34 results about "Recombinant baculovirus" patented technology

The invention discloses a swine fever and porcine parvovirus bivalent subunit vaccine and a preparation method thereof. The vaccine comprises a first recombinant protein encoded by a first gene, a second recombinant protein encoded by a second gene and a pharmaceutically acceptable carrier. The first gene has a sequence as shown in SEQ ID NO: 1 or an increased or reduced sequence thereof. And the second gene has a sequence as shown in SEQ ID NO: 2 or an increased or reduced sequence thereof. An antigen E2 protein of a hog cholera virus (CSFV) and a VP2 protein of a porcine parvovirus (PPV) are taken as double targets, a recombinant SC-E2 protein with a SpyCatcher tag and a recombinant ST-VP2 protein with a SpyTag tag are respectively expressed in insect cells through a recombinant baculovirus vector, double-antigen covalent assembly is realized in vitro, and the constructed bivalent subunit vaccine can be used for simultaneously preventing and controlling two epidemic diseases and has a good application prospect. And the vaccine has the advantages of high safety, strong immunogenicity, high prevention and control efficiency, easiness in large-scale production and the like.

The invention discloses a type 1 goose astrovirus virus-like particle and a preparation method and a pharmaceutical composition thereof, and the preparation method comprises the following steps: cloning a gene segment for coding type 1 goose astrovirus ORF2 protein to a baculovirus transfer vector, and constructing to obtain a recombinant plasmid; transforming the recombinant plasmid into a DH10Bac competent cell to obtain a recombinant baculovirus rod grain; transfecting the recombinant baculovirus stem into an insect baculovirus expression system to obtain a recombinant baculovirus; and inoculating the recombinant baculovirus into an insect baculovirus expression system, culturing, collecting a cell culture, and purifying to obtain the purified goose astrovirus type 1 virus-like particles. The GAstV-1 VLPs and the vaccine thereof provided by the invention not only solve the technical problem of research and development of the GAstV-1 vaccine, but also have the advantages of clear structure, high safety, good immunogenicity and exact protective efficacy, and have remarkable creativity and industrial application value.

To provide a method for stably and efficiently producing a protein in secretory production of a target protein using BEVS.SOLUTION: The present invention also provides a method for producing a fusion protein, comprising the steps of expressing the fusion protein in cultured insect cells using a recombinant baculovirus comprising a polynucleotide encoding the fusion protein, and recovering the fusion protein from the culture supernatant, wherein the fusion protein is derived from silkworm H1N1 influenza virus hemagglutinin or silkworm osteonectin.SELECTED DRAWING: None

The invention belongs to the technical field of bioengineering and the field of gene therapy. The invention discloses a baculovirus expression vector for optimizing Rep expression yield, proportion and time. Recombinant adeno-associated viruses with extremely low empty shell rate can be produced in insect cells. A baculovirus early promoter with corresponding strength is selected, and Rep78 / 68 and Rep52 / 40 are expressed at proper strength and proper proportion in the early stage of virus infection (2A self-cleavage polypeptide is used to adjust the promoter strength or virus infection titer). After the Rep expression cassette and AAV2 Cap controlled by a p10 promoter are jointly expressed in insect cells, rAAV particles with the vacant shell rate smaller than 1% can be obtained only by conducting one-step affinity chromatography on a cell lysis solution. Through testing, the strategy can be popularized to AAVs of other serotypes. The low-intensity promoter is used for driving the Rep, so that cell resources are saved, the yield of effective rAAV is remarkably improved due to reduction of the vacant shell rate, meanwhile, the step of removing vacant shell particles can be omitted in the production process due to the ultralow vacant shell rate, and finally the production cost of the rAAV is greatly reduced.

