292 results about "Shuttle plasmid" patented technology

The invention discloses an insect bioreactor capable of expressing multiple exogenous genes, and a construction method and application thereof. The construction method comprises the following steps: (1) introducing multicopy high-efficiency bacteria DNA (deoxyribonucleic acid) replication initiator into chitinase and cysteine proteinase genes of a baculovirus genome to obtain a baculovirus shuttle plasmid; (2) replacing virus duplicated essential gene downstream the polyhedrosis gene of the baculovirus shuttle plasmid with antibiotic gene to obtain a baculovirus plasmid DNA; and (3) replacingother virus duplicated and infected nonessential genes in the baculovirus shuttle plasmid with reverse selection marker gene to obtain the insect bioreactor. The antibiotic gene or reverse selection marker gene in the insect bioreactor which is constructed by replacing the exogenous target genes can express multiple exogenous genes in a host insect or insect cell. The insect bioreactor disclosed by the invention can efficiently expressing one or more exogenous genes in an insect body at the same time, and can produce massive recombinant proteins at low cost.

The invention provides an adenoviral vector system and a recombinant adenovirus construction method. The vector system comprises adenoviral plasmids pKAd5f11p-EF1aP and pKAd5f11pES-PmeI and shuttle plasmids pUC19-PM. The adenoviral plasmid contains an E1 / E3 deleted human adenovirus type 5 (HAdV-5) genome and the original HAdV-5 fiber gene is replaced by a fusion gene F5-11p of HAdV-5 and HAdV-11p.A PmeI site is an exogenous gene insertion site. Plasmids pKAd5f11p-EF1aP contain a human EF1a promoter in the original E1 region. The shuttle plasmids pUC19-PM are matched with the plasmids pKAd5f11pES-PmeI. The recombinant adenovirus construction method comprises: carrying out PCR amplification to obtain a desired gene fragment containing homologous overlapping regions on both sides and carrying out DNA assembling on the desired gene fragment and PmeI-linearized adenoviral plasmids to obtain adenovirus plasmids containing the desired exogenous gene, or cloning the multiple gene fragments tothe shuttle plasmids, shearing all the desired gene fragments through a restriction endonuclease and carrying out DNA assembling on the desired gene fragments and PmeI linearized pKAd5f11pES-PmeI toobtain the adenovirus plasmids containing the desired exogenous gene.

The invention relates to the field of biotechnology, and particularly relates to a shuttle plasmid vector, as well as a construction method and applications thereof. The shuttle plasmid vector can shuttle in yeast and Escherichia coli, and the nucleotide sequence of the shuttle plasmid vector is shown in SEQ ID NO.1. Compared with the prior art, he shuttle plasmid vector realizes the shuttling in yeast and bacteria, and has the capabilities of homologous recombination cloning, self replication and recombinant screening in yeast, as well as the characteristics of being stable in replication and maintenance and convenient for DNA purification and separation in bacteria.

The invention provides fowlpox virus vector shuttle plasmid pTGP3 which comprises recombinant arms TKL and TKR, a bidirectional promoter PE / L, a fluorescent protein expression cassette, and a resistant marker gene and replication origin ori; the upstream and the downstream of the bidirectional promoter PE / L are respectively provided with cloning sites MCSL and MCSR; and both ends of the fluorescent protein expression cassette are provided with loxp sequences. The plasmid of the invention has two different screening markers, and the recombinant fowlpox virus prepared with the plasmid can express 1 to 3 types of gene with different meshes in the whole processes of the early and the later periods; the strong composite promoter with expression activity in the early and the later periods is applied so as to realize the all-process high-efficiency expression of a target gene; and the loxp sequences are introduced into both ends of the fluorescent protein expression cassette, so as to knock out the exogenous recombinant fowlpox virus screening markers. The invention lays foundation for the series and the scale application of the recombinant fowlpox virus in vaccine and biological drug research and development fields.

