39 results about "Full length genome" patented technology

The present invention provides a method for replicating efficiently an RNA containing fulllength HCV genomic sequence and a method for producing HCV virus particles containing fulllength HCV replicon RNA or fulllength HCV genomic RNA by using a cell culture system. Further, the present invention relates to a method for producing hepatitis C virus particles which comprises culturing a cell, into which a replicon RNA comprising a nucleotide sequence comprising a fulllength genomic RNA sequence of hepatitis C virus of the genotype 2a, at least one selectable marker gene and / or at least one reporter gene and at least one IRES sequence or the fulllength genomic RNA of hepatitis C virus of the genotype 2a is introduced, and generating virus particles in the culture medium. Still further the present invention relates also to a hepatitis C vaccine and an antibody against hepatitis C virus particles.

The invention discloses a hydrophobia-canine distemper-canine parvovirus genetic recombination virus strain, a construction method and application thereof. Through a homologous recombination method, agene replacement method and a gene insertion method, a full-length genome of a virus is subjected to gene rearrangement, and a rearranged hydrophobia virus genome plasmid with a canine distemper G gene and a canine parvovirus VP2 gene is constructed and is named as a hydrophobia-canine distemper-canine parvovirus, which is pcDSRV9-NG-eGFP+CDV N-PM+VP2-L genome plasmid; the hydrophobia-canine distemper-canine parvovirus is rescued through a reverse genetic method, and is an rSRV9+CDV N+VP2 gene recombinant virus; a gene nucleotide sequence of a recombination hydrophobia virus is SEQ ID NO:1. The recombination gene virus provided by the invention can be applied to researching a trigeminy oral vaccine for hydrophobia, canine distemper and canine parvovirus of canine and other wild animals soas to prevent three infectious diseases.

An external culture system of hepatitis C totivirus for culturing complete multiplicative HCV virus by the molecular biology and gene recombination technique is disclosed. Its culture steps include amplifying the full-length genom containing 98 nucleotides after 3' PolyA in the serum of hepatitis C patient; artificial site-directed mutation to NS5A and NS5B in HCV genon; inserting the expression box of marker gene IRES-GFP at NS5B3' terminal in the mutated HCV genom; transfecting sensitive cell and culturing to obtain progeny HCV virus with infection activity.

The present disclosure relates to a method for obtaining rRNA sequence information, comprising the steps of calling an rDNA sequence from full-length genomic DNA sequence information, assembling the rDNA to form rDNA contigs; and extracting DNA sequence of 16S rRNA having high completeness from the rDNA contigs and correcting an assembly error, and a method for identifying a microorganism, using the rDNA information.

The invention discloses a construction method of HBV (Hepatitis B Virus) isolating strain replicating-form plasmid, comprising the following steps of: 1, extraction of HBV full-length genome, PCR (Polymerase Chain Reaction) amplification and clone construction; 2, construction of a recombinant transition vector; 3, enzyme cutting and dephosphorylation of the recombinant transition vector; and 4, construction of replicating-form plasmid, wherein the recombinant transition vector is synchronously and directionally connected by three connection segments, two reverse BspQI enzyme cutting sites are formed between two inserted HBV segments, and the clone of the HBV full-length genome and the bacterial colony cultivation and amplification of the replicating-form plasmid are carried out by an SOC culture medium. The invention applies a novel full-length genome clone vector, improves the enzyme cutting solution and adopts a novel bacterial colony culture medium. The invention has the advantages of simple performance, high efficiency and stability, can be applied to the comprehensive phenotype research on a China HBV clinic isolating strain whole genome and has very important application value.

The invention provides a quality control material for detecting respiratory pathogen nucleic acid. The quality control material comprises any one or more of the following respiratory pathogens: coronavirus, influenza virus, adenovirus, mycoplasma pneumoniae, streptococcus pneumoniae, rhinovirus or / and legionella pneumophila. A full-length genome sequence of the coronavirus is divided into six target fragments, and the length of each fragment ranges from 4000 bp to 5500 bp. The quality control material disclosed by the invention covers all detection target sequences (or target spots) of the coronavirus, the coverage is wide, the detection target spots are comprehensive, and the phenomenon of missing detection is avoided. The quality control material provided by the invention contains main respiratory tract infection pathogens, has a wide coverage range, and has more accurate detection targets by taking a real virus sample and a slow virus sample as raw materials. According to the coronavirus quality control material prepared by the invention, a target gene sequence is integrated into a host genome through a lentiviral vector, an autonomously replicated gene is knocked out in the preparation process, and the coronavirus quality control material has a self-inactivation capability, so that the recombinant lentivirus cannot be replicated in a target cell and infects other cells.

