1014 results about "Double chain" patented technology

The invention discloses a specie limitation-free eucaryote gene targeting method having no bio-safety influence and a helical-structure DNA sequence, and belongs to the field of gene engineering. The specie limitation-free eucaryote gene targeting method comprises the following steps of 1, designing and constructing CRISPR/Cas9 and chimeric RNA, and 2, carrying out Cas9mRNA internal translation so that Cas9 nuclease and the chimeric RNA are bonded, carrying out fixed point clipping so that DNA double-chain cleavage is realized after the clipping, and introducing an exogenous DNA by induction of a natural DNA restoration process which is a non-homologous end bonding process of cells so that cell endogenous gene modification is realized. The specie limitation-free eucaryote gene targeting method has simple processes, realizes flexible site recognition and has low energy consumption.

Compositions, methods, and kits are provided for efficiently generating and screening a library of highly diverse protein complexes for their ability to bind to other proteins or oligonucleotide sequences. In one aspect of the invention, a library of expression vectors is provided for expressing the library of protein complexes. The library comprises a first nucleotide sequence encoding a first polypeptide subunit; and a second nucleotide sequence encoding a second polypeptide subunit. The first and second nucleotide sequences each independently varies within the library of expression vectors. In addition, the first and second polypeptide subunit are expressed as separate proteins which self-assemble to form a protein complex, such as a double-chain antibody fragment (dcFv or Fab) and a fully assembled antibody, in cells into which the library of expression vectors are introduced. The library of expression vectors can be efficiently generated in yeast cells through homologous recombination; and the encoded proteins complexes with high binding affinity to their target molecule can be selected by high throughput screening in vivo or in vitro.

The invention relates to the field of gene engineering, and provides a system and a method for constructing a sequencing library. The system and the method comprise a joint element and a method for applying the joint element to the construction of a sequencing library. The joint element is a bifurcate double-chain nucleic acid joint with a bifurcate region and a paired region orderly included at the 5' and the 3' ends, wherein two single chains of the bifurcate region respectively comprise at least one amplification primer binding site, and the paired region comprises at least one restrictionenzyme site. The joint element of the invention can prevent the self-connection phenomenon of the joint element and the occurrence of a non-object joint element-DNA fragment compound during the library construction.

The invention discloses a double chain-type cross chain trading Internet of blockchains model (golden monkey model) core algorithm. Different from the traditional centralized architecture, the model has a fully-distributed and multi-chain network architecture. Due to the fully-distributed architecture, easier expansion, easier extension and easier fault tolerance are realized than the existing architecture. Each chain maintains the self consistency, the consistency between chains does not need to be managed by a central organization but is maintained by a fully-distributed mechanism. Limitations from the previous centralized Internet of blockchains (chain network, in short) are broken, all chains can operate in parallel on the model, and the trading efficiency and the network operation speed are improved. The model has a new financial market architecture, extensibility is realized, a financial unit can randomly and easily enter or leave the network, and the large-scale network and a high transaction volume are supported. The model comprises participant blockchains and inter-chain blockchains, the participant blockchains or the inter-chain blockchains can be one or one group of financial institutions, the inter-chain blockchain has a double-chain structure, a trading record and a balance account are separated, and cross chain trading is realized. A core algorithm for trading between chains, particularly, a protocol algorithm of enabling the inter-chain blockchains to assist two or more than two participant blockchains for mutual trading is provided, no centralized mechanism is needed, and the consistency of the whole network is maintained.

The invention provides a government affair data storage and query method and system based on a block chain double-chain structure. The government affair data storage method based on the block chain double-chain structure comprises the steps that a client uploads the data to a proxy server; the proxy server sends a data storage request to the identity chain and verifies the user identity and the data storage request; the identity chain verifies the identity of the user who makes the request and the storage operation authority, and returns a verification result to the proxy server; if the verification does not pass, the proxy server sends a data storage rejection request to the client; if the verification passes, the proxy server submits a data storage request to a service data chain, and the service data chain completes a consensus process according to the data storage request of the client and stores the data related information in a block chain system state database; and the service data chain feeds back a storage result to the client through the proxy server.