The invention relates to a genetic engineering strain for increasing the yield of ascomycin and a constructing method thereof. The genetic engineering strain is streptomyces hygroscopicus var.ascomyceticus TD01, and is deposited in the China General Microbiological Culture Collection Center with an accession number of CGMCC 10615. By cloning fkbN and frr genes, an fkbN-frr double-gene tandem overexpression vector is constructed by utilization of escherichia coli-streptomycete shuttle plasmid pIB139 and is transformed into escherichia coli ET12567, and streptomyces hygroscopicus var.ascomyceticus is transformed by a conjugal transfer method to allow the overexpression vector to be expressed in an original strain. The yield of the ascomycin of the constructed ascomycin genetic engineering strain TD01 reaches 500 mg / L, and is increased by 42% than that of the original strain. The genetic engineering strain and the constructing method have high potential and application value.

The invention relates to a preparation method of a CAR-T cell targeting B7H3. The preparation method includes first preparing a PBMC cell; then co-transfecting a 293T cell with a shuttle plasmid LV-B7H3 containing the CAR structure, a helper plasmid psPAX2 and an envelope plasmid VSV-G to obtain a packaged B7H3-CAR virus; then taking a PBMC cell, using anti-human CD3 and anti-human CD28 as activators, culturing and activating for 48 hours and adding the B7H3-CAR virus for infection. By means of the preparation scheme, the expression of IFN-gamma in the CAR-T cell is increased, and the cell killing activity is high. The CAR-T cell targeting B7H3 has a killing effect on various solid tumor cells, has high killing activity, is safe and effective, and can be used for immunotherapy of kidney cancer, lung cancer, liver cancer, glioma, ovarian cancer, breast cancer and the like.

The invention discloses a preparation method of a porcine epidemic diarrhea recombinant adenovirus vaccine. The preparation method provided by the invention comprises the following steps of inserting a DNA sequence of a zone S1 of a porcine epidemic diarrhea virus (PEDV) into an adenovirus shuttle plasmid pShuttle-CMV to obtain pShuttle-CMV-S1, carrying out linearization of the pShuttle-CMV-S1, transforming the linear pShuttle-CMV-S1 into a BJ5183 competent cell containing pAdEasy-1, carrying out homologous recombination, carrying out enzyme digestion, carrying out AD-293 cell transfection, carrying out packaging to obtain a recombinant adenovirus rAd-S1, and carrying out purification, amplification and sub-packaging. After oral immunization, the porcine epidemic diarrhea recombinant adenovirus vaccine can induce generation of mucosal immunity thereby preventing porcine epidemic diarrhea (PED) well.

The invention relates to a replication defective recombinant adenovirus Ad41 vector system and an application thereof in the field of gene therapy and recombinant vaccines. Specifically, the vector system comprises a backbone plasmid, a shuttle plasmid and a packaging cell line, wherein, the backbone plasmid contains an adenovirus Ad41 genome which deletes an encoding region required for packaging adenovirus, the shuttle plasmid contains multiple cloning sites used for inserting a target gene and an adenovirus Ad41 genomic fragment used for carrying out homologous recombination with the backbone plasmid, and the packaging cell line integrates genes required for packaging adenovirus Ad41 in the cell genome and can stably express the packaging genes. The generated recombinant Ad41 virus has broad application prospects in intestinal gene therapy and research of oral recombinant vaccines.

The invention relates to an unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination. The method comprises the following steps: temperature sensitive-type shuttle plasmid pSET4E and knock-out plasmid containing homologous fragments at upstream and downstream parts of target genes to be knocked out are constructed, the knock-out plasmid is subjected to electrotransformation into pediococcus acidilactici, and single commutators generating homologous recombination for the first time and double-exchange mutant strains generating homologous recombination for the second time are screened and identified. The method disclosed by the invention realizes the unmarked gene knock-out of pediococcus acidilactici for the first time, the obtained knock-out bacterial strain does not carry any resistant gene, can be taken as a original strain for subsequent and reconstruction, and also can be used for large-scale industrial production in a safe mode. The method is used for respective knock-out of L-lactate dehydrogenase gene and d-lactate dehydrogenase gene of the pediococcus acidilactici DQ2 (a preservation number is CGMCC NO.7471), the obtained knock-out bacterial strains are respectively named as pediococcus acidilactici ZP26 and TY112, the preservation numbers are CGMCC NO.8665 and CGMCC NO.8664 respectively, and optically pure D-lactic acid and L-lactic acid are respectively generated.