The invention relates to a method for improving PCV2 recombinant baculovirus protein expression, and belongs to the technical field of recombinant virus protein preparation. In order to solve the problem of low expression quantity of the existing PCV2 recombinant virus protein, the invention provides a method for efficiently expressing the PCV2 recombinant virus protein, the method comprises the step of carrying out H5 cell suspension culture for preparation, and we find that when glucose is lower than 5g / L in the culture process, glucose is added to 5-8g / L, glutamine is supplemented to 3-5mM, and beta-mercaptoethanol is added to 0.01-0.1 mM, the expression quantity of the PCV2 recombinant virus protein is increased, and the expression quantity of the PCV2 recombinant virus protein is increased. The expression quantity of the target protein can be obviously increased, and the expression quantity reaches 240 g / L or above. The method is simple in process, condition parameters are easy to control, cell culture is extremely easy to amplify, and the effects of cell culture, virus passage and target protein expression can be achieved without changing liquid in the cell culture period after virus inoculation.

The application discloses a bovine coronavirus virus-like particle, a preparation method and application, and the bovine coronavirus virus-like particle is assembled after co-infection of five kinds of recombinant baculoviruses capable of stably secreting bovine coronavirus E, M, N, S and HE proteins, and contains complete bovine coronavirus E, M, N, S and HE proteins, and BCoV VLPs similar to natural BCoV virus particles in morphology and size are constructed; after bovine coronavirus virus-like particles obtained by the application are used for immunizing mice and calves, high-titer antibody levels can be generated, and cellular immunity of the body can be stimulated.

The invention relates to a method for producing a recombinant baculovirus comprising n exogenous genes in an insect cell, by means of homologous recombination of a replication-deficient baculovirus genome and n transfer vectors, each comprising one of the n exogenous genes, n being an integer at least equal to 2.

The invention belongs to the technical field of biology, and discloses a recombinant turkey herpesvirus, and a tandem epitope expression cassette is inserted between UL45 and UL46 of the recombinant turkey herpesvirus; the tandem epitope expression cassette is used for expressing a plurality of B cell epitopes and a plurality of T cell epitopes. According to the recombinant herpesvirus of turkeys, a tandem epitope is inserted into an HVT (herpesvirus of turkeys) vector, and experiments prove that the recombinant herpesvirus of turkeys can induce higher HI antibody and neutralizing antibody titer, obviously stimulate spleen T lymphocyte response of immunized chicken, and can generate an obvious challenge protection effect in 3 days; the challenge protection effect is obviously superior to that of a combined immunization scheme of H9N2 AIV multi-epitope recombinant baculovirus (BV-BNT) and InV provided by the applicant; meanwhile, the recombinant turkey herpesvirus is verified to be capable of performing intraembryonic vaccination and generating an obvious immune protection effect. Meanwhile, the invention further provides a preparation method and application of the recombinant turkey herpesvirus.

The invention belongs to the field of gene engineering, and particularly discloses a recombinant baculovirus vector and an insect cell. The recombinant baculovirus vector comprises a nucleic acid molecule with an exogenous gene expression cassette, the exogenous gene expression cassette is located between a CNE sequence and an NAE sequence of the nucleic acid molecule, and the distance between the exogenous gene expression cassette and the CNE sequence and the distance between the exogenous gene expression cassette and the NAE sequence are both smaller than 5 kb; and the distance between the CNE sequence and the NAE sequence is greater than 1.5 kb. According to the application, the technical scheme that the exogenous gene expression cassette is arranged between the CNE sequence and the NAE sequence of the nucleic acid molecule is adopted, so that the production stability of the exogenous protein or the rAAV vector is better and the yield is higher in continuous passage.

A baculovirus vector and a use thereof in the preparation of a recombinant adeno-associated virus (rAAV) in an insect cell are provided. The baculovirus vector includes an exogenous gene expression cassette and a stable sequence. The stable sequence is located at a site 5 kb or less from the exogenous gene expression cassette, and the stable sequence is a conserved noncoding element (CNE) sequence or a nucleocapsid assembly-essential element (NAE) sequence. When an insect cell is infected with a recombinant baculovirus (rBV) constructed in this way, after multiple continuous passages, production levels of the rBV and the rAAV still remain relatively stable.

