136 results about "Alphabaculovirus" patented technology

The invention relates to a development method of an insect culture medium, which comprises the following steps: S1, constructing a double-promoter tandem vector by taking pFastBac plasmid as a skeleton; s2, establishing a baculovirus expression system; S21, transforming the double-promoter tandem vector into DH10Bac escherichia coli, so as to obtain recombinant Bacmid; s22, transfecting an Sf9 cell by using the recombinant Bacmid, so as to obtain a P0-generation baculovirus; s23, transfecting an Sf9 cell by using the P0-generation baculovirus to obtain a P1-generation baculovirus; s3, screening insect culture media: S31, providing culture media to be detected, and respectively inoculating Sf9 cells into the culture media to be detected; s32, respectively inoculating the P0-generation baculovirus or the P1-generation baculovirus into each to-be-detected culture medium for culturing; and S33, after culture is finished, detecting virus antibodies in the culture media to be detected, and screening the culture media. A comprehensive and quantitative evaluation system is established for development of the insect culture medium.

The invention discloses a method for preparing human metapneumovirus nucleocapsid protein based on an insect-baculovirus system. Comprising the following steps: 1) constructing a recombinant vector with a human metapneumovirus nucleocapsid protein coding sequence: sequentially connecting a sequence for promoting secretory expression, the human metapneumovirus nucleocapsid protein coding sequence and a tag sequence from 5'to 3 'ends in the recombinant vector; 2) converting the recombinant vector with the human metapneumovirus nucleocapsid protein coding sequence constructed in the step 1) into a competent cell containing a baculovirus genome plasmid Bacmid to obtain a recombinant baculovirus genome plasmid; and 3) transfecting insect cells with the recombinant baculovirus genome plasmid obtained in the step 2), culturing and purifying to obtain the human metapneumovirus nucleocapsid protein. The invention provides the preparation method of the human metapneumovirus nucleocapsid protein, and a material support is provided for subsequent further research.

The invention discloses chicken infectious anemia virus-like particles as well as a preparation method and application thereof. The invention discloses a virus-like particle vaccine for preventing chicken infectious anemia. The virus-like particle vaccine comprises VP1 and VP2 proteins of chicken infectious anemia viruses. Chicken infectious anemia VP1 and VP2 proteins are expressed by using a baculovirus expression system, immunoblotting shows that the two proteins are successfully expressed in sf9 cells, electron microscope observation finds that the expressed proteins can be autonomously assembled into complete chicken infectious anemia virus-like particles, and the chicken infectious anemia virus-like particles have a space structure similar to that of an original virus and can be used for preparing chicken infectious anemia virus-like particles. Meanwhile, the virus-like particles have the advantages of high titer, high safety, capability of stimulating humoral immunity and cellular immunity and the like. The preparation method disclosed by the invention is simple, can be used for preparing the antigen protein of the chicken infectious anemia virus on a large scale, is high in expression quantity and short in time consumption, greatly reduces the production cost, and is suitable for large-scale production.

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 relates to the technical field of oligosaccharide preparation through enzyme catalysis, in particular to a method for preparing a worm oligosaccharide product through cascade catalysis of glycosyltransferase. The method comprises the following steps: secreting and expressing truncated beta 1, 4-N-acetamino galactosyl transferase and alpha 1, 3-fucosyl transferase E by using an insect cell baculovirus expression system to obtain GalNAcT delta TM and FucT-E delta TM; gn [beta] GlyFmoc and Gn2Man3Gn2 [beta] AsnFmoc are used as substrates, and continuous catalysis is carried out, so as to prepare an oligosaccharide intermediate containing LDN epitopes and an oligosaccharide product containing LDN-F epitopes. The method solves the problem of high production cost of an LDN-F epitope-containing oligosaccharide product.

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.

The invention relates to a baculoviral vector system for improved delivery of DNA, RNA and protein into mammalian cells, including primary cells, tissues, and whole mammalian organisms.

Provided are a nucleic acid construct for improving production of adeno-associated virus and a method for constructing the same, the nucleic acid construct comprising: an adeno-associated virus (AAV) element, a polynucleotide encoding an IE protein; the AAV element comprising a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis-acting element. The method for constructing comprises integrating the AAV element carrying an exogenous target gene and the polynucleotide encoding the IE protein into a baculovirus vector skeleton. The recombinant adeno-associated virus (rAAV) obtained by the method has a lower empty shell rate, while improving the rAAV production of a single cell and a unit volume of culture, reducing the production cost, and being easy to scale up.