The invention relates to a low-temperature alkaline protease and a preparation method thereof, belongs to a method of performing site-directed mutagenesis to a wild type alkaline protease gene by utilizing a recombinant DNA (deoxyribonucleic acid) technology to improve the characteristics of the gene, connecting the mutated gene with escherichia coli-bacillus subtilis shuttle plasmid pBE2S and expressing the mutated gene in the bacillus subtilis at high efficiency, and relates to a low-temperature alkaline protease with cold adaptability and alkali stability and a preparation method thereof. The invention solves the problem that the alkaline protease has so low activity in a low-temperature environment that the application is limited. The invention adopts a technical scheme that: the wildtype alkaline protease gene is separated from a microbe, in particular bacillus alcalophilus; amino acid residues of Glu110 and Glu134 of the gene are mutated; the enzyme activity of fermentation fluid of the gene is 1985 U / mL after the gene is expressed in the bacillus subtilis at high efficiency; at 40 DEG C, the activity of the low-temperature alkaline protease (Glu110 and 134 Ala) is improvedby 28 percent, compared with that of the wild type alkaline protease; and at 10 DEG C, the activity is improved by 62 percent, compared with that of the wild type alkaline protease.

The invention provides an electrotransformation method for introducing shuttle plasmid into corynebacterium acetoacidophilum. The method is mainly used for rapidly and effectively introducing shuttle-expression recombinant plasmid in escherichia coli and corynebacterium acetoacidophilum into corynebacterium acetoacidophilum through electrotransformation. Through controlling composition of a culture medium, cell culture time, ampicillin processing time, methylation of the plasmid, heat shock time and temperature and electric shock conditions, a high efficiency electrotransformation competent cell is prepared, a blocking effect of the cell wall of corynebacterium acetoacidophilum on the shuttle plasmid is weakened, restriction enzyme digestion of corynebacterium acetoacidophilum on the shuttle plasmid is reduced, so the shuttle plasmid can be highly efficiently introduced into corynebacterium acetoacidophilum through electrotransformation, and electrotransformation efficiency reaches 6.4 * 10<4> cfu / mu g DNA.

The invention discloses a genetically engineered bacterium capable of increasing the yield of spinosads as well as a construction method and an application. The construction method of the genetically engineered bacterium capable of increasing the yield of spinosads comprises the following steps: (1) inserting an spnk expression cassette into an escherichia coli-saccharopolyspora spinosa shuttle plasmid pOJ260 to construct a recombinant plasmid pLU101; (2) guiding the recombinant plasmid pLU101 into saccharopolyspora spinosa ATCC49460, and integrating the recombinant plasmid with a genome through homologous recombination, thus obtaining the genetically engineered bacterium capable of increasing the yield of spinosads and naming the genetically engineered bacterium LU101. The genetically engineered bacterium constructed by the method can increase the yield of spinosads.

The invention provides a preparation method of avian influenza virus HA gene recombinant adenovirus, which creatively comprises the following steps: carrying out a series of intermediate processes on plasmid pCAGGS, adenovirus shuttle plasmid pShuttle, adenovirus framework plasmid pAdEasy-1 and the like to obtain a gene expression plasmid and other intermediate products, and transfecting the obtained recombinant adenovirus plasmid with 293 cell; and carrying out immunohistochemical screening on the recombinant virus according to the adenovirus-infected cytopathy and specific cells. By using the CAG as the promoter to express the target gene, the method obviously enhances the expression level of the target gene. The hemagglutinin recombinant adenovirus for respectively expressing H5N1 and H9N2 subtype avian influenza viruses provides a virus model for development of the H5 / H9 subtype avian influenza virus bivalent nucleic acid vaccine, and also lays the foundation for development of the AIV (avian influenza virus) adenovirus live vector vaccine.

