20 results about "Cloning vector" patented technology

The present invention provides gene therapies for the treatment of Friedreich's ataxia. Specifically, the present invention provides a nucleic acid, cloning vector and transfer vector for the production of an adeno-associated virus (AAV) vector. The nucleic acid comprises (i) a nucleic acid sequence encoding frataxin, (ii) a phospho-glycerate-kinase (PGK) promoter, and (iii) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The present invention also provides a pharmaceutical composition which comprises the AAV vector or nucleic acid. Also, the AAV vector, nucleic acid or pharmaceutical composition can be used as a medicament, specifically as a medicament for the treatment of Friedreich's ataxia.

The invention discloses an mRNA (messenger Ribonucleic Acid) quality control product of human papilloma virus as well as a preparation method and application thereof, and belongs to the technical field of in-vitro diagnosis quality control. The method comprises the following steps: chemically synthesizing an HPV E6 / E7 gene DNA sequence, respectively cloning the HPV E6 / E7 gene DNA sequence into a cloning vector to construct a synthetic plasmid template, further preparing a nucleic acid fragment containing E6 / E7 mRNA through in-vitro transcription, carrying out absolute quantification through a digital PCR technology, mixing the nucleic acid fragment with human cervical cancer cell line RNA without any HPV sequence, and screening to obtain the HPV E6 / E7 gene DNA sequence. And the mRNA quality control product with stable structure and accurate value is prepared through a vacuum freeze drying technology. The quality control product can simulate HPV E6 / E7 mRNA in a clinical sample, has comprehensive types, is suitable for accurate evaluation of sensitivity, specificity, precision and other analysis performances of an HPV E6 / E7 mRNA detection kit, provides a key quality control tool for accurate risk stratification of cervical cancer, and is suitable for various detection platforms.

The application provides a standard plasmid for detecting the copy number of exogenous genes of a recombinant CVA10 vaccine, and a preparation method and application thereof. The standard plasmid of the application comprises an endogenous gene MOX fragment SEQ ID No. 1, an exogenous gene P1 fragment SEQ ID No. 2 and an exogenous gene 3CD fragment SEQ ID No. 3 which are connected in series in a cloning vector. The application constructs the standard plasmid, adopts Taqman probe-fluorescence quantitative PCR method, and simply, quickly, absolutely quantitatively and accurately analyzes the copy number of inserted exogenous genes, so that the detection time of the determination of the copy number of exogenous genes of the recombinant CVA10 vaccine is effectively shortened, repeated operation is facilitated, and the determination of the copy number of exogenous genes of the recombinant CVA10 vaccine based on the Hansenula platform and the verification of the genetic stability of the exogenous genes of the recombinant CVA10 vaccine have a wide application prospect.

The application of Trifolium repens TrWRKY7c1 gene in improving plant cold resistance belongs to the technical field of plant genetic engineering and crop genetic breeding. In order to solve the technical blank of the existing technology about the research on low-temperature stress related genes of Trifolium repens, the application of Trifolium repens TrWRKY7c1 gene in improving plant cold resistance is provided, the nucleotide sequence of the gene is shown as SEO ID NO. 1, and the amino acid sequence encoded by the gene is shown as SEO ID NO. 2; a recombinant cloning vector, a plant expression vector or a recombinant glycerol preservation strain containing the gene are also provided, and a transgenic plant is obtained through an agrobacterium-mediated genetic transformation method. The TrWRKY7c1 gene provided in the application can be applied to improve plant cold resistance, cultivate cold-resistant plants, and provide a new gene resource for plant cold resistance breeding, and has important significance for the improvement of cold resistance and the research on the regulation mechanism of plant growth and development.

It provides an inhibitor of complement activation. [Solution] An isolated monoclonal antibody and its antigen-binding fragment are provided that specifically bind to an epitope within the serine protease domain of human MASP-2. In some embodiments, the antibody or its antigen-binding fragment inhibits lectin pathway complement activation. Polynucleotides encoding the monoclonal antibody or its antigen-binding fragment, and cloning vectors or expression cassettes containing such polynucleotides are also provided. Methods for inhibiting lectin pathway complement activation and methods for treating diseases and disorders of the lectin pathway are further provided.

