5 results about "PBR322" patented technology

The invention provides a gene VII type NDV recombinant virus for expressing IBV S protein as well as a preparation method and application of the gene VII type NDV recombinant virus, and the method comprises the following steps: inserting a modified S gene between a P gene and an M gene of a pBR322-DHN3mF virus genome to obtain any one of a recombinant virus rDHN3mF-MH20S1-IBVS2, a recombinant virus rDHN3mF-H120-S6P and a recombinant virus rDHN3mF-H120-S6P-GSAS. According to the present invention, the S2 region of IBV-P65 adapted to a cell strain is replaced with the S2 region of QX type IBV MH20, such that the recombinant virus rDHN3mF-MH20S1-IBVS2 is rescued; the preparation method comprises the following steps: mutating hydrophobic amino acids at the 692nd site, the 768th site, the 772nd site, the 815th site, the 862nd site and the 863rd site on an H120-S protein into proline to rescue a recombinant virus rDHN3mF-H120-S6P; on the basis that six hydrophobic amino acids are mutated into proline, amino acids from the 533th site to the 537th site are mutated into glycine, serine, alanine and serine in sequence, and then the recombinant virus rDHN3mF-H120-S6P-GSAS is rescued. The three rescued recombinant viruses are respectively infected with BHK-21 cells, and high-efficiency expression of S protein and NDV holoprotein can be detected through WB verification.

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.