The invention discloses a preparation method and system of recombinant adeno-associated virus (rAAV), and recombinant bacmid. The method comprises: (1) separately preparing shuttle plasmids and a corresponding recombinant bacmid containing baculovirus genome; (2) integrating a rAAV core expression element which has a heterologous functional gene fragment with other expression cassettes which produce functional protein components necessary for rAAV so as to obtain a recombinant bacmid containing recombinant baculovirus genome producing the rAAV; and (3) transfecting the obtained recombinant bacmid into a host cell line for culturing. The system comprises the shuttle plasmids and the corresponding recombinant bacmid containing the baculovirus genome. The recombinant bacmid comprises at leastone expression cassette that produces functional protein components necessary for rAAV. The system has flexibility, compatibility, and higher passage stability.

By selecting a high-expression promoter, a shuttle plasmid capable of stably expressing green fluorescent protein and red fluorescent protein is constructed, so that various staphylococcus aureus bacterial strains which can be used for stably expressing fluorescent protein at a high level are obtained. The conventional dye labeling is substituted, so that real-time dynamic observation can be realized for observing and dynamically researching biological behaviors of staphylococcus aureus in a biological film formation process, a cell infection process and animal infection process in real time. Fluorescent labels in different colors can be used for effectively analyzing interaction among different bacterial strains, so that technical support is provided for researching the similarities and differences of different staphylococcus aureus in the biological film formation process and a host interaction process.

The invention belongs to the technical field of vaccine manufacturing. The invention discloses a recombinant baculovirus rBac-[delta]Cap expressing porcine circovirus type 3 truncated Cap protein, with the accession number of the recombinant baculovirus being CGMCC No. 15691. The invention also discloses primers used for constructing the recombinant baculovirus, and a constructing method of the recombinant baculovirus. The method comprises the following steps: 1, constructing pSK-SPCV3 containing PCV3 whole genome; 2. Constructing a transfer vector; 3. constructing a shuttle plasmid; 4. obtaining recombinant baculovirus, wherein the positive shuttle plasmid Bacmid-[delta]Cap transfects Sf9 cells to obtain the recombinant baculovirus rBac-[delta]Cap expressing the porcine circovirus type 3truncated Cap protein. The method can conveniently and quickly construct the recombinant baculovirus expressing the PCV3 truncated Cap protein, and can confirm that the expressed truncated Cap proteinhas good biological activity, and immunizes mice to find out that the recombinant baculovirus has good immunogenicity.

The invention discloses a Zika virus vaccine based on a replication-defective recombinant adenovirus vector. A nucleotide sequence of a JE signal peptide, and an optimized nucleotide sequence encodingZika virus membrane protein prM and envelope glycoprotein E are cloned into a shuttle plasmid vector pShutle2-CMV-Flag to obtain a recombinant shuttle plasmid, so that the expression of foreign proteins is significantly increased, and at the same time immunogenicity of an antigen is improved. Meanwhile, a recombinant adenovirus expression vector SAd23-L is used to escape pre-existing immune responses against common adenovirus vectors, and the humoral and cellular immunity with a higher level can be induced to generate in animals. After the recombinant adenovirus as a Zika virus vaccine is used to immunize animals, the humoral and cellular response to Zika virus is quickly induced to generate, especially a neutralizing antibody with a high level is induced to generate, and the specific cellular response to Zika virus antigen M and E protein is induced to generate in mice. Therefore, the Zika virus vaccine can be used to prevent large-scale outbreak and epidemic of Zika virus.