The disclosure provides for methods of constructing synthetic chromosomes including the steps of providing host cells with an endogenous chromosome, transforming a cloning vector and a cloning cassette into the host cells, excising target genomic nucleic acids from the endogenous chromosome, recombining the excised target genomic nucleic acids with the cloning cassette via homologous recombination to form heterologous vectors comprising cloned sequences, extracting the heterologous vectors containing the cloned sequences from the host cells, digesting the heterologous vectors with a restriction endonuclease to release the cloned sequences from the heterologous vectors to provide released cloned sequences, and introducing the released cloned sequences, a centromere cassette, and a yeast artificial chromosomes or bacterial artificial chromosomes into a second host cell, such that the released cloned sequences, the centromere cassette, and the YAC or BAC recombine with one another via homologous recombination to produce the synthetic chromosome.

The invention discloses an infectious pseudo-ginseng virus A cloning vector, the nucleotide sequence of the infectious pseudo-ginseng virus A cloning vector is shown as SEQ ID NO: 1, the infectious pseudo-ginseng virus A cloning vector belongs to the field of plant virology and genetic engineering, and the infectious pseudo-ginseng virus A cloning vector is obtained by cloning CP gene and RdRp gene of pseudo-ginseng virus A into a pCB301 vector; on the basis of the vector, a PnVA infectious cloning vector pPnVA-GFP carrying a green fluorescent protein GFP is constructed; a PnVA infectious cloning vector is transferred into an agrobacterium GV3101 strain through a freeze-thaw method, the agrobacterium GV3101 strain is transferred into nicotiana benthamiana and pseudo-ginseng through an injection method for expression, and results show that pCB301-PnVA and pPnVA-GFP can complete PnVA gene replication in pseudo-ginseng and tobacco and trigger typical virus disease symptoms.

The invention discloses a sequencing library construction method and application of the sequencing library construction method in genome typing and high-density physical map construction. The method comprises the following steps: respectively connecting genome DNA fragments covering the whole genome of a target species to a cloning vector, then carrying out enzyme digestion by using restriction endonuclease, screening fragments of which the length is greater than the length of the cloning vector from the enzyme digestion fragments, and adding ligase to carry out catalytic connection to obtain a self-ligation product; carrying out PCR (Polymerase Chain Reaction) amplification by using the primer pair self-ligation product as a template, wherein an obtained PCR product is a genome sequencing library of the target species; in combination with a PacBio HiFi sequencing technology, the method provided by the invention omits the processes of BAC library construction and monoclonal selection, and the marker generation and genomic typing accuracy are significantly improved. The method can be applied to development of high-resolution bin markers of polyploidy species and genome assembly quality, and can promote breeding and improvement of polyploidy crops.

The invention discloses an application of an HbCESTA1 gene in regulating and controlling a plant to respond to low-temperature stress, and the application comprises the step that the HbCESTA1 gene is introduced into the plant through overexpression so as to improve the cold resistance of the plant. The method comprises the following steps: by taking hevea brasiliensis cDNA as a template, designing a primer for amplification, connecting the primer to a cloning vector, transforming, screening and detecting to obtain an HbCESTA1 gene, connecting a gene segment to a vector, introducing arabidopsis thaliana, and screening and detecting to obtain an HbCESTA1 transgenic strain with improved cold resistance. According to the invention, the HbCESTA1 gene is cloned and identified for the first time, and by applying the HbCESTA1 gene in biology, the self cold resistance adaptation mechanism of the plant can be activated, and the low-temperature tolerance of the plant is remarkably improved, so that a new breakthrough in the field of cold resistance of the plant is obtained.

The application provides a method for sequencing small circular DNA molecules, comprising: constructing recombinant transposon fragments; enriching small circular DNA molecules; constructing recombinant small circular DNA molecules; and verifying positive clone vectors. Antibiotic resistance genes are randomly inserted into each small circular DNA molecule by using transposons, so that at least one antibiotic resistance gene is obtained in each recombinant small circular DNA molecule. Since an antibiotic is added to the culture medium in the subsequent culture process, only the clone vectors successfully transformed with the recombinant small circular DNA molecules can survive and reproduce in the culture medium, so that the antibiotic resistance gene becomes a "conserved sequence" of the recombinant small circular DNA molecule, and a corresponding PCR amplification can be designed for the antibiotic resistance gene. In addition, the antibiotic resistance gene sequence can be removed by using software when analyzing the sequence of the small circular DNA molecule.

