292 results about "Shuttle plasmid" patented technology

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.

The invention relates to the field of biotechnology, and particularly relates to a shuttle plasmid vector, as well as a construction method and applications thereof. The shuttle plasmid vector can shuttle in yeast and Escherichia coli, and the nucleotide sequence of the shuttle plasmid vector is shown in SEQ ID NO.1. Compared with the prior art, he shuttle plasmid vector realizes the shuttling in yeast and bacteria, and has the capabilities of homologous recombination cloning, self replication and recombinant screening in yeast, as well as the characteristics of being stable in replication and maintenance and convenient for DNA purification and separation in bacteria.

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.

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.

The invention relates to a replication defective recombinant adenovirus Ad41 vector system and an application thereof in the field of gene therapy and recombinant vaccines. Specifically, the vector system comprises a backbone plasmid, a shuttle plasmid and a packaging cell line, wherein, the backbone plasmid contains an adenovirus Ad41 genome which deletes an encoding region required for packaging adenovirus, the shuttle plasmid contains multiple cloning sites used for inserting a target gene and an adenovirus Ad41 genomic fragment used for carrying out homologous recombination with the backbone plasmid, and the packaging cell line integrates genes required for packaging adenovirus Ad41 in the cell genome and can stably express the packaging genes. The generated recombinant Ad41 virus has broad application prospects in intestinal gene therapy and research of oral recombinant vaccines.

The invention discloses a genetically engineered bacterium capable of increasing the yield of spinosads as well as a construction method and an application. The construction method of the genetically engineered bacterium capable of increasing the yield of spinosads comprises the following steps: (1) inserting an spnk expression cassette into an escherichia coli-saccharopolyspora spinosa shuttle plasmid pOJ260 to construct a recombinant plasmid pLU101; (2) guiding the recombinant plasmid pLU101 into saccharopolyspora spinosa ATCC49460, and integrating the recombinant plasmid with a genome through homologous recombination, thus obtaining the genetically engineered bacterium capable of increasing the yield of spinosads and naming the genetically engineered bacterium LU101. The genetically engineered bacterium constructed by the method can increase the yield of spinosads.

The invention provides a preparation method of avian influenza virus HA gene recombinant adenovirus, which creatively comprises the following steps: carrying out a series of intermediate processes on plasmid pCAGGS, adenovirus shuttle plasmid pShuttle, adenovirus framework plasmid pAdEasy-1 and the like to obtain a gene expression plasmid and other intermediate products, and transfecting the obtained recombinant adenovirus plasmid with 293 cell; and carrying out immunohistochemical screening on the recombinant virus according to the adenovirus-infected cytopathy and specific cells. By using the CAG as the promoter to express the target gene, the method obviously enhances the expression level of the target gene. The hemagglutinin recombinant adenovirus for respectively expressing H5N1 and H9N2 subtype avian influenza viruses provides a virus model for development of the H5/H9 subtype avian influenza virus bivalent nucleic acid vaccine, and also lays the foundation for development of the AIV (avian influenza virus) adenovirus live vector vaccine.

The invention discloses a preparation method and system of recombinant adeno-associated virus (rAAV), and recombinant bacmid. The method comprises: (1) separately preparing shuttle plasmids and a corresponding recombinant bacmid containing baculovirus genome; (2) integrating a rAAV core expression element which has a heterologous functional gene fragment with other expression cassettes which produce functional protein components necessary for rAAV so as to obtain a recombinant bacmid containing recombinant baculovirus genome producing the rAAV; and (3) transfecting the obtained recombinant bacmid into a host cell line for culturing. The system comprises the shuttle plasmids and the corresponding recombinant bacmid containing the baculovirus genome. The recombinant bacmid comprises at leastone expression cassette that produces functional protein components necessary for rAAV. The system has flexibility, compatibility, and higher passage stability.

The invention discloses a Zika virus vaccine based on a replication-defective recombinant adenovirus vector. A nucleotide sequence of a JE signal peptide, and an optimized nucleotide sequence encodingZika virus membrane protein prM and envelope glycoprotein E are cloned into a shuttle plasmid vector pShutle2-CMV-Flag to obtain a recombinant shuttle plasmid, so that the expression of foreign proteins is significantly increased, and at the same time immunogenicity of an antigen is improved. Meanwhile, a recombinant adenovirus expression vector SAd23-L is used to escape pre-existing immune responses against common adenovirus vectors, and the humoral and cellular immunity with a higher level can be induced to generate in animals. After the recombinant adenovirus as a Zika virus vaccine is used to immunize animals, the humoral and cellular response to Zika virus is quickly induced to generate, especially a neutralizing antibody with a high level is induced to generate, and the specific cellular response to Zika virus antigen M and E protein is induced to generate in mice. Therefore, the Zika virus vaccine can be used to prevent large-scale outbreak and epidemic of Zika virus.