The invention discloses an sgRNA directing sequence for specific targeting of a human ABCG2 gene and an application thereof, and belongs to the field of gene engineering application. The sgRNA directing sequence has a nucleotide sequence of GCTGCAAGGAAAGATCCAAG. According to the sgRNA directing sequence, two single-stranded oligo sequences are designed and synthesized and then are annealed to form a double chain, and then the double chain is connected with a Cas9 vector; with use of the Cas9 vector, sgRNA and a CRISPR system are introduced into target cells, a Cas9 protein can find out a DNA sequence matched with the Cas9 protein under guidance of the sgRNA, shearing is performed, and the ABCG2 gene is knocked out. According to the sgRNA directing sequence provided by the invention, the ABCG2 gene is knocked out or edited by the CRISPR-Cas9 system, then the expression of the ABCG2 is inhibited or eliminated, and the problem of multi-drug resistance generated in treatment of tumor can be effectively solved.

The invention provides an automatic plug tray recovering and stacking device and a method thereof and belongs to the field of agricultural machinery. The device comprises a machine frame, a conveying device, a lifting mechanism, a raising mechanism, a locking mechanism and a control system, wherein the control system is electrically connected with the conveying device, the lifting mechanism and the raising mechanism respectively. According to the device, the control system controls a double-chain transmission mechanism of the conveying device to convey a used empty plug tray, stacked empty trays are lifted by the lifting mechanism, the empty tray is raised by the raising mechanism, and the raised empty tray is locked by the locking mechanism; the device can automatically convey, recover and stack used plastic or foam plug trays in sequence so that the plug trays are reused later, can greatly reduce workers' labor intensity in plug tray recovering and stacking, and increases the working efficiency; and the device is simple in structure and reliable in service performance and can be applied to plug tray seedling transplanting machinery.

The invention discloses an automatic disassemble device of a circuit board components. A melting solder furnace, a steel wire rolling brush, a turnover transfer mechanism, a vibratory shock device and a device used for leading the welding surface of a circuit board to contact with the melting solder in the melting solder furnace and the like are arranged in an incubator. Two double-chain conveyors which are respectively driven by different motors are respectively positioned at the front and the rear of the turnover transfer mechanism; and the rear ends of the corresponding melting solder furnace and the vibratory shock device are respectively provided with a position-triggering sensor. The automatic disassemble device also includes an device which is independent, has displace elasticity and is used for clamping the fixed circuit board; the device used for clamping the fixed circuit board is provided with a slot and is positioned on the chain of the double-chain conveyor. The device can not only remain the completeness of usable components in the disassembling process, but also can guarantee that the circuit board has better disassembling efficiency, and also can effectively prevent the disassembling process of the circuit from causing pollution to human bodies and environment.

The invention provides a supply chain finance realization method and a control system thereof based on a block chain double-chain structure, which adopts the block chain double-chain structure, namely, the generation process information of financing assets is recorded through an asset chain, including key state information such as order generation, warehousing change, logistics transportation, order signing-in and the like; the entire financing process information of the financing assets is recorded through the capital chain, including notes, asset ownership circulation, factoring services andother information, so that participants can trace the original information generated by the assets, reduce investment and financing risks, reduce human and material costs. In addition, the cross-validation of asset generation information and asset flow information, combined with the identity information and transaction content of participants, together are used as the basis for risk control and credit granting of financial institutions to promote the rapid operation of funds and business development. At the same time, the double-chain structure of asset chain and fund chain is adopted, whichis convenient for the administrator to set the authority and improves the security and confidentiality of the data.

