs to the technical field of gene engineering, particularly relates to a test tube cleaning device for gene engineering, and aims to provide a test tube cleaning device which is capable of more thoroughly cleaning a test tube, avoids residues in the test tube after cleaning and is fast in cleaning and high in working efficiency. The test tube cleaning device comprises a box, first sliding rails, first sliders, a first motor, a rotating shaft, a placing plate, a clamping device, a handle, second sliding rails, second sliders, a horizontal plate, hairbrushes and the like, the first sliding rails are symmetrically arranged on the left side and the right side of the inner middle of the box and connected with the left side and the right side of the inner middle of the box through bolts, and the first sliders are slidably arranged on the first sliding rails and slidably matched with the first sliding rails. According to the test tube cleaning device for the gene engineering, the hairbrushes vertically move to clean test tubes.

The present invention relates to the cloning and high level expression of novel cellulase proteins or derivatives thereof in the in a host cell. Further aspects of the present invention relate to transformants that express the novel cellulases, and expression vectors comprising the DNA gene fragments or variants thereof that code for the novel cellulases derived from Actinomycete using genetic engineering techniques. The present invention is also directed to novel cellulase compositions and methods of use therefore in industrial processes. In particular, the present invention is related to treating textiles with a novel cellulase derived from Actinomycete spp. The present invention also relates to the use of cellulase derived from Actinomycete spp. to enhance the digestibility of animal feed, in detergents, in the treatment of pulp and paper and in the production of starch and treatment of by-products thereof.

The invention relates to an application of CRISPR / Cas 9 technology in obtaining of bombyx zinc finger protein gene mutation, and belongs to the technical field of bombyx breeding and gene engineering. In the invention, the CRISPR / Cas 9 gene editing technology is applied to obtain bombyx zinc finger protein gene mutation, a set of method for performing gene edition on zinc finger protein and exploring the gene function is realized, and thus a set of effective method for the batch research and transcription factors in future is provided.

The present invention provides a recombinant fusion protein which stimulates the rejuvenation and reactivation of skin and epidermal cells for improving skin appearance, smoothing wrinkles and freckles, and whitening skin. Particularly, the present invention provides various types of products for improving skin, which contain recombinant fusion protein of human serum albumin (HSA) with cytokine peptides (EGF, FGF, KGF, HGH, HGF, PDGF, GCSF, interferon, IL-11 or IGF) by genetic engineering technology. The fusion protein can be used independently or in a combination or combination with yeast fermentation products, or with varied emulsifiers, thickeners, moisturizer, preservatives, yeasts and ferments.

The invention relates to a construction method of a homologous repair vector based on a CRISPR / Cas9 system, and belongs to the technical field of genetic engineering. The construction method comprisesthe following steps: with a plasmid PCBC and wild-type arabidopsis genome as templates, amplifying a target band AS-gRNA containing a target sequence and an AS homologous repair template segment by virtue of PCR, implementing electrophoresis and gel cutting, and recovering the target band; implementing enzyme digestion on a plasmid PHDE-mCH by virtue of Bsa1; assembling and linking a PHDE-mCh vector, which undergoes complete enzyme digestion, with the ASgRNA by virtue of homologous recombinase, so that a recombinant plasmid PHDE-ASgRNA is formed, implementing transformation and sequencing identification, then linking the PHDE-ASgRNA plasmid, which is correct in sequencing and is subjected to enzyme digestion by virtue of EcoR1, with AS homologous repair template segment homologous recombinase, and implementing transformation and sequencing identification, so that the PHDE-ASgRNA-AS homologous repair vector is constructed. According to the technique provided by the invention, the method for constructing the CRISPR / Cas9 system which has a site-specific editing function on target biology genome is actually implemented; the vector is constructed just by conducting PCR by one step, sothat the method is simple and easy to implement; and the method, which is unnecessary to purify or recover a digestion vector and a PCR product, is high in assembly efficiency.

The invention relates to a method for biosynthesis preparation of human glucagon-like peptide-1 (GLP-1) and an analogue thereof. With adopting of a gene engineering technology, a recombinant escherichia coli expressed GLP-1 fusion protein is constructed, and a protein enzyme digestion site is designed in the fusion protein; a fusion gene has a gene sequence with a form of A-B-C structure, wherein A is a chaperonin gene, B is a nucleotide sequence encoding a connection peptide containing the enzyme digestion site, and C is a gene encoding the GLP-1 or the analogue thereof. After recombinant engineering bacteria is subjected to induced expression, the fusion protein is purified and subjected to enzyme digestion, and then the GLP-1 and the analogue thereof are obtained and are detected to have biological activity. The preparation method of the GLP-1 and the analogue thereof provided by the invention is simple and quick, the production conditions are mild, the product is convenient to separate and extract, the process is simple, and the industrialization prospect is good.