An allergen-free transgenic peanut seed is produced by recombinant methods. Peanut plants are transformed with multiple copies of each of the allergen genes, or fragments thereof, to suppress gene expression and allergen protein production. Alternatively, peanut plants are transformed with peanut allergen antisense genes introduced into the peanut genome as antisense fragments, sense fragments, or combinations of both antisense and sense fragments. Peanut transgenes are under the control of the 35S promoter, or the promoter of the Ara h2 gene to produce antisense RNAs, sense RNAs, and double-stranded RNAs for suppressing allergen protein production in peanut plants. A full length genomic clone for allergen Ara h2 is isolated and sequenced. The ORF is 622 nucleotides long. The predicted encoded protein is 207 amino acids long and includes a putative transit peptide of 21 residues. One polyadenilation signal is identified at position 951. Six additional stop codons are observed. A promoter region was revealed containing a putative TATA box located at position −72. Homologous regions were identified between Ara h2, h6, and h7, and between Ara h3 and h4, and between Ara h1P41B and Ara h1P17. The homologous regions will be used for the screening of peanut genomic library to isolate all peanut allergen genes and for down-regulation and silencing of multiple peanut allergen genes.

The invention relates to construction and application of SHIV (simian / human immunodeficiency virus) infectious clone. Firstly, the overall length genome of a virulent strain SHIV-KB9 embedded with a B subtype env gene is cloned to a low copy number carrier pBR322, the obtained plasmid SHIV-KB9 / pBR322 is taken as a genome skeleton, the env gene of Chinese main prevalent strain CRF08_BC of HIV-1 is used for substituting for corresponding gene segments so as to obtain the infectious clone SHIV-KBQJ-12, the replacing env gene is from the infectious clone strain QJ001 of HIV-1CRF08_BC and comprises the whole intracellular region, a transmembrane region and part of an extracellular region of gp120 and gp41, and the position of the replacing env gene, corresponding to the position of the nucleotide of a standard reference strain HXB2, is 6103-8249. The construction is simple and convenient to operate, has high success rate, can obtain the infectious clone in a short time, and can be used for establishing an SHIV / Chinese rhesus infection model.

The invention relates to the field of biological virus construction and particularly discloses a recombinant oncolytic Newcastle disease virus for expressing human GM-CSF. The recombinant oncolytic Newcastle disease virus is obtained by inserting a human GM-CSF coding gene into a full-length genome plasmid of a Newcastle disease virus Italien strain and reviving the virus in vitro. The human GM-CSF gene is as shown in SEQ ID NO.1. A GM-CSF molecule is used for recruiting DC cells from mononuclear cells, activating neutrophil and promoting differentiation and maturation of the DC cells, especially has an important effect on promoting processing and presentation of antigens, combines a characteristic of selective replication of tumors of the oncolytic Newcastle disease virus, directly kills tumor cells, causes inflammation, recruits more DC cells, promotes maturation of the DC cells and presentation of tumor antigens, enhances local and whole-body anti-tumor immune responses of tumors, improves a treatment effect of single or combined use of the oncolytic virus in treating solid tumors, and brings a new treatment thought for tumor immunotherapy.

The present invention provides a rabies virus CTN strain whole genome cDNA infectious clone, as well as a method of using reverse genetics technology to save the active rabies virus. The present inventive method includes: using the single restriction enzyme sites in the CTN strain of rabies virus full-length genome to divide the full-length virus into four segments to amplify, simultaneously using the green fluorescent protein gene to substitute 423bp base at the pseudo-gene point, cloning in the pVAX-R expression vector, to construct a recombinant full-length plasmid of the virus genome; performing PCR amplification of nucleoprotein, phosphoprotein, glycoprotein and transcription large protein gene sequence of the CTN strain of the rabies virus, and cloning the amplified fragment into a vector pVAX1, to constitute four auxiliary plasmids in the reverse heredity system; and then using the five plasmids to jointly transfect cells, to rescue the rabies virus. The present invention successfully saves the rabies virus, and has a great significance in the study of the pathogenic mechanism of rabies virus, development of new vaccines, viral vectors and the like areas.

The invention relates to a preparation method of a HPV full-length genome quality control product, an amplification primer and a detection reagent. Wherein, the preparation method of the HPV full-length genome quality control product comprises the following steps: (1) obtaining a DNA template; (2) using amplification primers to perform PCR amplification; (3) using HD Cloning connection technology to connect the target DNA fragment to the carrier ; (4) introducing the plasmid into competent cells, and then extracting the plasmid; (5) diluting the plasmid with human genome. The preparation method of the human papillomavirus full-length genome quality control product of the present invention can successfully obtain the HPV gene detection quality control product that has no biological infection risk type, can simulate clinical samples, can be produced in large quantities, has a controllable concentration, and is stably stored. Has good applicability.

The purpose of the present invention is to determine the nucleotide sequence of the full-length genomic RNA of papaya leaf-distortion mosaic virus. The present invention provides the full-length genomic RNA of papaya leaf-distortion mosaic virus, a method for diagnosing infection with papaya leaf-distortion mosaic virus using the full-length genomic RNA, a method for producing a papaya leaf-distortion mosaic virus-resistant plant, and a method for producing a foreign protein in a plant body.

