16 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 CAV-VP1-P2A-VP2 recombinant adenovirus vector live vaccine and application of the CAV-VP1-P2A-VP2 recombinant adenovirus vector live vaccine, and belongs to the technical field of vaccines. According to the characteristics of a natural protective antigen of the chicken anemia virus, fragments beneficial to vaccine preparation are artificially screened, after 305aa-320aa and 78aa-84aa regions with low KU645520.1 protein antigenicity are deleted, a GGGGS connecting arm is used for connection to obtain the VP1-VP2 recombinant protein, and the VP1-VP2 recombinant protein only retains a high-antigenicity core region, can be more efficiently recognized by an immune system, and has a good application prospect. The immune response intensity is enhanced; and the cross-host characteristic of the shuttle plasmid is adapted. The virus titer level of the recombinant adenovirus vector live vaccine obtained on the basis is greater than or equal to 4.74 * 10 < 10 > pfu / mL, and the immune effect on the chicken anemia virus is outstanding.

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.

The invention discloses a recombinant adenovirus carrying a porcine circovirus type 3 Cap gene as well as a construction method and application thereof. The recombinant adenovirus is obtained by homologous recombination of a replication-defective adenovirus skeleton plasmid and a recombinant adenovirus shuttle plasmid carrying a target gene, a replication-defective adenovirus skeleton plasmid is pBHGlox [delta] E1, 3Cre; the target gene comprises one or more copied PCV3 Cap genes, the sequence of the PCV3 Cap genes is obtained through codon optimization and is as shown in SEQ ID NO.1, tPA signal peptide is fused at the N end of the PCV3 Cap genes, and the sequence of the target gene is as shown in SEQ ID NO.2. The vaccine prepared from the recombinant adenovirus not only can effectively promote mice to generate strong body fluid and cellular immunity, but also can induce the mice to generate high-level SIgA, thereby helping to resist the attack of the porcine circovirus type 3, and having a wide application prospect.

The invention discloses a recombinant chimpanzee oncolytic adenovirus Adsimian-delta24-IL21 and a construction method and application thereof, the vector is based on serotype 25 chimpanzee adenovirus, the immunogenicity is reduced by deleting an E3 region, and the integrin targeting is enhanced by inserting an RGD peptide fragment; a skeleton plasmid pAdsimian-deltaE3 is in seamless cloning connection with a shuttle plasmid pSRK17-delta24-CMV carrying delta24 mutation E1A, a recombinant oncolytic adenovirus Adsimian-delta24-IL21 and a control virus Adsimian-delta24-EGFP are constructed, the delta24 mutation enables the virus specificity to be copied in Rb defect tumor cells, the IL-21 gene remodels a tumor immune microenvironment by activating CD3 + / CD8 + T cells, and the tumor immune microenvironment of the Rb defect tumor cells is improved. The invention provides an efficient carrier and a method for oncolytic-immune combined treatment of colorectal cancer.

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 present application relates to a kind of recombinant coryneform bacteria and its method for fermenting production of spermidine with glucose as raw material.The present application first knocks out snaA gene in the genome of coryneform bacteria, constructs knockout strain SP-1.Spermidine transporter gene cluster, S-adenosylmethionine synthetase, spermidine synthase, ornithine decarboxylase, S-adenosylmethionine decarboxylase derived from Escherichia coli are expressed in previously constructed knockout strain SP-1 using shuttle plasmid pEC-XK99E and pXMJ19 plasmid between Escherichia coli and coryneform bacteria as expression vector.The recombinant coryneform bacteria of the present application can directly utilize glucose to directly produce spermidine.In the absence of exogenous methionine, 2.61g / L spermidine can be accumulated in 250mL shake flask fermentation for 72h.Spermidine yield can reach 45.2g / L in 5L fermentor by substrate feeding.

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.

The invention belongs to the technical field of gene engineering, and particularly discloses a pig pasteurella multocida gene editing vector based on CRISPR-Cas9 as well as a construction method and application of the pig pasteurella multocida gene editing vector based on CRISPR-Cas9, escherichia coli-pasteurella multocida shuttle plasmid pMI28 is taken as a skeleton, plasmids pCas9, psodc-tpia and the like are taken as templates, and pasteurella multocida conjugational transfer type gene editing plasmid pSCTcas9 is constructed; the CRISPR-Cas9 gene editing system constructed by the invention can efficiently construct the pasteurella multocida gene deletion strain, and compared with the traditional method, the operation is simpler and more convenient, the consumed time is shorter, the in-depth study on the gene function of the pasteurella multocida is facilitated, and a foundation is laid for revealing the pathogenic mechanism and the drug resistance mechanism of the pasteurella multocida; through targeted elimination of drug-resistant genes and direct sterilization, the drug resistance of the pasteurella multocida can be reduced, the sensitivity of the pasteurella multocida to antibiotics can be recovered, a new strategy is provided for clinical treatment of pasteurella multocida infection, and the current antibiotic resistance crisis can be relieved.

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.