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 relates to an ELISpot tuberculosis infection diagnostic kit and the application, which adopts the genetic engineering technology to obtain a CFP10-ESAT6 fusion protein antigen from a mycobacterium tuberculosis by cloning, expression and purification, a tuberculosis specific cell stimulator is used for stimulating the peripheral blood T lymphocyte cell of the detected person to secrete a specific IFN-Gamma, then the invention is detected by ELISpot, and the whole process needs to take two days. The usage of the invention can be used for detecting whether the detected person is infected by mycobacterium tuberculosis by using the naked eye or instrument to determine the result according to the number of the generated purple blue spots, so as to assist the diagnosis of tuberculosis and differential diagnosis.

The invention is confluens protein of SARS virus S protein and N protein, and the manufacturing, application and the gene project technology, vaccine and diagnosing reductant. Through analyzing the cistine series of SARS s protein and N protein, sifts out SARS virus S protein segment with strong antigen epitope, namely the 162nd cistine to 460nd cistine, and the N protein segment of SARS virus with strong antigen epitope, anmely the first cistine to 258nd cistine. It synthesizes the new SARS virus S protein segment and N protein segment gene series, connects the two gene segments in series. Expresses the confluens protein of the two protein segments, the two are connected by glycine and serine, inserts a glycine adjoining to the frist cistine methionine of N protein segment, the confluens proteiní» length is 560 cistines.

The invention is applicable to the technical field of gene engineering, and provides a method for constructing a graph in a short sequence assembly and a system thereof. The method comprises the following steps: receiving an order-checking sequence; carrying out sliding cutting on each base of the received order-checking sequence to obtain a short string with a fixed base length and a left and right connecting relation of the short string; storing a sequence value of the obtained short string, the left and right connecting relation and a connection number as a node of a de Bruijn graph. In the invention, the method for constructing the graph in the short sequence assembly can be realized by slidingly cutting the base of the received order-checking sequence one by one to obtain the short string with the fixed base length and the left and right connecting relation of the short string, and storing the sequence value of the obtained short string, the left and right connecting relation and the connection number as the node of the de Bruijn graph. The method can assemble a large genome with small occupied memory and fast speed.

The invention belongs to the technical field of genetic engineering, and particularly relates to an experimental pesticide spraying device for transgenic plants. The invention aims to solve the technical problem of providing an experimental pesticide spraying device for the transgenic plants, which can save labor, is high in working efficiency and can reduce labor strength. In order to solve the technical problem, the invention provides the experimental pesticide spraying device for the transgenic plants, which comprises a cart, a left side plate, a compression cylinder, a right side plate, a piston, a stirring tank, a bearing seat, a rotating rod, stirring blades, a first gear, a rack, a push rod, a top plate and the like. The left side plate, the compression cylinder and the right side plate are sequentially arranged at the top of the cart from left to right; and the left side plate and the right side plate are connected with the top of the cart in a welding mode. The experimental pesticide spraying device for the transgenic plants, which is provided by the invention, is manually pushed to spray a pesticide, so that labor can be saved and labor intensity can be reduced.

Disclosed are novel proteins, referred to as tumor necrosis factor binding proteins, that modulate the activity of tumor necrosis factor. Also disclosed are processes for obtaining the tumor necrosis binding proteins by recombinant genetic engineering techniques.

Immunization of human antibody-producing transgenic mice, which have been created using genetic engineering techniques, with AILIM molecule as an antigen resulted in various human monoclonal antibodies capable of binding to AILIM and capable of controlling a variety of biological reactions (for example, cell proliferation, cytokine production, immune cytolysis, cell death, induction of ADCC, etc.) associated with AILIM-mediated costimulatory signal (secondary signal) transduction. Furthermore, it has been revealed that the human monoclonal antibody is effective to treat and prevent various diseases associated with AILIM-mediated costimulatory signal transduction, being capable of inhibiting the onset and / or advancement of the diseases.

The invention relates to the technical field of plant gene engineering, in particular to design, verification and application of a rice disease resistance related DNA molecule of 21nt. An artificial microRNA sequence of the 21nt is artificially designed, and an artificial microRNA precursor is constructed by using a natural microRNAosa-mi528 precursor as a skeleton. In transgenic rice, the artificial microRNA precursor is specifically expressed by using a leaf specific promoter of a rice or arabidopsis thaliana source so that the resistance of the transgenic rice to bacterial leaf blight can be enhanced and important agronomic traits of the transgenic rice such as fertility and the like is not affected at the same time.