29 results about "Shuttle plasmid" patented technology

The invention discloses an escherichia coli-bacillus subtilis shuttle plasmid and application, and belongs to the field of gene engineering. The shuttle plasmid comprises the following elements: a promoter sequence, a coding sequence of bacillus subtilis signal peptide, a replication initiation sequence of bacillus subtilis, a replication initiation sequence of escherichia coli and an antibiotic resistance gene. According to the plasmid design, a modular structure is adopted, and quick replacement of key elements such as a promoter, a signal peptide and RBS is supported through optimized enzyme cutting site layout; a 6 * His tag introduced at the tail end of a multiple cloning site (MCS) can be adapted to nickel column affinity chromatography, and the purification process of the recombinant protein is simplified. The plasmid has the characteristics of small molecular weight and convenience in operation, can be efficiently cloned and constructed in escherichia coli and then directly converted into bacillus subtilis for protein expression, is particularly suitable for development of a gram-positive bacterium secretory expression system, and provides an efficient tool for large-scale production of industrial enzyme preparations.

The invention discloses a streptomyces Targetron gene targeting vector as well as a construction method and application of the streptomyces Targetron gene targeting vector. According to the invention, an II-type intron Ll.LtrB from lactococcus lactis is cloned to an escherichia coli-streptomyces shuttle plasmid to construct a streptomyces genetic manipulation tool-vector pSC30 based on targeting of the II-type intron, and gene targeting in streptomyces coelicolor is successfully realized by using the tool. Screening a biosynthetic gene cluster of red pigment in streptomyces roseosporus; and activating a biosynthetic gene cluster of janus doxorubicin in streptomyces spinosus. According to the invention, the conversion rate and the gene targeting efficiency of the Ll.LtrB intron in streptomyces with high GC content are effectively improved, and a new tool is provided for construction of a gene mutation library.

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 recombinant adenovirus shuttle plasmid and a preparation method thereof, a recombinant adenovirus and an application thereof, and a Japanese encephalitis recombinant adenovirus plasmid, belonging to the field of biomedicine. The Japanese encephalitis recombinant adenovirus shuttle plasmid provided by the invention uses pShuttle as a carrier, carries a Japanese encephalitis virus wild-type E protein gene or a Japanese encephalitis virus E protein mutant gene, the 5' end of the Japanese encephalitis virus wild-type E protein gene or the Japanese encephalitis virus E protein mutant gene is connected to a signal peptide gene, the 5' end of the signal peptide gene is connected to a Kozak sequence, and the 5' end of the Kozak sequence is connected to a restriction site 1. The recombinant shuttle plasmid, recombinant plasmid, and recombinant adenovirus provided by the invention can be applied to the preparation of a Japanese encephalitis virus adenovirus vaccine, and have broad development and application prospects.

The invention relates to the technical field of gene engineering, in particular to akabane disease pseudovirus particles and a preparation method and application thereof.The akabane disease pseudovirus particles comprise recombinant lentiviral vectors and packaging plasmids, the recombinant lentiviral vectors comprise lentiviral shuttle plasmids and S whole-genome fragments of akabane disease viruses, the nucleotide sequence of the S whole-genome fragments is SEQ ID NO: 1, and the nucleotide sequence of the S whole-genome fragments is SEQ ID NO: 2. The preparation method comprises the following steps: (1) preparing a recombinant lentiviral vector; (2) preparing a mixture of a recombinant lentiviral vector and a transfection reagent; (3) culturing an HEK293T cell; (4) transfecting an HEK293T cell; and (5) obtaining the pseudovirus, the pseudovirus solution containing the AKAV S gene prepared by the invention can be used as a positive quality control product for AKAV nucleic acid detection, plays an effective monitoring role in the whole process from nucleic acid extraction to nucleic acid detection, and lays a foundation for preparing akabane disease nucleic acid diagnostic reagents.

