4 results about "PBR322" patented technology

The invention belongs to the field of biology, and discloses a proteus mirabilis homologous recombination system expression plasmid which comprises a pBR322 replication starting point, a resistance gene, an arabinose inducible promoter and a proteus mirabilis-derived homologous recombination operon. The homologous recombination operon is used for coding a TPM66325 protein, an EPM66325 protein, a single-chain binding protein SSB and a fake release protein. The expression plasmid has relatively high recombination efficiency in proteus mirabilis GDMCC 66325 and can also play a recombination role in a proteus mirabilis standard strain ATCC35659, and the expression plasmid is obtained by knocking out a related gene bcsB formed by a hemolysin related gene hpmA and a biological membrane of the proteus mirabilis GDMCC 66325 and directly knocking in a heterologous gene after a related gene cluster is adjusted by flagellum. The effectiveness of the expression plasmid in recombinant operation of proteus mirabilis is verified; meanwhile, the invention further discloses a proteus mirabilis homologous recombination system, application and a gene knockout / knockin method.

The application discloses a construction method of a human adenovirus type 4 protein V knockout mutant strain and application thereof, and belongs to the technical field of biotechnology. The application precisely knocks out a protein V coding sequence in a human adenovirus type 4 full-genome infectious clone by means of a Red / ET homologous recombination system combined with a ccdB reverse screening technology, and obtains a recombination plasmid pBR322-Ad4-△V-EGFP which is clear in genetic background and highly homozygous in sequence. After the plasmid is transfected into HEK-293 cells, the human adenovirus type 4 protein V knockout mutant strain obtained exhibits a typical “replication-deficient” phenotype: although a particle containing a viral genome can be packaged, the physical titer (4.76×10 6 copies / μL) of the particle is about one order of magnitude lower than that of a wild type, and the progeny virus completely loses secondary infection ability. Transcriptome analysis reveals that the deletion mutation can specifically down-regulate a host proteasome pathway and activate a natural immune response. The mutant strain is high in safety and strong in immunogenicity, and can be used as an ideal attenuated vaccine carrier or a gene therapy tool.

The invention provides a method for efficiently producing noottanone through biological fermentation, and relates to the technical field of microbial fermentation, the method comprises the following steps: (1) seed preparation: connecting a gene of UDP-glucosyltransferase to a PBR322 plasmid vector, transducing the PBR322 plasmid vector loaded with the gene of UDP-glucosyltransferase to competent state of saccharomyces cerevisiae, and preparing seeds; inoculating into a culture medium for culturing to obtain saccharomyces cerevisiae A; (2) seed culture: inoculating the saccharomyces cerevisiae A in the step (1) into a culture medium according to the inoculum size of 1-10% in volume ratio, and continuously culturing to obtain a seed culture solution; (3) fermentation production: inoculating the seed culture solution into a culture medium according to the inoculum size of 1-10% by volume ratio, and fermenting to obtain fermentation liquor; and (4) separation and purification: standing the fermentation liquor obtained in the step (3), and centrifuging. The noottanone prepared by the invention has the advantages of environmental protection, low cost, high benefit and the like.

A single plasmid vector system for packaging recombinant human adenovirus type 4. The vector system contains an E3 region-deleted human adenovirus type 4 (HAdV-4 or Ad4) genome, a vector sequence for amplifying plasmids in bacteria, a pBR322 replication origin, a kanamycin resistance gene, and a replication control sequence; and an exogenous gene embedding site is located behind a packaging signal of the human adenovirus type 4 and in front of an E1 region. The present invention further provides a method for packaging the recombinant human adenovirus type 4 by the single plasmid vector system and an application in vaccine and drug preparation. The vector system can be used for rapidly and efficiently preparing a human adenovirus type 4 vector recombinant virus for stably expressing an exogenous gene, and has a good application prospect in the fields of preparation of a diagnostic kit, a vaccine, a gene therapy kit and / or a tumor therapy drug, etc.