The invention relates to plication-deficient recombinant adenovirus which contains the expression sequence of siGNA of the gene APE1 of human being. The sequence of siGNA of the gene APE1 of human being at which the two ends are provided with restriction site is chemically synthesized and inserted under the prompter U6 of the shuttle plasmid of adenovirus after restriction enzyme, then shuttle plasmid pDC316-EGFP-U6-APE1siRNA is constructed, and then shuttle plasmid pDC316-EGFP-U6-APE1siRNA and the bone plasmid of adenovirus pBHG-fiber5 / 35 are simultaneously cotransfected into cell 293, consequently the recombinant adenovirus Ad5 / F35-APE1siRNA with the expression sequence of siGNA of the gene APE1 of human being is achieved through the fixed-point recombination produced by the system function of Cre / loxP. The recombinant adenovirus is capable of effectively preventing the expression of the gene APE1 and effectively strengthening the sensibility of radiotheraphy and chemotherapy of tumour cell.

The invention discloses an improved recombinant bacillus calmetter Guerin (BCG), belonging to the technical field of new medicaments. The immune protecting effect of a unique anti-tuberculosis vaccine BCG for tuberculosis is not exact and the tuberculosis morbidity gradually rises over the past 10 years. Therefore, the development of a more effective anti-tuberculosis vaccine is important. Gene sequences of mycobacterium tuberculosis ESAT6 and Ag85A are inserted into shuttle plasmids of colon bacillus-mycobacterium tuberculosis for forming recombinant plasmids; and the recombinant plasmids are converted into the BCG to form a recombinant anti-tuberculosis vaccine. Proved by research, the recombinant BCG is used for expressing introduced foreign genes and is a novel anti-tuberculosis vaccine.

The invention provides a recombinant fowl adenovirus FAdV-4 vector system. The recombinant fowl adenovirus FAdV-4 vector system comprises a framework plasmid pKFAV4, a middle plasmid pKFAV4AP and a shuttle plasmid pKFAV4APNM. A target gene coding region is cloned to a multiple cloning site of the shuttle plasmid, NheI digestion is carried out on the shuttle plasmid carrying a target gene expression cassette, and the obtained fragment replaces the corresponding part of the middle plasmid; AvrII or PacI digestion is carried out on the middle plasmid carrying a target gene, the obtained fragmentreplaces the corresponding part of the framework plasmid by virtue of the ligation reaction or the DNA assembly reaction, and then a recombinant adenovirus plasmid is obtained; a PmeI linearized adenovirus plasmid is used for transfecting LMH cells, and the recombinant FAdV-4 adenovirus realizing control over the expression of the target gene by virtue of a human cytomegalovirus promoter (CMVp) can be prepared. In the prepared recombinant adenovirus, the Orf1-Orf2 regions of the FAdV-4 genome are replaced by the target gene expression cassette, and the other regions of the adenovirus are reserved. The recombinant fowl adenovirus FAdV-4 vector system is expected to have the broad application prospect in the research and development of oral vaccines for birds.

The invention relates to a method for constructing highly expressed trehalose synthase engineering bacteria by using a Pcry3Aa promoter. For a recombinant carrier, a Pcry3Aa-PhoD fragment by which a Pcry3Aa promoter fragment and a PhoD signal peptide fragment are connected by an overlap PCR (Polymerase Chain Reaction) is inserted at the upstream of a restriction enzyme cutting site BamHI of a shuttle plasmid PHT01, and a target protein trehalose synthase TreS fragment is inserted between two restriction enzyme cutting site, i.e., BamHI and AatII. The invention further relates to a method for constructing the highly expressed trehalose synthase engineering bacteria by using the recombinant carrier. According to the method disclosed by the invention, the Pcry3Aa promoter is adopted to naturally induce the synthesis of trehalose synthase; because the Pcry3Aa promoter contains a special STAB-SD structure, the stability of the Pcry3Aa promoter to transcribe mRNA is improved, the half-life period of mRNA is prolonged, the mRNA translation level of a downstream target gene is improved, and therefore the trehalose synthase is highly expressed.