The invention relates to a method for increasing the saponin content of panax notoginseng cells, and belongs to the technical field of biologication.The method comprises the following steps that 1, a CRISPR expression vector is constructed, specifically, target sites are designed and knocked out according to panax notoginseng target genes, a cloning vector plasmid pUC57-DT1T2 is used as a template to amplify a target fragment, an obtained fragment gel recovery product is connected with a pH SE401 linear vector subjected to enzyme digestion, and a CRISPR expression vector is obtained; transforming escherichia coli TOP10 to obtain the positive clone, and extracting recombinant plasmids, so as to obtain the CRISPR expression vector; and (2) performing agrobacterium tumefaciens-mediated panax notoginseng cell genetic transformation. The method specifically comprises the following steps: S1, preparing an agrobacterium tumefaciens liquid containing the CRISPR expression vector; s2, performing infection; s3, co-culture is carried out; and S4, degerming and screening. According to the invention, the CRISPR expression vector is constructed and introduced into agrobacterium, the vector is efficiently transformed into the panax notoginseng cells through agrobacterium mediation, and screening and identification are carried out, so that the panax notoginseng cells with significantly improved saponin content are efficiently and stably obtained.

The invention relates to the field of PDRN preparation in the technical field of biology, in particular to a rapid preparation method of sequence-controllable PDRN. The invention discloses a rapid preparation method of sequence-controllable PDRN, which comprises the following steps: firstly, establishing a PDRN library, selecting a PDRN sequence from the library, performing gene synthesis, and connecting the PDRN sequence into a cloning vector; and carrying out amplification by using a Taq enzyme and a specific primer pair. By optimizing and adjusting a reaction system, microgram-level, milligram-level and gram-level amplification reactions are realized, and amplification can be completed within 4 hours. And then carrying out ultrafiltration, alcohol precipitation, drying and the like to obtain a PDRN product. The PDRN prepared by the method is single in length, controllable in sequence and high in base composition consistency, breaks through the tradition that the PDRN is a mixture, is high in safety, simple in production process and high in preparation speed, greatly improves the production efficiency, and can remarkably improve the economic benefit.

The invention discloses an infectious clone plasmid of a human fiveleaf virus or a human norovirus, a reverse genetic virus rescue system and application of the infectious clone plasmid and the reverse genetic virus rescue system. The infectious clone plasmid is based on a clone vector and is inserted into a cDNA genome of a human fiveleaf virus or human norovirus; the 5'end of the cDNA genome is connected in series with two T7 promoters, and an internal ribosome entry site is inserted between the two T7 promoters; an HDV ribozyme sequence and a T7 terminator sequence are sequentially connected in series behind a PolyA tail of the cDNA genome. The reverse genetic virus rescue system comprises a human fiveleaf virus or human norovirus infectious clone plasmid, a plasmid for expressing T7RNA polymerase and a BHK-T7 cell. The reverse genetic virus rescue system provided by the invention can efficiently and quickly rescue filial generation virions of human fiveleaf viruses or human noroviruses, and can quickly obtain a large number of viruses.

The invention belongs to the technical field of biology, and discloses a method for determining a multivalent antigen sequence of an anti-periodontitis red complex, which comprises the following steps: firstly, screening out key virulence factor antigens (FimA-II, RgpA, Msp and BspA) from three core pathogenic bacteria of the red complex: porphyromonas gingivalis, treponema denticola and fusisterone; then, independently cloning each antigen gene to a pUC57 cloning vector, constructing a single antigen recombinant plasmid, and avoiding intergene interference by designing a specific enzyme cutting site; and finally, through plasmid transformation, positive clone screening and double enzyme digestion verification, the plasmid quality is ensured. The obtained single antigen plasmid can be flexibly combined and used, is beneficial to subsequent multivalent vaccine development, and has the advantages of wide antigen coverage, high expression reliability, flexible construction strategy, easiness in industrialization and the like.

This invention belongs to the field of genetic engineering technology and provides the SmHRD3A gene and its application in increasing the tanshinone content in *Salvia miltiorrhiza*. This invention comprehensively applies transcriptome data analysis, gene cloning, vector construction, molecular detection, RT-qPCR analysis, target exploration, protein-protein interaction, and compound content determination to obtain the encoding gene of the ERAD protein SmHRD3A, which interacts with the rate-limiting enzyme SmHMGR in the MVA pathway. This gene can significantly increase the tanshinone content in *Salvia miltiorrhiza*. It provides new ideas and target genes for the production of active ingredients in *Salvia miltiorrhiza* and for molecular design breeding of *Salvia miltiorrhiza*, and has high application and research value.