The invention relates to the technical field of gene editing, in particular to a sphingomonas genome editing vector based on a CRISPR-Cas12a system, an editing method and application. According to the invention, an escherichia coli-sphingomonas shuttle plasmid pBRSL capable of realizing stable genetic expression in sphingomonas is constructed, and a library with 15 endogenous promoters is obtained through red fluorescence characterization; on the basis of a plasmid and a promoter, a CRISPR-Cas12a single plasmid, a sphingomonas genome editing vector based on a CRISPR-Cas12a system and an editing method are further constructed. The editing vector can be used for editing a sphingomonas genome, by utilizing the editing method disclosed by the invention, the genome editing time of a traditional double-exchange method can be shortened from 10-12 days to less than 3 days, and gene editing can be carried out through one-time electrotransfection of CRISPR-Cas12a single plasmids only by assembling a target site guide sequence and a proper repair homologous sequence; the short-time high-efficiency traceless editing of the sphingomonas is realized, the efficiency is high, the time and the cost can be saved, and the operation is simple and convenient.

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.

According to the double-plasmid packaging system for improving the AAV2 virus titer, the packaging system is co-transfection of an AAV producing cell and two plasmids, and the two plasmids are shuttle plasmids which are provided with two AAV ITRs, are respectively inserted with FRT sequences on two sides and are loaded with target genes; the invention relates to a helper plasmid which is provided with rep-cap, can express different serotypes, is inserted into a human miRNA (micro Ribonucleic Acid) sequence and a CAG-Flpo-wpre-pa sequence and provides a helper gene separated from an adenovirus. According to the system disclosed by the invention, the AAV2 virus with the titer of 1.0 * 10 < 10 > can be packaged by using a double-plasmid co-transfection system. The difficulty of plasmid cotransfection is reduced, and a product route is provided for commercial production of AAV2 virus packaging.

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.

An african swine fever virus vaccine includes five groups of antigens in total, and each group is respectively obtained by constructing recombinant adenovirus vectors co-expressing four antigen genes of african swine fever virus, and packaged by 293TD37 cells. The four antigenic genes of African swine fever virus in each group are 1, P72, B602L, P30 and P54; 2, CP129Rubiqutin, MGF5L6L, CP312R, and MGF110-4L; 3, L8Lubiqutin, I215L, I73RHBsAgHBsAg and E146L; 4, EP402R, EP153R, I177L, and K205Rubiqutin; 5, F317L, A151R, P34, and pp62. The construction of the recombinant adenovirus vector for co-expression of four antigen genes of the african swine fever virus mainly includes: knocking out E1, E3, E2a and E4 genes of the adenovirus vector by CRISPR / cas9 technology, constructing an ORF6 / 7 expression frame of E4 in an E2a region, and constructing shuttle plasmids in E1 and E4 regions for appropriately expressing four antigen genes, thereby obtaining a completely new adenovirus vector.

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.

A fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system Ad5MixPlus and an application thereof are provided. The system is composed of three adenovirus recombinant plasmids. The core technology of the system is that two sets of different site recombination sequences are skillfully loaded on a first 5-type adenovirus right arm backbone plasmid large vector, then two small shuttle plasmids respectively provide a right arm-modified Hexon / E3 / Fiber sequence and an E1a expression cassette controlled by a left arm tumor-specific promoter, and the difficulties and obstacles to the modification of the adenovirus backbone large vector are overcome. After two rounds of site-specific recombination, the ideal oncolytic adenovirus is packaged accurately and quickly.