The invention discloses a transgenic mesenchymal stem cell which can treat radiation enteritis and a preparation method. The CXCR4 protein can be expressed highly on the surface of transgenic mesenchymal stem cell; under the chemotaxis of stromal cell derived factor which is released from the injured intestine, the homing and colonization of a large amount of transgenic mesenchymal stem cells can promote proliferation and repair of the intestinal mucosa and inhibit apoptosis; the radiation enteritis is cured. The steps of the preparation method of the transgenic mesenchymal stem cell are that : (1)the separation and cultivation of mesenchymal stem cell; (2) the preparation of CXCR4 recombinant adenovirus: full length cDNA of CXCR4 is amplified by RT-PCR; the construction of CXCR4 recombinant shuttle plasmid and skeleton plasmid; the CXCR4 recombinant adenovirus is packaged, amplified, purified and determined by titer in HEK293T cell; (3) the transfect ion of mesenchymal stem cell by using CXCR4 recombinant adenovirus.

An embodiment of the invention discloses a pig circovirus III-type virus-like particle. A preparation method of the virus-like particle includes the steps: amplifying pig circovirus III-type Cap protein genes; constructing recombinant shuttle-plasmid pFB-Cap by the aid of the genes; constructing rB-Cap recombinant rod granules by the aid of the recombinant shuttle-plasmid pFB-Cap; transfecting therB-Cap recombinant rod granules into SF9 cells to obtain recombinant baculovirus rBV-PCV3 Cap expressing pig circovirus III-type Cap genes; enabling the recombinant baculovirus rBV-PCV3 Cap to infectHigh Five cells, and purifying the recombinant baculovirus rBV-PCV3 Cap infected by the High Five cells to obtain the pig circovirus III-type virus-like particl PCV3 VLP. A nucleotide sequence of theCap protein gene is as shown in SEQ ID NO.1. According to the particle, based on a baculovirus-insect cell expression system, preparation is implemented by the aid of High Five cell expression of serum-free suspension culture, and the PCV3 virus-like particle is acquired by combining sucrose cushion ultracentrifugation and sucrose density gradient centrifugation purification. The virus-like particle is good in immunogenicity and high in safety and has good development and application prospects.

Gene of an E.coli-Lactobacillus shuttle plasmid expressing and secreting beta-galactosidase is composed of the gene encoding beta-galactosidase and its upstream regulatory sequence containing SD sites. Experiments proves that the recombinant plasmid can be transformed into Lactococcus lactis, and highly express the non-fused beta-galactosidase in E.coli and Lactococcus lactis, which is applicable in the lactic acid bacteria strain expressing beta-galactosidaseion. The construction method includes the following steps: taking Lactobacillus Bulgaria subspecies genomic DNA as template to amplify and obtain the beta-galactosidase gene containing the upstream regulatory sequence, digesting the gene to conjugate with the dephosphorylated vector digested with the same two enzymes and transform E.coli, then screening the positive one to extract the recombinant for enzyme identification and sequencing the inserted sequence.

The invention discloses a recombinant adenovirus and tetravalent adenovirus vaccine and a preparation method thereof. The tetravalent recombinant adenovirus vaccine contains a recombinant type 3 adenovirus strain, a recombinant type 7 adenovirus strain, a recombinant type 14 adenovirus strain and a recombinant type 55 adenovirus strain. The preparation method disclosed by the invention comprises the following steps: preparing recombinant shuttle plasmids containing hexon gene segments, and performing in-bacteria homologous recombination with a recombinant human type 3 adenovirus strain, thereby obtaining a recombinant adenoviral genome in which the hexon gene segments are replaced by type 7, type 14 and type 55; transfecting cells, rescuing to obtain recombinant human type 3, 7, 14 and 55 recombinant adenoviruses with different main capsid protein-hexon proteins; purifying, mixing according to the same protein content, and inactivating by using beta-propiolactone, thereby obtaining the tetravalent adenovirus vaccine. The tetravalent adenovirus vaccine is capable of inducing neutralizing antibody responses to four types of serotype adenoviruses, and the neutralizing titer is 500-1000.