The application discloses a Chinese Sida herb yellow leaf virus infectious clone carrier combination and application thereof in preparation of a model plant. The application provides a recombinant plasmid combination, which is composed of a recombinant plasmid A and a recombinant plasmid B; the recombinant plasmid A has a DNA molecule shown in SEQ ID NO: 3; and the recombinant plasmid B has a DNA molecule shown in SEQ ID NO: 4. The application further provides a recombinant agrobacterium combination, which is composed of a recombinant agrobacterium A obtained by introducing the recombinant plasmid A into agrobacterium and a recombinant agrobacterium B obtained by introducing the recombinant plasmid B into agrobacterium. The application further provides a method for preparing a model plant, which comprises the following steps: infecting leaf blades of a plant of a receptor plant with the recombinant agrobacterium combination to obtain the model plant. The model plant is a plant with the following phenotypes: leaf blade undercurling and / or leaf blade crumpling. The model plant can be used for drug screening and mechanism research.

The invention discloses a sugarcane yellow leaf virus infectious cloning vector as well as a construction method and application thereof, and belongs to the technical field of biology. The recombinant plasmid provided by the invention contains a whole genome of the SCYLV, and the nucleotide sequence of the whole genome is the 771th nucleotide to the 6676th nucleotide of SEQ ID No.1. The recombinant plasmid is obtained by taking a pCB301 vector as a skeleton and then inserting the skeleton into a whole genome of SCYLV, and the recombinant agrobacterium tumefaciens (pCB301-SCYLV infectious clone) prepared from the recombinant plasmid can successfully infect sugarcane and has efficient infection ability; the infectious clone of the recombinant expression plasmid prepared from the recombinant plasmid can successfully infect nicotiana benthamiana, has efficient infection ability and can express foreign proteins.

The application discloses a kind of SlGRP gene and the application of coding protein in regulating plant resistance tomato brown rugose fruit virus and transgenic plant cultivation method, wherein the transcript sequence of SlGRP gene is as shown in SEQ ID NO:1.The application constructs the cloning vector of pCAMBIA1300-SlGRP-GFP using homologous recombination system, amplifies gene SlGRP using the cDNA of tomato, connects SlGRP into pCAMBIA1300-GFP vector by homologous recombination, so as to obtain the overexpression vector containing SlGRP, imports gene into tomato, tobacco and other plants by the method of agrobacterium transformation and obtains stable inheritance overexpression strain, and through disease resistance determination, it is shown that the overexpression plant of SlGRP can significantly inhibit the infection of tomato brown rugose fruit virus.

The invention relates to the technical field of recombinant viruses, and particularly discloses a recombinant porcine Seneca virus for expressing a porcine circovirus 2d genotype Cap gene as well as a preparation method and application of the recombinant porcine Seneca virus. According to the invention, an SVA virus HeN-1 / 2018 is taken as a vector, an infectious cloning vector of a full-length genome of the SVA virus HeN-1 / 2018 is constructed, a PCV2d Cap-P2A gene synthesized by a PCV2d Cap gene without a nuclear localization signal and a P2A gene is recombined into the infectious cloning vector through a seamless cloning technology to obtain a connection product, the connection product is converted into a competent cell, and the recombinant PCV2d Cap-P2A gene with the nuclear localization signal removed is obtained. A recombinant plasmid pSVA-PCV2d-Cap is obtained after culture and verification, then an SVA recombinant virus rSVA-PCV2d-Cap for expressing PCV2d Cap protein is obtained through rescue based on a CMV polymerase transcription system, and the whole gene sequence of the recombinant virus is shown as SEQ ID NO: 1; an inactivated vaccine is prepared from the recombinant virus, it is determined that a vaccine composition produced by the recombinant virus rSVA-PCV2d-Cap strain can induce immune animals to generate neutralizing antibodies with high titers for SVA and PCV2, and a certain theoretical and practical foundation is laid for clinical use of the inactivated vaccine for the SVA and PCV2.

The present application relates to a method for producing a target DNA sequence and a cloning vector, comprising the steps of amplifying and extracting a DNA construct in a host cell and a protelomerase-type IIS restriction endonuclease and / or meganuclease-DNA exonuclease catalyzed three-step isothermal enzymatic reaction step, where the construct is autonomously replicating and contains (a) one or more type IIS restriction endonuclease and / or meganuclease recognition sequences; (b) a target DNA sequence; and (c) protelomerase recognition sequences at the two terminal flanks of the target DNA sequence.