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292 results about "Shuttle plasmid" patented technology

The Shuttle Plasmid: The shuttle plasmid is usually cloned by the investigator and contains the gene of interest driven by the desired promoter and any other elements needed for a single or bicistronic construct. Reporter genes such as eGFP may be cloned into this plasmid or may come from the viral backbone plasmid.

Adenoviral vector system and recombinant adenovirus construction method

The invention provides an adenoviral vector system and a recombinant adenovirus construction method. The vector system comprises adenoviral plasmids pKAd5f11p-EF1aP and pKAd5f11pES-PmeI and shuttle plasmids pUC19-PM. The adenoviral plasmid contains an E1/E3 deleted human adenovirus type 5 (HAdV-5) genome and the original HAdV-5 fiber gene is replaced by a fusion gene F5-11p of HAdV-5 and HAdV-11p.A PmeI site is an exogenous gene insertion site. Plasmids pKAd5f11p-EF1aP contain a human EF1a promoter in the original E1 region. The shuttle plasmids pUC19-PM are matched with the plasmids pKAd5f11pES-PmeI. The recombinant adenovirus construction method comprises: carrying out PCR amplification to obtain a desired gene fragment containing homologous overlapping regions on both sides and carrying out DNA assembling on the desired gene fragment and PmeI-linearized adenoviral plasmids to obtain adenovirus plasmids containing the desired exogenous gene, or cloning the multiple gene fragments tothe shuttle plasmids, shearing all the desired gene fragments through a restriction endonuclease and carrying out DNA assembling on the desired gene fragments and PmeI linearized pKAd5f11pES-PmeI toobtain the adenovirus plasmids containing the desired exogenous gene.
Owner:中国疾病预防控制中心病毒病预防控制所 +1

Fowlpox virus vector shuttle plasmid and application thereof

InactiveCN101775410APreserve immune efficiencyRetain the ability to replicateGenetic material ingredientsGenetic engineeringShuttle plasmidFowlpox virus
The invention provides fowlpox virus vector shuttle plasmid pTGP3 which comprises recombinant arms TKL and TKR, a bidirectional promoter PE/L, a fluorescent protein expression cassette, and a resistant marker gene and replication origin ori; the upstream and the downstream of the bidirectional promoter PE/L are respectively provided with cloning sites MCSL and MCSR; and both ends of the fluorescent protein expression cassette are provided with loxp sequences. The plasmid of the invention has two different screening markers, and the recombinant fowlpox virus prepared with the plasmid can express 1 to 3 types of gene with different meshes in the whole processes of the early and the later periods; the strong composite promoter with expression activity in the early and the later periods is applied so as to realize the all-process high-efficiency expression of a target gene; and the loxp sequences are introduced into both ends of the fluorescent protein expression cassette, so as to knock out the exogenous recombinant fowlpox virus screening markers. The invention lays foundation for the series and the scale application of the recombinant fowlpox virus in vaccine and biological drug research and development fields.
Owner:MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI

Preparation method and application of CAR-T cell targeting B7H3

The invention relates to a preparation method of a CAR-T cell targeting B7H3. The preparation method includes first preparing a PBMC cell; then co-transfecting a 293T cell with a shuttle plasmid LV-B7H3 containing the CAR structure, a helper plasmid psPAX2 and an envelope plasmid VSV-G to obtain a packaged B7H3-CAR virus; then taking a PBMC cell, using anti-human CD3 and anti-human CD28 as activators, culturing and activating for 48 hours and adding the B7H3-CAR virus for infection. By means of the preparation scheme, the expression of IFN-gamma in the CAR-T cell is increased, and the cell killing activity is high. The CAR-T cell targeting B7H3 has a killing effect on various solid tumor cells, has high killing activity, is safe and effective, and can be used for immunotherapy of kidney cancer, lung cancer, liver cancer, glioma, ovarian cancer, breast cancer and the like.
Owner:XUZHOU MEDICAL UNIV

Unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination

The invention relates to an unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination. The method comprises the following steps: temperature sensitive-type shuttle plasmid pSET4E and knock-out plasmid containing homologous fragments at upstream and downstream parts of target genes to be knocked out are constructed, the knock-out plasmid is subjected to electrotransformation into pediococcus acidilactici, and single commutators generating homologous recombination for the first time and double-exchange mutant strains generating homologous recombination for the second time are screened and identified. The method disclosed by the invention realizes the unmarked gene knock-out of pediococcus acidilactici for the first time, the obtained knock-out bacterial strain does not carry any resistant gene, can be taken as a original strain for subsequent and reconstruction, and also can be used for large-scale industrial production in a safe mode. The method is used for respective knock-out of L-lactate dehydrogenase gene and d-lactate dehydrogenase gene of the pediococcus acidilactici DQ2 (a preservation number is CGMCC NO.7471), the obtained knock-out bacterial strains are respectively named as pediococcus acidilactici ZP26 and TY112, the preservation numbers are CGMCC NO.8665 and CGMCC NO.8664 respectively, and optically pure D-lactic acid and L-lactic acid are respectively generated.
Owner:EAST CHINA UNIV OF SCI & TECH

A kind of low temperature alkaline protease and preparation method thereof

The invention relates to a low-temperature alkaline protease and a preparation method thereof, belongs to a method of performing site-directed mutagenesis to a wild type alkaline protease gene by utilizing a recombinant DNA (deoxyribonucleic acid) technology to improve the characteristics of the gene, connecting the mutated gene with escherichia coli-bacillus subtilis shuttle plasmid pBE2S and expressing the mutated gene in the bacillus subtilis at high efficiency, and relates to a low-temperature alkaline protease with cold adaptability and alkali stability and a preparation method thereof. The invention solves the problem that the alkaline protease has so low activity in a low-temperature environment that the application is limited. The invention adopts a technical scheme that: the wildtype alkaline protease gene is separated from a microbe, in particular bacillus alcalophilus; amino acid residues of Glu110 and Glu134 of the gene are mutated; the enzyme activity of fermentation fluid of the gene is 1985 U / mL after the gene is expressed in the bacillus subtilis at high efficiency; at 40 DEG C, the activity of the low-temperature alkaline protease (Glu110 and 134 Ala) is improvedby 28 percent, compared with that of the wild type alkaline protease; and at 10 DEG C, the activity is improved by 62 percent, compared with that of the wild type alkaline protease.
Owner:TIANJIN UNIV OF SCI & TECH

Fowl adenovirus 4 (FAdV-4) vector system and applications thereof

The invention provides a recombinant fowl adenovirus FAdV-4 vector system. The recombinant fowl adenovirus FAdV-4 vector system comprises a framework plasmid pKFAV4, a middle plasmid pKFAV4AP and a shuttle plasmid pKFAV4APNM. A target gene coding region is cloned to a multiple cloning site of the shuttle plasmid, NheI digestion is carried out on the shuttle plasmid carrying a target gene expression cassette, and the obtained fragment replaces the corresponding part of the middle plasmid; AvrII or PacI digestion is carried out on the middle plasmid carrying a target gene, the obtained fragmentreplaces the corresponding part of the framework plasmid by virtue of the ligation reaction or the DNA assembly reaction, and then a recombinant adenovirus plasmid is obtained; a PmeI linearized adenovirus plasmid is used for transfecting LMH cells, and the recombinant FAdV-4 adenovirus realizing control over the expression of the target gene by virtue of a human cytomegalovirus promoter (CMVp) can be prepared. In the prepared recombinant adenovirus, the Orf1-Orf2 regions of the FAdV-4 genome are replaced by the target gene expression cassette, and the other regions of the adenovirus are reserved. The recombinant fowl adenovirus FAdV-4 vector system is expected to have the broad application prospect in the research and development of oral vaccines for birds.
Owner:中国疾病预防控制中心病毒病预防控制所 +1