A virus-like particle (VLP) includes: an extracellular domain of a hemagglutinin having an amino acid sequence, or the extracellular domain of a spike protein; a matrix protein 1 having an amino acid sequence; and a transmembrane-cytoplasmic domain of the hemagglutinin. A method for preparing the VLP includes: constructing a recombinant plasmid; generating a recombinant bacmid by transformating the recombinant plasmid into a competent cell; generating a recombinant baculovirus by transfecting the recombinant bacmid into a first cell; and harvesting the VLP by transfecting the recombinant baculovirus into a second cell. Also provided is the use of the VLP in preparing a vaccine.

The invention discloses a micropterus salmoides rhabdovirus gene engineering vaccine preparation method, which comprises: S1, recombinant plasmid construction: carrying out PCR amplification on a micropterus salmoides rhabdovirus G protein gene with a 6 * His tag, cloning the amplified gene between BamH I and Hind III restriction enzyme cutting sites of a pVL1393 vector to obtain a pVL1393-G-His recombinant plasmid, S2, preparing a transfection system, and S3, carrying out purification on the transfection system to obtain the micropterus salmoides rhabdovirus gene engineering vaccine. S3, recombinant baculovirus preparation and identification, S31, cell transfection, S32, P1-generation virus harvesting, and S33, virus identification, the G protein of the micropterus salmoides rhabdovirus (MSRV) is directionally expressed through a genetic engineering technology, a large number of pathogenic viruses do not need to be cultured, the biological safety risk is greatly reduced, meanwhile, the defect that a prokaryotic expression system lacks protein post-translational modification is overcome, and the method is suitable for industrial production. The sf9 insect cell is used as an expression host, so that the recombinant G protein can be correctly folded to form a functional structure domain, and the immunogenicity is remarkably improved.

The invention provides a gene capable of influencing the insecticidal virulence effect of baculovirus and application of the gene, a beet armyworm SeCaspase-4 gene is obtained through screening, a genome bacmid of a recombinant virus carrying the gene is constructed, and a recombinant virus polyhedrosis is successfully obtained through cell transfection. A large amount of expression of the SeCaspase-4 gene can promote cell apoptosis in the baculovirus infection process, and can improve the insecticidal toxicity of the recombinant virus polyhedrosis to lepidoptera insects. Therefore, the recombinant virus is obtained by constructing the recombinant baculovirus bacmid carrying SeCaspase-4 and transfecting insect cells, and the insecticidal toxicity of the recombinant baculovirus on lepidoptera insects can be improved. The invention provides an effective way for improving the insecticidal toxicity of the baculovirus on the lepidoptera insects, and has important significance for efficiently utilizing the baculovirus to carry out biological control on the lepidoptera insects.

The invention discloses a fever with thrombocytopenia syndrome virus-like particle, a vaccine and a preparation method of the fever with thrombocytopenia syndrome virus-like particle. Based on SFTSV genome analysis, glycoproteins Gn and Gc and capsid protein NP are selected as core antigens, potential T cell epitopes of the Gn protein are predicted by utilizing bioinformatics, and the chimeric antigen Mosaic-Gn protein is designed. The optimized antigen gene is cloned to a baculovirus transfer vector, and three recombinant baculoviruses are constructed through a homologous recombination technology. The three recombinant viruses co-infect suspended insect cells, and the SFTSV virus-like particles self-assembled in the cells can be harvested after 72 hours. And mixing and emulsifying with an adjuvant to prepare the vaccine after ultra-filtration and ultra-separation concentration purification. The prepared virus-like particle vaccine can induce a body to generate a specific antibody when immunizing a BALB / c mouse, and has the advantages of strong immunogenicity, good safety, high hereditary stability and the like. And the vaccine is verified to have broad spectrum.

