17 results about "Recombinant baculovirus" patented technology

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 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.

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.

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 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.

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 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.

The present invention provides a method for preparing coronavirus-like particles and a vaccine comprising the same. The method comprises: constructing a recombinant baculovirus carrying a coronavirus spike protein gene; transducing mosquito cells with the recombinant baculovirus; and culturing transduced cells in serum-free medium to produce coronavirus-like particles, wherein the resulting coronavirus-like particles contain coronavirus spike protein. Furthermore, the transduced cells are preferably C6 / 36 cells, and the coronavirus spike protein gene may be from SARS-CoV, MERS-CoV, or SARS-CoV-2. Additionally, the present invention provides a vaccine containing the coronavirus-like particles prepared by the aforementioned method, which can be administered via intranasal or intramuscular routes.

The present invention provides for a production system based on baculovirus vectors for parvovirus vectors. Highly stable baculovirus clones were provided comprising parvovirus vector genomes. By reducing sequence identity between the first and second ITR, i.e. of the first ITR sequence to both the second ITR sequence, and the reverse complement of the second ITR sequence, stable baculovirus vectors are obtained. These allow for large scale production and high passage numbers, while substantially retaining parvoviral vector genome integrity, which is in particular advantageous for large scale clinical manufacturing.

This oral vaccine composition against feline viral infection virus comprises pupae or cells of a baculovirus-infectable insect that has been: infected with recombinant baculovirus into which DNA that encodes viral proteins causing feline viral infections has been introduced; and dried.