The invention discloses a high strength and high toughness self-repairing thermoplastic polyurethane urea elastomer and a preparation method thereof. The self-repairing thermoplastic polyurethane ureaelastomer is synthesized by double chain extension of prepolymers generated from diisocyanate and polyether diols separately in the presence of 2,6-diaminopyridine and cysteamine. A pyridine ligand unit is introduced onto a main chain, and coordination bonds are formed between molecular chains through the coordination effect of metal ions, thereby forming interchain reversible molecular healing;at the same time, the coordination bonds act as dynamic crosslinking points and achieve the effects of limiting molecular chain slip and improving mechanical properties; a disulfide bond is introducedinto a macromolecular main chain through double chain extension, and the self-healing ability is improved in an association manner through a reversible exchange reaction of the main chain. The elastomer has the self-repairing efficiency being more than 85%, the tensile strength being not less than 8 MPa and the toughness being not less than 40 MJ/m<3>. The elastomer is capable of both improving mechanical properties and self-repairing performance, can maintain the integrity and functionality of materials against complex deformation, and has extremely high application value in the field of flexible electronic equipment.

The invention discloses a disinfectant for animals and a preparation method thereof, belonging to the field of medicines for veterinary use. The disinfectant for animals takes glutaric dialdehyde, single-chain quaternary ammonium salt and double-chain quaternary ammonium salt as active ingredients. The preparation method comprises the following steps: 1) adding glutaric dialdehyde, alcohol and surfactant to a container to stir uniformly and place statically; 2) adding single-chain quaternary ammonium salt and double-chain quaternary ammonium salt; 3) using a part phosphoric acid buffer solution to dissolve the chelating agent and oxidant completely and mixing with the above ingredient; and 4) continuously adding phosphoric acid buffer solution to the formulation amount to obtain a disinfectant stock solution with pH value of 2.5-4.0. The invention has low toxicity, low irritation, small smell, small corrosiveness, high efficiency of disinfection and side disinfecting spectrum. The liquid disinfectant with wide bactericidal spectrum and some washing efficiency is specifically suitable for daily disinfection of colony house, and is suitable for largely popularizing and using.

The invention discloses a novel reaction system for rapidly constructing a plant gene site-directed knockout carrier. The system comprises 1) based on a CRISPR-Cas system carrier, the carrier comprises the DNA molecules having the following characteristics: an agrobacterium infection method is used for carrier conversion to the plant, a carrier capable of simultaneously realizing driving of SgRNA by an OsU3 promoter for transcription and driving of Cas9 protein by an Ubiquitin promoter for expression; 2) restriction enzyme AarI; 3) buffer and Oligo required by a reaction of the restriction enzyme AarI; 4) target gene site specific DNA double chain; and 5) T4DNA ligase and ATP required by an enzyme reaction. The system only requires 5-10 reaction cycles for rapidly and efficiently completing a construction process of plasmid, greatly simplify the operation flow, and satisfies the requirement of rapid construction with high flux for plant gene site-directed knockout plasmid.

The invention discloses a sgRNA sequence for specifically targeting an arabidopsis ILK2 gene and application of the sgRNA sequence. The nucleotide sequence of the sgRNA sequence is represented by SEQ ID NO.1. Two single-chain oligo DNA sequences are designed and synthesized according to a sgRNA guide sequence, a double chain is formed through annealing and is linked with a Cas9 carrier, a sgRNA coding sequence and a CRISPR system are introduced into Arabidopsis by using an agrobacterium-mediated genetic transformation technique, a target sequence is shorn by a Cas9 protein under the guidance of sgRNA, and therefore, the knockout of an ILK2 gene is realized. According to the sgRNA sequence provided by the invention, the ILK2 gene can be knocked out or edited by virtue of a CRISPR-Cas9 system, so as to analyze the functions of the arabidopsis ILK2 gene.