The invention discloses preparation of an akabane disease virus N protein monoclonal antibody and a double-antibody sandwich ELISA (Enzyme-Linked Immunosorbent Assay) detection method, in order to prepare the akabane disease virus nucleocapsid protein monoclonal antibody, an eukaryotic recombinant plasmid pFastBac-AKAV-N is constructed and is converted into a DH10bac competent cell, and a recombinant stem Bacmid-AKAV-N for expressing AKAV N is successfully obtained. After the recombinant baculovirus is transfected to an sf9 insect cell, the recombinant baculovirus capable of stably expressing the AKAV N protein is rescued. A mouse and a rabbit are immunized with the identified and purified recombinant N protein, and a monoclonal antibody and a polyclonal antibody are prepared respectively. The titer of the polyclonal antibody is measured through an ELISA method, and two positive cell strains are obtained through screening and are named as 2G4 and 6C7 respectively. Furthermore, the prepared polyclonal antibody is used as a coating antigen, an HRP-labeled 2G4 monoclonal antibody is used as a detection antibody, and a double-antibody sandwich ELISA detection method for AKAV N protein antigen detection is optimized and established. According to the method, the AKAV N antigen can be specifically recognized, and reliable technical support is provided for clinical antigen monitoring of AKAV. The monoclonal antibody preparation system and the double-antibody sandwich ELISA detection method established by the invention lay an important foundation for pathogen detection and epidemiological investigation of akabane disease.

The invention relates to a method for generating in a single step a library of recombinant baculoviruses for screening for a protein of interest, starting from a pooled-library of poly-nucleotides each encoding a different protein. Insect cells are infected with a pooled-library of baculoviruses at a very low multiplicity of infection (MOI) leading to the situation where the majority of cells are infected by a single baculovirus. The pool of cells is then treated with a viral inhibitor which prevents secreted baculoviruses from penetrating other cells without affecting, either the capacity of the cell to express the target protein, nor intracellular viral replication and accumulation.

A method for preparing protein-based nanoparticle for self-packaging and delivering mRNA includes the following steps. A first donor plasmid, a second donor plasmid and a third donor plasmid are provided. A plasmid transposing step is performed. A recombinant virus preparing step is performed so as to obtain a first recombinant baculovirus, a second recombinant baculovirus and a third recombinant baculovirus. A transducing step is performed, wherein the first recombinant baculovirus, the second recombinant baculovirus and the third recombinant baculovirus are used to infect a producer cell so as to express a nucleocapsid protein, an envelope protein, an engineered envelope protein and a target RNA, and the nucleocapsid protein, the envelope protein, the engineered envelope protein and the target RNA are self-assembled to form a protein-based nanoparticle for self-packaging and delivering mRNA.

The invention provides a stirring type reactor culture process of a porcine circovirus type 2 recombinant baculovirus, which comprises the following steps: preparing a seed virus of the porcine circovirus type 2 recombinant baculovirus by using an insect cell Sf9 full-suspension culture process, and inoculating an insect cell High Five by using the seed virus prepared by using an Sf9 cell to perform antigen expression of the porcine circovirus type 2 recombinant baculovirus. A full-suspension culture mode is utilized, so that the labor intensity is reduced, the production cost is reduced, and pollution is not easily caused; 2) the risk of porcine virus pollution can be reduced by using insect cells and a baculovirus expression system; and 3) the characteristics (Sf9 and High Five) of different insect cells are simultaneously utilized to respectively carry out seed virus preparation and antigen expression, so that higher virus titer can be obtained, the virus titer is greater than or equal to 7.50 Log (TCID50 / mL), higher recombinant protein expression is obtained, and the protein expression quantity is greater than or equal to 150 mu g / mL.