The invention discloses a prawn white spot comprehensive virus capsule membrane protein vp28 and genetic engineering strain of carp growth hormone GH fused protein and constructing method and appliance, which comprises the following steps: extracting total RNA from glandula pituitaria of prawn; reverse- transcripting; getting GH gene; extracting WSSV from ill prawn polypide; preparing vp28 gene through PCR method; constructing shuttle plasmid pPIC6alphac-GH-vp28 with growth hormone GH gene and WSSV vp28 gene; cutting pPIC6alphac-GH-vp28 with enzyme; making to linearization; inverting yeast X33 competence cell; inducing with methanol; expressing genetic engineering fused protein GH+VP28 protein in pichia. This protein can either accelerate growth of prawn, or increases immunity and disease resistance for WSSV.

The invention relates to plication-deficient recombinant adenovirus which contains the expression sequence of siGNA of the gene APE1 of human being. The sequence of siGNA of the gene APE1 of human being at which the two ends are provided with restriction site is chemically synthesized and inserted under the prompter U6 of the shuttle plasmid of adenovirus after restriction enzyme, then shuttle plasmid pDC316-EGFP-U6-APE1siRNA is constructed, and then shuttle plasmid pDC316-EGFP-U6-APE1siRNA and the bone plasmid of adenovirus pBHG-fiber5/35 are simultaneously cotransfected into cell 293, consequently the recombinant adenovirus Ad5/F35-APE1siRNA with the expression sequence of siGNA of the gene APE1 of human being is achieved through the fixed-point recombination produced by the system function of Cre/loxP. The recombinant adenovirus is capable of effectively preventing the expression of the gene APE1 and effectively strengthening the sensibility of radiotheraphy and chemotherapy of tumour cell.

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.

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.

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.

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.

The invention discloses a method for simultaneously removing bacillus anthracis virulence megaplasmids pXO1 and pXO2. The invention provides a DNA molecule having a nucleotide sequence 1 shown in the sequence table. The invention also provides a recombinant vector, a transgenic cell line or recombinant bacteria containing the DNA molecule. The recombinant vector is prepared by inserting the DNA molecule between recognition sites HindIII and EcoRI of a shuttle plasmid. An experiment proves that according to a plasmid incompatibility principle, an incompatiple plasmid is constructed and a strain A16QT which does not contain virulence megaplasmids is obtained. The method has the characteristics of simpleness, speediness, good singularity and high safety. The method has a very important meaning for construction of a plasmid-less bacillus anthracis strain and a research on the interaction between chromosome and megaplasmids pXO1 and pXO2, provides a novel experimental means for construction of a novel vaccine, and provides a novel idea for preventing and controlling bacillus anthracis.

The invention discloses a preparation method of a novel coronavirus pneumonia bivalent vaccine. The preparation method comprises the following steps: amplifying shRNA of a 2019-nCoV targeted interfering gene, performing enzyme digestion on the PCR product, constructing an interfering vector pSilencer-shRNA, performing DH5a transformation, constructing a shuttle vector pDC312-shRNA, co-transfectingHEK293 by the shuttle vector pDC312-shRNA and adenovirus framework plasmid pBHGloxAEl to prepare Ad-shRNA, and then preparing Ad-nCoVdsRNA. The preparation method further comprises the following steps: amplifying a 2019-nCoV antibody expression gene, performing enzyme digestion on the PCR product, constructing a shuttle plasmid pShuttle-nCoV, performing DH5a transformation, performing PCR amplification, co-transfecting HEK293 by the amplified shuttle plasmid pShuttle-nCoV and adenovirus skeleton plasmid pAd-nCoV to prepare a recombinant adenovirus Ad-nCoV, and further preparing Ad-nCoVDNA, and then preparing the bivalent vaccine by using the Ad-nCoVds RNA, the Ad-nCoVDNA and H20 according to a ratio of 1:1:19. After spray inoculation through a respiratory tract, the adenovirus (Ad) introduces nCoVds RNA and nCoVDNA into the cells.