11 results about "Shuttle plasmid" patented technology

This invention discloses a plasmid for gene overexpression in non-pattern *Bacillus laterosporus*. Its leader region contains a methyltransferase expression cassette derived from the *Bacillus laterosporus* i83 genome, enabling stable existence in specific hosts containing the RM system. Based on this, a PidR2 promoter is added to obtain the pMCPidR2 shuttle plasmid. The PidR2 promoter effectively drives target gene expression, the pWV01 replicon ensures plasmid replication in non-pattern hosts, and the chloramphenicol resistance gene is used for positive colony selection. The plasmid contains a TraJ element, allowing it to be introduced into non-pattern microbial hosts via conjugation transfer. This invention also provides a target gene expression strategy in non-pattern microorganisms, achieving high transformation efficiency and high strain yield through conjugation transfer transformation of donor bacteria, conjugation transfer helper bacteria, and recipient bacteria, resulting in stable expression of the target protein. This invention provides an effective vector for efficient and stable gene overexpression in specific non-pattern microorganisms, helping to solve the problem of difficult genetic manipulation of industrial production strains.

The invention relates to the technical field of medicine and biology, and particularly discloses a stress-inducible virus and application thereof.The stress-inducible virus comprises a stress-inducible adenovirus or a stress-inducible lentivirus, the nucleotide sequence of the stress-inducible adenovirus or the stress-inducible lentivirus is shown as SEQ ID NO: 1, and the nucleotide sequence of the stress-inducible adenovirus or the stress-inducible lentivirus is shown as SEQ ID NO: 2. The preparation method comprises the following steps: constructing a virus vector, wherein the virus vector has a nucleotide sequence as shown in SEQ ID NO: 1; and virus packaging: transfecting HEK293T cells by adopting shuttle plasmids containing SEQ ID NO: 1 and skeleton plasmids containing viral genome sequences, and collecting a virus stock solution to obtain the stress induced virus. The invention also discloses application of the stress-induced virus in preparation of medicines for preventing and treating oral tumors. Controllable release of the foreign protein under a specific signal is realized. The therapeutic protein produced by the invention can maximize the curative effect of drugs such as CD47 targeted protein and the like, and meanwhile, the toxic and side effects of the drugs on other tissues of the whole body are reduced.

The application discloses a recombinant adenovirus shuttle plasmid and a preparation method thereof, a recombinant adenovirus and application thereof, and a Zika virus recombinant adenovirus plasmid, and belongs to the field of biological medicines. The Zika virus recombinant adenovirus shuttle plasmid provided by the application takes pShuttle as a carrier, carries a Zika virus E protein mutant gene, and the 5' end of the Zika virus E protein mutant gene is connected with a signal peptide gene; the 5' end of the signal peptide gene is connected with a Kozak sequence; and the 5' end of the Kozak sequence is connected with an enzyme cutting site 1. The recombinant shuttle plasmid, the recombinant plasmid and the recombinant adenovirus provided by the application can be applied to the preparation of a Zika virus adenovirus vaccine, and have wide development and application prospects.

The invention discloses a DNA structure for saccharomyces cerevisiae single-cell barcode labeling and chromatin state conjoint analysis and application thereof, and belongs to the technical field of molecular biology and single-cell multi-omics analysis. From the 5'end to the 3 'end, a fluorescent protein gene, a Tn5ME structure, a reverse transcription primer binding region, a CYC1 terminator fragment and a linearized shuttle plasmid skeleton are subjected to homologous recombination, and a pCM carrier is obtained; after NdeI / BamHI enzyme digestion, oligonucleotides with random sequences, which are subjected to annealing, amplification, Klenow filling-in and enzyme digestion, are connected, and transformation extraction is carried out, so that the DNA structure for saccharomyces cerevisiae single-cell barcode labeling and chromatin state combined analysis is obtained. On the basis of the constructed DNA structure, ATAC-seq and a bar code reverse transcription method are combined, important data related to the transcription state and chromatin state of a single cell can be obtained at the same time through bar code information, single cell pedigree tracking of the saccharomyces cerevisiae is achieved, and research on single cell epigenomics of the saccharomyces cerevisiae and other microorganisms is promoted.

Recombinant BCG, a preparation method therefor and an application thereof. A shuttle plasmid that can express a FimH protein on the surface of BCG is constructed, and a gene fragment that expresses the FimH protein is transformed into wild-type BCG to thereby obtain recombinant BCG. Upon verification, the recombinant BCG can express the FimH protein on the surface of BCG; therefore, the BCG can specifically bind to subjects that can selectively bind to the FimH protein. The recombinant BCG can also enhance the local innate immune effect and adaptive immune effect induced by BCG and the anti-tumor effect of peripheral blood mononuclear cells, and also significantly improves the effect of BCG against bladder tumors, and is used to treat bladder tumors.