The invention discloses a restructured infectious cloning plasmid of a low virulent strain of a Japanese encephalitis virus, which contains a full-length genome cDNA sequence of the low virulent strain 10S3 of the Japanese encephalitis virus, or a full-length cDNA sequence of a marked cleavage site introduced in the full-length genome cDNA sequence of the low virulent strain 10S3 of the Japanese encephalitis virus. The invention also discloses an infectious cloning low virulent strain of the Japanese encephalitis virus. The low virulent strain is a cloning strain of the low virulent strain 10S3 of the Japanese encephalitis virus, and the full-length genome cDNA sequence of the low virulent strain 10S3 is shown in SEQ ID NO.1. The restructured infectious cloning plasmid of the low virulent strain of the Japanese encephalitis virus and the infectious cloning low virulent strain of the Japanese encephalitis virus rescued from cells can provide complete immunoprotection for immunized animals resisting Japanese encephalitis viruses, thereby extremely facilitating the prevention and control of Japanese encephalitis viruses.

The invention discloses an adenovirus vector and its application in preparing HCV (hepatitis C virus) cells and mouse models. The invention provides a recombinant adenovirus vector which is obtained by inserting nucleotides shown by the sequence 1 in the sequence table in the adenovirus vector HDAd (helper-dependent adenovirus). The experiments prove that the invention provides a third generation adenovirus vector of a recombinant HCV 2a type full-length genome, the prepared adenovirus can be used for preparing HCV whole-genome cell models or mouse models, and the obtained models can be used for drug screening of anti-HCV drugs, so as to be used for HCV vaccine evaluation and researches of the pathogenic mechanism.

Belonging to the technical field of biological genetic engineering, the invention in particular relates to a hordeum vulgare calmodulin gene HvCAM1 and salt tolerant application thereof. The hordeum vulgare calmodulin gene HvCAM1 has a nucleotide sequence shown as SEQ ID No:1 and a full-length genome sequence 2124 bp. According to the invention, firstly a hordeum vulgare calmodulin coded gene is obtained by cloning and is named as HvCaM1, and the transcript cDNA full-length nucleotide sequence is shown as SEQ ID No:2, and the full-length cDNA sequence is 728 bp. The invention also utilizes RNAi interference method to silence the HvCAM1 gene successfully in hordeum vulgare Golden Promise, also utilizes the material to verify the negative regulation effect of HvCAM1 gene under salt stress can effectively enhance the salt tolerance of hordeum vulgare. At the same time, overexpression of the HvCAM1 gene in hordeum vulgare Golden Promise cannot enhance the salt tolerance of hordeum vulgare.

The invention discloses a GII.4 type norovirus genome amplification primer and an amplification method. In the present invention, six pairs of primers are used as the upstream and downstream primers of the amplification primers, and the RNA of GII.4 type norovirus is used as the template to carry out RT-PCR amplification to obtain amplification products respectively, and then perform nucleic acid sequence sequencing on the amplification products , and then spliced ​​and compared to obtain the full-length sequence of the GII.4 norovirus genome. According to the main popular genotypes of GII.4 Norovirus, the present invention designs a "4+1+1" segmentation amplification strategy according to the three open reading frames contained in the genome, and designs corresponding amplification according to the conserved region Primer; it can effectively amplify the whole genome sequence of GII.4 type norovirus. The invention can be widely used in medical and health care, inspection and quarantine, etc., which have norovirus detection requirements and corresponding scientific research fields.

The invention discloses primers for cloning SPFMV (sweet potato feathery mottle virus) O strain and full length genome sequence of SPVC (sweet potato virus C). The primers are respectively a primer for cloning the SPFMV O strain, a primer for cloning the full length genome sequence of SPVC, and the primer for simultaneously cloning the SPFMV O strain and the full length genome sequence of SPVC. The SPFMV and the SPVC generally infect sweet potatoes in a mixed way, the separation and the purification are very difficult; the content of the virus in the sweet potatoes is low; the full length genome sequence is difficult to obtain by a conventional virus purification cloning method. The principle that SPCSV can promote the content rise of the SPFMV and the SPVC in the sweet potato plants is used; the sweet potato plants simultaneously infected with SPCSV, SPFMV-O and SPVC viruses are used as materials; the total RNA (ribonucleic acid) of the virus-infecting sweet potato plant leaves is extracted; the virus full length genome sequence cloning is performed; through a great amount of PCR (polymerase chain reaction) amplification and sequence testing, the genome full length sequence of SPVC and SPFMV is obtained through comparison and splicing.

The invention relates to the technical field of plant molecular biology and gene engineering, and in particular relates to a rice protein (rice OsPIMT2) related to low nitrogen stress resistance as well as cloning, functional verification and application of a coding gene thereof. Full-length cDNA is cloned out by using a Smarter RACE technology to further obtain a full-length genomic sequence of OsPIMT2. Then, an over-expression and dry interference expression vector of an OsPIMT2 gene is constructed, and the agrobacterium mediated transformation of keng rice (Nipponbare) and the analysis of low nitrogen stress of a transgenic plant show that an over-expression transgenic plant of the OsPIMT2 gene, compared with a wild plant, has a lower lipid preoxidation level and a higher biomass (such as dry weight, plant height and the like). Therefore, the expression of the gene can enable the rice to use limited nitrogen to obtain and generate a higher biomass, thereby providing a certain theory and a certain basis for culturing a rice variety with a high nitrogen utilization ratio in the future.