The invention belongs to the technical field of biology, and particularly relates to a recombinant baculovirus capable of simultaneously expressing gene type 1 and gene type 3 duck hepatitis A virus VP1 protein and application of the recombinant baculovirus in preparation of a duck hepatitis A divalent subunit vaccine. The recombinant baculovirus co-expressing the gene type 1 and gene type 3 duck hepatitis A virus VP1 protein is constructed, the virus titer is high, and the two proteins can be expressed. Therefore, the recombinant baculovirus provided by the invention can be used for research and development and epidemic disease prevention and control of novel vaccines for duck viral hepatitis A.

This invention relates to a botulinum toxin A1 precursor molecule, a nucleic acid molecule, an engineered recombinant baculovirus, a method for preparing the same, and a method for preparing natural botulinum toxin A using the precursor molecule. The precursor molecule comprises a light chain and a heavy chain, and a linker region connecting the light and heavy chains. The amino acid sequence of the precursor molecule is shown in SEQ ID NO. 2. The light chain is the amino acid sequence from position 1 to position 444 of natural botulinum toxin A, as shown in SEQ ID NO. 5. The heavy chain sequence is the amino acid sequence from position 449 to position 1296 of natural botulinum toxin A, as shown in SEQ ID NO. 6. The amino acid sequence of the linker region is shown in SEQ ID NO. 11. This invention provides a safe process, and the resulting recombinant botulinum toxin not only has a sequence identical to the active protein of natural botulinum toxin but also exhibits high activity, providing a low-cost, high-activity botulinum toxin product for clinical use.

The application belongs to the technical field of biology, and discloses virus-like particles of H5N6, H5N8 and H7N9 and purposes and vaccines thereof, wherein the virus-like particles of H5N6, H5N8 and H7N9 are obtained by jointly connecting HA, NA and M1 genes of H5N6, H5N8 and H7N9 to plasmids and then being transferred into competent cells to obtain recombinant baculovirus plasmids, and then the recombinant baculovirus plasmids are transfected and expressed to obtain corresponding virus-like particles, and in addition, the trivalent vaccine prepared by using the virus-like particles does not produce antagonism between different antigens.

The invention discloses a sheep omega interferon mutant, a recombinant vector as well as a preparation method and application of the recombinant vector. Based on the problems of expression efficiency and the like caused by poor secretion efficiency of the sheep omega interferon, a signal peptide mutant with an amino acid sequence shown as SEQ ID NO.3 is obtained by mutating a signal peptide of the sheep omega interferon, and the antiviral activity of the signal peptide mutant is obviously improved compared with that of the sheep omega interferon; the signal peptide mutant is further subjected to single-site, double-site or multi-site mutation to respectively obtain the mutant with improved antiviral activity. The invention also discloses a method for preparing the sheep omega interferon mutant, which comprises the following steps: obtaining recombinant baculovirus by using insect cells, and expressing the sheep omega interferon mutant by using an insect (cell) bioreactor infected by the virus; the antiviral activity of the expressed sheep omega interferon mutant is greatly improved. The sheep omega interferon mutant provided by the invention has an application prospect in preparation of drugs or reagents for preventing or treating sheep viral diseases.

The invention belongs to the technical field of biology, and discloses virus-like particles of H5N6, H5N8 and H7N9 and application and vaccine.The virus-like particles of H5N6, H5N8 and H7N9 are jointly connected to plasmids through HA, NA and M1 genes of H5N6, H5N8 and H7N9 and transferred into competent cells to obtain recombinant baculovirus plasmids, then the recombinant baculovirus plasmids are transfected and expressed to obtain the corresponding virus-like particles, and in addition, the virus-like particles of H5N6, H5N8 and H7N9 can be used for preparing the vaccine. The trivalent vaccines prepared from the virus-like particles do not generate antagonism among different antigens.

A virus-like particle (VLP) includes: an extracellular domain of a hemagglutinin having an amino acid sequence, or the extracellular domain of a spike protein; a matrix protein 1 having an amino acid sequence; and a transmembrane-cytoplasmic domain of the hemagglutinin. A method for preparing the VLP includes: constructing a recombinant plasmid; generating a recombinant bacmid by transformating the recombinant plasmid into a competent cell; generating a recombinant baculovirus by transfecting the recombinant bacmid into a first cell; and harvesting the VLP by transfecting the recombinant baculovirus into a second cell. Also provided is the use of the VLP in preparing a vaccine.