The invention provides a real-time fluorescence quantitative PCR (Polymerase Chain Reaction) kit for detecting the expression level of HER2 genes and relates to a PCR kit. The real-time fluorescence quantitative PCR kit comprises PCR reaction liquid of the HER2 genes, PCR reaction liquid of ACTB genes, a Taq enzyme system, a control product and a packaging object; and the adopted primer is an annular primer, 3-8 self-designed basic groups are at the 5' end, and can be combined with the sequence at the 3' end under proper conditions so as to form double chains. The real-time fluorescence quantitative PCR kit has the advantages that the non-specific amplification products especially primer dimer generated by nonspecific amplification products especially the primer can be effectively prevented from being formed, the specificity is increased; and a relative-quantitative RT-PCR method is utilized for detecting the expression level of the HER2 genes of a patient with the breast cancer, adopts a 2-delta deltaCt value method for quantifying the detected result, and can be used for diagnosis of early stage and transferring of the breast cancer and assisting multiple fields such as clinicalmedicine selection and prognosis.

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 and application thereof. A lentivirus of the CRISPR/Cas9 recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 gene Delta 32 region is constructed that the lentivirus can introduce cells into a CRISPR/Cas9 system specific to CCR5, double-chain breakage occurs to a specific site of CCR5 gene, a random mutation is introduced to a breakage site after repairing by means of nonhomogeneous recombinant terminal binding, and the mutation rate reaches 90% and above. As gRNA is a nonhomogeneous region of CCR5 and CCR2, detection shows that the missing efficiency of the two gRNAs is lower than 0.2%. Cells modified via the recombinant lentivirus have significantly decreased efficiency of HIV (human immunodeficiency virus) infection. The system is quick to construct, simple and low in price, and is applicable to gene therapy of acquired immune deficiency syndrome.

The invention discloses a method of packaging a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-associated 9) system by using a temperate phage vector. The method comprises the steps of: (1) constructing suicide genes, target spot sequences bound with specific gRNA (guide ribonucleic acid), and downstream PAM (protospacer adjacent motif) sequences into pSTK (protein serine threonine kinase) plasmids, (2) transforming the pSTK plasmids into escherichia coli host bacteria, (3) transforming CRISPR-Cas9 sequence recombination template double-chain DNA (deoxyribonucleic acid) linear fragments carrying phage sequence homologous arms at the two ends into the host bacteria, (4) inducing expression of homologous recombination related enzymes and the suicide genes SacB, (5) screening the host bacteria subjected to homologous recombination, and (6) inducing temperate phages to crack the host bacteria, and harvesting the recombined temperate phages packaging the CRISPR-Cas9 system, wherein chromosomes of the escherichia coli host bacteria are integrated with the temperate phages; and plasmids capable of expressing the homologous recombination related enzymes are transformed into the escherichia coli host bacteria. According to the packaging method, a secondary recombination step of deleting a resistance marker is removed, and the technical support is provided for the phage vector presenting CRISPR-Cas9 system to resist drug-resistance bacteria efficiently and quickly.

The invention relates to a method for achieving HMGCR gene knockout based on the CRISPR/Cas9 technology. The method is characterized in that two CRISPR/Cas9 target sequence aiming at the HMGCR gene isdesigned, a gRNA single chain is synthesized in vitro, annealing is performed to obtain two gRNA double-chain DNA target insertion fragments, the insertion fragments are inserted into PX459 (pSpCas9(BB)-2A-Puro)V2.0 vectors to obtain the two different-locus plasmids of the target HMGCR gene; the two plasmids are transfected into PK15 cells, puromycin is used to process the cells, the processed cell genome DNA is extracted to perform PCR amplification, the PCR product is denatured, annealing is performed, and then T7E1 is used to perform HMGCR gene knockout identification. The method has the advantages that method can be used for analyzing the expression conditions of sequence and mRNA after the HMGCR gene knockout, whether an off-target phenomenon exists or not can be verified by using amethod combining PCR and T7E1 enzyme treatment, and accordingly the specificity based on target sequence HMGCR-gRNA can be determined; the method is applicable to cell and animal models to achieve fixed-point HMGCR gene knockout, has a reference value to the knockout of other genes, and is good in effect, simple, economical, short in time and the like.