Method for constructing highly expressed trehalose synthase engineering bacteria by using Pcry3Aa promoter

The invention relates to a method for constructing highly expressed trehalose synthase engineering bacteria by using a Pcry3Aa promoter. For a recombinant carrier, a Pcry3Aa-PhoD fragment by which a Pcry3Aa promoter fragment and a PhoD signal peptide fragment are connected by an overlap PCR (Polymerase Chain Reaction) is inserted at the upstream of a restriction enzyme cutting site BamHI of a shuttle plasmid PHT01, and a target protein trehalose synthase TreS fragment is inserted between two restriction enzyme cutting site, i.e., BamHI and AatII. The invention further relates to a method for constructing the highly expressed trehalose synthase engineering bacteria by using the recombinant carrier. According to the method disclosed by the invention, the Pcry3Aa promoter is adopted to naturally induce the synthesis of trehalose synthase; because the Pcry3Aa promoter contains a special STAB-SD structure, the stability of the Pcry3Aa promoter to transcribe mRNA is improved, the half-life period of mRNA is prolonged, the mRNA translation level of a downstream target gene is improved, and therefore the trehalose synthase is highly expressed.
Owner:山东开盾生物科技有限公司

Pig circovirus III-type virus-like particle and preparation method thereof

An embodiment of the invention discloses a pig circovirus III-type virus-like particle. A preparation method of the virus-like particle includes the steps: amplifying pig circovirus III-type Cap protein genes; constructing recombinant shuttle-plasmid pFB-Cap by the aid of the genes; constructing rB-Cap recombinant rod granules by the aid of the recombinant shuttle-plasmid pFB-Cap; transfecting therB-Cap recombinant rod granules into SF9 cells to obtain recombinant baculovirus rBV-PCV3 Cap expressing pig circovirus III-type Cap genes; enabling the recombinant baculovirus rBV-PCV3 Cap to infectHigh Five cells, and purifying the recombinant baculovirus rBV-PCV3 Cap infected by the High Five cells to obtain the pig circovirus III-type virus-like particl PCV3 VLP. A nucleotide sequence of theCap protein gene is as shown in SEQ ID NO.1. According to the particle, based on a baculovirus-insect cell expression system, preparation is implemented by the aid of High Five cell expression of serum-free suspension culture, and the PCV3 virus-like particle is acquired by combining sucrose cushion ultracentrifugation and sucrose density gradient centrifugation purification. The virus-like particle is good in immunogenicity and high in safety and has good development and application prospects.
Owner:ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE

Recombinant adenovirus and tetravalent adenovirus vaccine and preparation method thereof

ActiveCN106318916ANo recombinationHigh neutralization potencyViral antigen ingredientsVirus peptidesHuman typeSerotype
The invention discloses a recombinant adenovirus and tetravalent adenovirus vaccine and a preparation method thereof. The tetravalent recombinant adenovirus vaccine contains a recombinant type 3 adenovirus strain, a recombinant type 7 adenovirus strain, a recombinant type 14 adenovirus strain and a recombinant type 55 adenovirus strain. The preparation method disclosed by the invention comprises the following steps: preparing recombinant shuttle plasmids containing hexon gene segments, and performing in-bacteria homologous recombination with a recombinant human type 3 adenovirus strain, thereby obtaining a recombinant adenoviral genome in which the hexon gene segments are replaced by type 7, type 14 and type 55; transfecting cells, rescuing to obtain recombinant human type 3, 7, 14 and 55 recombinant adenoviruses with different main capsid protein-hexon proteins; purifying, mixing according to the same protein content, and inactivating by using beta-propiolactone, thereby obtaining the tetravalent adenovirus vaccine. The tetravalent adenovirus vaccine is capable of inducing neutralizing antibody responses to four types of serotype adenoviruses, and the neutralizing titer is 500-1000.
Owner:GUANGZHOU GIR MEDICINE CO LTD +1
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