Disclosed herein are methods and uses for improving production of adeno-associated virus (AAV) using baculovirus systems. The recombinant baculovirus vector comprises an AAV capsid protein gene expression cassette (Cap), an AAV Rep gene expression cassette (Rep), and an AAV inverted terminal repeat (ITR) AD sequence (AD), which can be located after the Rep gene expression cassette, or between the Rep gene expression cassette and the Cap gene expression cassette, and after the Cap gene expression cassette. The level of baculovirus and AAV production in insect cells carrying the recombinant baculovirus comprising Cap, Rep, and AD sequence is higher than that of the control comprising Cap and Rep but without the AD sequence. The methods of the present invention are useful for producing recombinant adeno-associated virus (rAAV) and have high versatility and efficiency.

The present invention relates to an expression system for the extracellular production of recombinant tri-fucosylated N-glycoproteins in insect cells. The invention also relates to an expression cassette comprising a glyco-module comprising polynucleotide sequences of a nematode galactosyltransferase, fucosyltransferase, and mannosidase; and a polynucleotide sequence encoding one or more tri-fucosylated N-glycoproteins derived from a nematode; a recombinant baculovirus comprising said expression cassette, a method for producing said tri-fucosylated N-glycoproteins in transfected insect cells, and a transfected insect cell. The invention also relates to an immunological composition comprising said tri-fucosylated N-glycoproteins, which can be administered to ruminants to induce protective anti-nematode immunity.

The application discloses a CSFV-PPV bivalent subunit vaccine and a preparation method thereof. The vaccine comprises a first recombinant protein encoded by a first gene, a second recombinant protein encoded by a second gene and a pharmaceutically acceptable carrier. The first gene has a sequence shown in SEQ ID NO:1 or an increased or reduced sequence thereof. The second gene has a sequence shown in SEQ ID NO:2 or an increased or reduced sequence thereof. The application takes the antigen E2 protein of CSFV (CSFV) and the VP2 protein of PPV (PPV) as double targets, expresses the recombinant SC-E2 protein with a SpyCatcher label and the recombinant ST-VP2 protein with a SpyTag label in insect cells through a recombinant baculovirus vector, realizes in-vitro covalent assembly of the double antigens, and constructs the bivalent subunit vaccine which can simultaneously prevent and control two diseases, and has the advantages of high safety, strong immunogenicity, high prevention and control efficiency and easiness in large-scale production.

The invention discloses a function and an application of virus source tyrosine phosphatase in development of male testis of plutella xylostella, namely an application of a protein tyrosine phosphatase gene CvBV22-9 coded by cotesia vestalis virus in induction of apoptosis of testis cells of larvae of plutella xylostella. According to the invention, the CvBV22-9 gene is screened and identified as a main factor for inducing testis apoptosis, and the gene is continuously and highly expressed in a parasitized testis tissue and has tissue specificity. After the expression of the CvBV22-9 is knocked down, the testis apoptosis rate is obviously reduced; and about 90.80% of testis cell apoptosis can be induced by using recombinant baculovirus to over-express the gene. The low-concentration recombinant baculovirus can also destroy the testis structure and lead to decrease of the egg laying amount of mating female insects. According to the invention, a transgenic fruit fly model is successfully constructed, specific expression of CvBV22-9 in fruit fly testis is realized by using a UAS-Gal4 system, and 97.30% of testis apoptosis is induced.

An oral vaccine composition against porcine circovirus associated diseases, comprising the pupa or cell of a baculovirus infectious insect, which is subjected to an infection treatment with a recombinant baculovirus, into which porcine circovirus type 2 protein-encoding DNA is introduced, and a freeze-drying treatment.