The invention belongs to the technical field of biology, and specifically relates to Cas9 nuclease and an application. The Cas9 nuclease (Cas9-R919P) has the activity as Cas9 nuclease, fits to a CRISPR/Cas9 system, and is made by mutating the 919 location arginine of wild Cas9 nuclease to proline. A protruding fracture terminal is generated by cutting a DNA double chain with the Cas9 nuclease (Cas9-R919P), and a basic group complementary to the protruding fracture terminal is added in a filling-in manner, so that accurate addition at a specific position of the genome DNA segment can be carried out.

The invention provides a gene, recombinant plasmid and polypeptide for an anti-tumor binary polypeptide. The gene of the recombinant anti-tumor binary polypeptide is obtained by connecting in an operable way the gene of a coding antibody simulator with a recombinant bacillus anthraci protein antigen gene. The recombinant plasmid of the invention is formed by inserting the gene of the coding antibody simulator by double-chain oligomeric nucleotide directed mutagenesis method into the recombinant bacillus anthraci protein antigen gene. The obtained recombinant plasmid is infected into engineering bacillus coli BL-21 to get engineering bacillus coli cell of anti-tumor binary polypeptide; the anti-tumor binary polypeptide can be obtained by expanding the bacillus coli, settling in centrifugal way the bacillus coli body, crushing in altrasonic way, settling and crushing bacillus coli body by hi-speed centrifuging and treating the upper clean solution. The anti-tumor binary polypeptide is of special targeting characteristic, higher efficiency in killing special physical tumor than prior anti-tumor medicine, and will not attack normal cells, and has much lower toxicity and poor-reaction than prior anti-tumor medicine.

An automatic seedling plate van based on double-chain transmission comprises a machine frame, a fetching device, a lifting device, a placement mechanism, a traveling mechanism and a control system. The fetching device comprises grabbing fingers, a transmission chain mechanism, an unpowered roller mechanism and the like and is used for achieving fetching of seedling plates. The lifting device comprises a guide mechanism, a chain transmission mechanism, a tensioning mechanism and the like and is used for achieving vertical movement of the fetching device and three-dimensional placement of the seedling plates. The placement mechanism comprises a support and supporting boards, and the paired supporting boards are arranged at equal intervals. The traveling mechanism is of a symmetrically distributed four-wheel system structure, and two driving wheels are controlled by a direct current geared motor in a directional differential mode to travel and turn. The control system comprises a loading system and a traveling system and is composed of a single-chip microcomputer, various sensors and the like. Automatic loading and tracking moving of the van are achieved by detecting signals of the control system. Integration of loading, unloading and transportation of the seedling plates in industrialized seedling production is achieved, and automatic carrying of the seedling plates can be completed.

The invention discloses a double chain adaptor, application thereof and a method for constructing end-pairing DNA library. The double chain adaptor comprises an adaptor A and an adaptor B, and the adaptor A and the adaptor B respectively comprise a LoxP sequence and an auxiliary sequence for linking the LoxP sequence to the two ends of a to-be-cyclized DNA fragment in a same direaction. By applying the technical scheme provided by the invention, based on the characteristics of the Cre recombinase/LoxP action system (when two LoxP sequences are disposed at one DNA chain and has the same direction, Cre recombinase is capable of effectively excising the sequence between the two LoxP sites, and the excised sequence automatically becomes annularity, so that the cyclization of a DNA fragment is realized), the double chain adaptor is designed and is efficiently linked to the two ends of a DNA fragment for constructing the end-pairing DNA library in a T-A clone manner, and the Cre recombinase is employed for efficiently enabling the DNA fragment for constructing the end-pairing DNA library to be cyclized.