The invention discloses a method for simultaneously removing bacillus anthracis virulence megaplasmids pXO1 and pXO2. The invention provides a DNA molecule having a nucleotide sequence 1 shown in the sequence table. The invention also provides a recombinant vector, a transgenic cell line or recombinant bacteria containing the DNA molecule. The recombinant vector is prepared by inserting the DNA molecule between recognition sites HindIII and EcoRI of a shuttle plasmid. An experiment proves that according to a plasmid incompatibility principle, an incompatiple plasmid is constructed and a strain A16QT which does not contain virulence megaplasmids is obtained. The method has the characteristics of simpleness, speediness, good singularity and high safety. The method has a very important meaning for construction of a plasmid-less bacillus anthracis strain and a research on the interaction between chromosome and megaplasmids pXO1 and pXO2, provides a novel experimental means for construction of a novel vaccine, and provides a novel idea for preventing and controlling bacillus anthracis.

The invention discloses a preparation method of a novel coronavirus pneumonia bivalent vaccine. The preparation method comprises the following steps: amplifying shRNA of a 2019-nCoV targeted interfering gene, performing enzyme digestion on the PCR product, constructing an interfering vector pSilencer-shRNA, performing DH5a transformation, constructing a shuttle vector pDC312-shRNA, co-transfectingHEK293 by the shuttle vector pDC312-shRNA and adenovirus framework plasmid pBHGloxAEl to prepare Ad-shRNA, and then preparing Ad-nCoVdsRNA. The preparation method further comprises the following steps: amplifying a 2019-nCoV antibody expression gene, performing enzyme digestion on the PCR product, constructing a shuttle plasmid pShuttle-nCoV, performing DH5a transformation, performing PCR amplification, co-transfecting HEK293 by the amplified shuttle plasmid pShuttle-nCoV and adenovirus skeleton plasmid pAd-nCoV to prepare a recombinant adenovirus Ad-nCoV, and further preparing Ad-nCoVDNA, and then preparing the bivalent vaccine by using the Ad-nCoVds RNA, the Ad-nCoVDNA and H20 according to a ratio of 1:1:19. After spray inoculation through a respiratory tract, the adenovirus (Ad) introduces nCoVds RNA and nCoVDNA into the cells.

The present invention discloses one attenuated vaccinia virus Tiantan plant carrier and its preparation process and application, and the attenuated vaccinia virus is high attenuated TT.WHU.ZCI, CCTCC: V200417. The preparation process includes the following steps: A. constituting shuttle plasmid containing flanking sequence homogenous with the coding region sequence or coding region lateral sequences of the vaccinia virus Tiantan plant HA gene, or containing flanking sequence homogenous with the promoter sequence or lateral sequences of the vaccinia virus Tiantan plant HA gene; and B. homogenously recombining the shuttle plasmid and the vaccinia virus Tiantan plant to deactivate vaccinia virus HA gene part for high attenuating the vaccinia virus Tiantan plant. The attenuated vaccinia virus Tiantan plant carrier is used in preparing vaccine for treating or preventing infectious diseases.

The invention discloses a shuttle plasmid and a derivative plasmid thereof. A nucleotide sequence of the shuttle plasmid which is provided by the invention is a sequence 1 in a sequence table and a 4872-5664th deoxyribonucleotide from 5'end of the sequence 1 in the sequence table is a kanamycin resistance gene and a promoter thereof. The invention also discloses the derivative plasmid thereof, which is obtained through inverting the kanamycin resistance gene of the 4872-5664th deoxyribonucleotide from the 5'end of the sequence 1 in the sequence table and the promoter of the kanamycin resistance gene and through inserting transcription terminators of a reporter gene and a corynebacterium glutamate LeuB gene in multiple cloning sites, the direction of the reporter gene is opposite to the direction of the kanamycin resistance gene and the promoter of the kanamycin resistance gene, and the transcription terminators of the corynebacterium glutamate LeuB gene are located between the reporter gene and the kanamycin resistance gene. The two plasmids have high stability and can obtain wide application on stic engineering.