The present application relates to the technical field of genetic engineering, in particular to a kind of gene medicine for treating PNPLA2 mutation arrhythmogenic cardiomyopathy and its preparation method and application.The effective component of gene medicine provided in the present application includes shuttle plasmid;The shuttle plasmid includes AAV9 carrier and PNPLA2 gene.The present application delivers normal PNPLA2 gene by AAV9 carrier, repairs the lipid metabolism disorder caused by mutation, reduces the fat deposition in myocardium, and then improves the pathological changes of ACM.By restoring the function of PNPLA2 gene, the process of myocardial fibrosis can be effectively inhibited, and the heart function can be improved.The present application uses myocardial specific promoter cTnT to ensure that PNPLA2 gene is only expressed in myocardium, so as to improve the targeting of gene therapy, and avoid the side effects caused by systemic expression.This strategy can effectively reduce the side effects of gene therapy, and ensure the accuracy and safety of treatment.

This invention provides a chimeric adenovirus vector Ad5F35 and a recombinant CCR5-32 mutant gene, its construction method, and its application, belonging to the field of gene therapy technology. The construction method provided by this invention includes the following steps: (1) inserting the CCR5Δ32 mutant gene into the multiple cloning site of a shuttle vector to obtain a shuttle plasmid; (2) co-transfecting cells with the shuttle plasmid and the Ad5F35 adenovirus backbone plasmid to generate an Ad5F35 chimeric recombinant adenovirus capable of expressing the CCR5Δ32 gene. By inducing the inability of the CCR5 protein on the host cell to be normally expressed on the cell membrane surface, the binding of HIV-1 gp 120 to the CCR5Δ32 mutant gene is effectively prevented, thus preventing the HIV-1 virus from entering the host cell for replication, thereby achieving the goal of treating AIDS.

The application discloses a preparation method and application of a recombinant adenovirus spontaneously generating virus-like particles, and relates to the technical field of biology. The preparation method of the recombinant adenovirus comprises the following steps: connecting a virus membrane protein gene and a budding-promoting EARP gene to construct a recombinant exogenous virus membrane protein gene; inserting the recombinant exogenous virus membrane protein gene into an E3 region of an adenovirus skeleton plasmid to obtain a recombinant skeleton plasmid; inserting the recombinant exogenous virus membrane protein gene into an E1 region of an adenovirus shuttle plasmid to obtain a recombinant shuttle plasmid; and co-transfecting the recombinant skeleton plasmid and the recombinant shuttle plasmid into a host cell to obtain a recombinant adenovirus spontaneously generating virus-like particles (VLP). The recombinant adenovirus constructed by using the preparation method can spontaneously assemble VLPs containing virus outer membrane proteins in an in-vivo or in-vitro infection process, and thus the immunogenicity of a vaccine can be greatly improved, the VLP production process is simplified, and the production cost of the vaccine is reduced.

This invention discloses an engineered Trichoderma reesei strain that secretes and expresses lysostaphin, its construction method, and its applications, belonging to the field of genetic engineering technology. This invention obtains the lysostaphin proenzyme and its encoding gene by modifying lysostaphin with a secretory peptide. The encoding gene, promoter, and terminator are then ligated into a shuttle plasmid to obtain a lysostaphin expression plasmid. This plasmid is transformed into Trichoderma reesei, and the endogenous protein encoding gene cbh1, which has the highest secretion expression level in Trichoderma reesei, is replaced with the aforementioned encoding gene, thus constructing an engineered Trichoderma reesei strain that secretes and expresses lysostaphin. This invention is the first successful construction of an engineered Trichoderma reesei strain capable of secreting and expressing lysostaphin, which simplifies the production process of lysostaphin, significantly reduces its production cost, and lays the foundation for large-scale industrial production of lysostaphin.

This invention provides a recombinant plasmid for expressing the Bacillus subtilis resistance marker gene without antibiotic resistance, its construction method, and its application. This invention utilizes Bacillus subtilis SCK6 supercompetent cells to directly construct a Bsggt recombinant expression plasmid that does not contain the Ori replication origin site of Escherichia coli or the ampicillin resistance gene. This results in a smaller free plasmid expression system, providing a stable expression tool for heterologous proteins in production applications. The use of Cas9n-AID base editing technology in this invention simplifies the strain screening process. The constructed resistance marker-free expression plasmid is smaller than traditional shuttle plasmids, and no inducing agent is needed during expression. Because the expression plasmid is free of antibiotic resistance markers, the growth rate is significantly increased during culture due to the absence of antibiotic pressure. High expression without antibiotic introduction is more suitable for food safety-grade production applications.