The invention discloses a double chain nucleic acid fragment joint adding method, a library constructing method and a kit. According to the double chain nucleic acid fragment joint adding method, a 3'terminal lateral joint is connected to the 3' terminal of a double chain target nucleic acid fragment; the double chain target nucleic acid fragment comprises a connection site, the connection site comprises the 3' terminal, which contains 3'-hydroxyl, the connection site is an incision, notch, or a 5' terminal embossment; the 3' terminal lateral joint comprises the 5' flat terminal and non-connection 3' terminal of 5'-phosphoric acid; and the 3' terminal lateral joint connection method comprises a step of adopting ligase to connect a double chain target nucleic acid fragment and the 3' terminal lateral joint. According to provided method, the 3' terminal lateral joint is connected to the 3' terminal of a double chain target nucleic acid fragment; based on the method, a library is established, the library is applied to cPAL and synthesis sequencing, and is suitable for genomic sequence or whole exon sequencing; the primary amount of nucleic acid for library construction is reduced, the process for library construction is simplified, the sequencing covering rate of a GC enriched area is improved, and the sequencing performance is enhanced.

The invention discloses an intelligent garbage collection path planning method based on a quantum cuckoo search algorithm. The method comprises the following steps: S1, the state of a garbage can is detected, whether the garbage can is full is determined, and the full garbage can data are obtained; S2, the transmitted data of each garbage can are subjected to path planning through the quantum cuckoo search algorithm; and S3, according to path planning, a garbage collection navigation map is drawn. The quantum cuckoo search algorithm is designed for planning the collection path, a quantum double chain encoding mode is adopted to improve the cuckoo search algorithm to plan the best collection path, and the garbage collection navigation map is finally made according to the collection path. The method has the advantages of good intelligence and high efficiency, the collection path accuracy is improved, the cost manpower and mateiral resurces for garbage collection management in a certain range can be effectively saved, and the operation cost is greatly reduced.

The invention discloses a long-distance line type wireless sensor network cross-layer communication method. The long-distance line type wireless sensor network cross-layer communication method includes the following steps that: at first, a wireless sensor network adopts a double-chain topologic structure, and each aggregation node only communicates with two sensor nodes; and then, the aggregation nodes perform whole-network periodic time synchronization; and finally, the sensor nodes synchronously perform periodic monitoring and sleep, wherein the periodic monitoring and sleep includes a monitoring phase and a sleep stage, in the monitoring phase, the sensor nodes compete time slot deployment of the whole-network sensor nodes through transmitting time slot deployment requests, and in the sleep stage, the sensor nodes perform data transmission according to the time slot deployment of the sensor nodes their own. According to the long-distance line type wireless sensor network cross-layer communication method of the invention, the characteristics of the long-distance line type wireless sensor network are considered; a cross-layer design method is adopted, and communication decision threshold and time slot deployment modes are adopted in combination, and therefore, the network can be more suitable for cross-layer protocols; and the problem of long time delay caused by periodical monitoring and sleep can be solved.

The invention discloses a preparation method of an electrochemical sensor capable of simultaneously detecting two acute leukemia markers. The method comprises the following steps: respectively standing a lysozyme report probe and a gamma-interferon report probe in a liquid containing trichloroethyl phosphate (TCEP), so as to open a disulfide bond and respectively form a double-chain structure together with a lysozyme aptamer and a gamma-interferon aptamer; preprocessing a gold electrode, and immersing the processed gold electrode into the pre-processed mixed liquid of lysozyme report probe-aptamer double chains and gamma-interferon report probe-aptamer double chains; standing at room temperature over the night, and then cleaning by using secondary distilled water and a cleanout fluid; immersing the electrode into the liquid containing MCH to seal the electrode, and then cleaning the secondary distilled water and the cleanout fluid; and taking the electrode processed by the above steps as a work electrode to be connected on a chemical work station together with a reference electrode and a counter electrode, so as to obtain the electrochemical sensor. The electrochemical sensor can be applied to simultaneous detection of two acute leukemia markers, namely lysozyme and gamma-interferon.