The invention discloses a recombinant plasmid based on a nuclear localization signal and a PP7 RNA (Ribonucleic Acid) stem-loop structure, the recombinant plasmid comprises NLS-cas9-NLS + 6 * PP7 RNA, and the gene sequence of the NLS-cas9-NLS + 6 * PP7 RNA is as shown in SEQ ID NO. 2. The invention also discloses an application of the recombinant plasmid in VLP, exosome, lentivirus or AAV delivery vectors. The invention also discloses an efficient VLP carrier system, which comprises the recombinant plasmid, and also comprises a plasmid for fusion expression of PP7 bacteriophage capsid protein and gag structural protein, a plasmid for expression of membrane protein, an auxiliary plasmid, a shuttle plasmid carrying gRNA and an integrase mutated pMDLg-pRRE plasmid. The invention also discloses application of the vector system in treatment of clinical diseases. The vector system can improve the gene editing efficiency.

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 invention discloses a patchouli alcohol synthase mutant as well as a coding gene and application thereof, the amino acid sequence of the patchouli alcohol synthase is as shown in SEQ ID NO.1, and the patchouli alcohol synthase mutant is one of the following: leucine at the 410 site of the amino acid sequence as shown in SEQ ID NO.1 is mutated into alanine or isoleucine at the 451 site of the amino acid sequence as shown in SEQ ID NO.1 is mutated into alanine. According to the invention, pogostemon cablin alcohol synthase PcPTS sourced from pogostemon cablin is subjected to single amino acid site mutation, is loaded on a shuttle plasmid and is expressed in saccharomyces cerevisiae, and it is found that the ratio of the yield of alpha-guaiacol in fermentation products of recombinant bacteria to the yield of sesquiterpenoids total products is increased.

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 invention relates to the technical field of gene engineering, in particular to a gene medicine for treating arrhythmic cardiomyopathy caused by PNPLA2 mutation as well as a preparation method and application of the gene medicine. Effective components of the gene drug provided by the invention comprise shuttle plasmids; the shuttle plasmid comprises an AAV9 vector and a PNPLA2 gene. The PNPLA2 gene with normal function is delivered through the AAV9 carrier, lipid metabolism disorder caused by mutation is repaired, fat deposition in cardiac muscle is reduced, and then the pathological change of ACM is improved. By recovering the function of the PNPLA2 gene, the process of myocardial fibrosis can be effectively inhibited, and the cardiac function can be improved. The cardiac muscle specific promoter cTnT is adopted to ensure that the PNPLA2 gene is only expressed in cardiac muscle, so that the targeting property of gene therapy is improved, and side effects caused by systemic expression are avoided. The strategy can effectively reduce the side effects of gene therapy and ensure the accuracy and safety of therapy.

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 discloses a preparation method and application of recombinant adenovirus capable of 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 with 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 to a host cell to obtain the recombinant adenovirus capable of spontaneously generating virus-like particles (VLP). The recombinant adenovirus constructed by the preparation method can spontaneously assemble the VLP containing the virus outer membrane protein in the in-vivo and in-vitro infection process, so that the immunogenicity of the vaccine can be greatly improved, the VLP production process is simplified, and the production cost of the vaccine is reduced.

The invention relates to the technical field of oncolytic adenovirus construction, and discloses an oncolytic adenovirus construction method based on shRNA of an adenovirus-loaded c-Re l gene, and the method comprises the following steps: designing shRNA sequences of the c-Re l gene, and screening out a target shRNA sequence from the shRNA sequences according to the silencing effect of the shRNA sequences; the preparation method comprises the following steps: constructing a pDC315 plasmid, an hTERT promoter, a U6 promoter and a target shRNA sequence to obtain a shuttle plasmid; according to the shuttle plasmid, adenovirus packaging is carried out to obtain the oncolytic adenovirus of shRNA loaded with the c-Re 1 gene, due to the introduction of shRNA of the c-Re 1 gene of a myeloid immune checkpoint, tumor cells can be dissolved through the oncolytic adenovirus, and by knocking down the expression of the c-Re 1 gene in tumor-related myeloid cells, the purposes of changing the immunosuppression microenvironment and improving the immunosuppression efficiency are achieved. The anti-tumor immune response of an organism is remodeled, so that tumors are treated more effectively.

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.