The present invention relates to synthetic designs or chimeric proteins for pseudotyping baculovirus (Autographa californica nuclear polyhedrosis virus) with the rabies virus glycoprotein G (gG) on its surface (Bac::gG-FL), which can be used in anti-rabies vaccine formulations. The chimeric protein is designed from a gene cassette containing gene fragments of the ectodomain of the G glycoprotein of the Pasteur strain rabies virus, a linker of 7 amino acids (GGGGSGG), as well as transmembrane (TM) and cytoplasmic (CT) regions of the gp64 baculovirus protein, with the arrangement of the sequences in the designed gene cassette being shown in figure 1.

The present invention discloses a recombinant virus-like particle of Akabane disease virus and a preparation method thereof, and belongs to the field of biotechnology. The recombinant virus-like particle of Akabane disease virus is obtained by recombinantly expressing the structural protein Gn and the structural protein Gc of Akabane disease virus in an insect cell-baculovirus expression system and self-assembly; the amino acid sequence of the structural protein Gn is shown in SEQ ID NO.1; the amino acid sequence of the structural protein Gc is shown in SEQ ID NO.2. The present invention optimizes the genes encoding the structural proteins Gn and Gc according to the codon preference of insect cells, and successfully prepares safer AKAV virus-like particles through the baculovirus-insect cell expression system. The AKAV virus-like particles provided by the present invention have application potential in various fields such as AKAV vaccines and immunotherapy, and provide technical reserves for biosafety monitoring.

The application discloses a complex preparation containing bovine lactoferrin and lyticase and application thereof, and belongs to the technical field of biology. The preparation is composed of bovine lactoferrin and streptococcal lyticase, and both are prepared through a baculovirus-insect cell eukaryotic expression system. Researches show that the complex preparation has a significant synergistic antibacterial effect. The complex preparation not only can effectively inhibit a plurality of gram-positive bacteria, but also can overcome the limitation that single streptococcal lyticase has weak activity on gram-negative bacteria, and significantly enhances the inhibiting effect on escherichia coli and salmonella. Especially, the complex preparation still has clear in-vitro antibacterial activity on multi-drug resistant escherichia coli such as ciprofloxacin and doxycycline, thereby providing a new way for developing a new type of drug for resisting drug-resistant bacteria.

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 application discloses a recombinant FCV antigen and a construction method and application thereof. The construction method of the recombinant FCV antigen comprises the following steps: fusing a T cell epitope coding sequence of a non-structural protein NS7 of FCV and a coding sequence of a SpyTag peptide segment through a coding sequence of a linker, then cloning into a baculovirus transfer vector to obtain a recombinant plasmid, and finally integrating the T cell epitope coding sequence into Bacmid through Tn7 transposition to finally obtain the recombinant FCV antigen. The application selects NS7 as a core immunogen, guides the immune system to produce a high cellular immune response, and thus makes up for the deficiency of an existing vaccine in clearing intracellular viruses; meanwhile, a specific T cell epitope is selected in the sequence of NS7 as an immunogen, which can avoid the immunological escape caused by the variation degree of antigens among different strains and virus antigen drift, and thus provides broader protection.

The invention discloses an intestinal symbiotic bacterium for enhancing baculovirus resistance of spodoptera frugiperda and application thereof, and relates to the technical field of microorganisms, the intestinal symbiotic bacterium is derived from the midgut of the spodoptera frugiperda, is separated and purified and is subjected to 16S rDNA (ribosomal deoxyribonucleic acid) gene sequencing identification, the sequence length is 1397 bp, and the sequence homology with the known Glutamicibacter soli is 99.93%. The strain is preserved in Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No.34972. The invention provides intestinal symbiotic bacteria for enhancing baculovirus resistance of spodoptera frugiperda and application of the intestinal symbiotic bacteria, and provides an experimental basis for further revealing an interaction mechanism among a host, a pathogen and the symbiotic bacteria. And meanwhile, a technical basis is provided for realizing a comprehensive virus prevention and control strategy of targeted biological prevention and control and non-target beneficial insect protection of the lepidoptera pests.

The present application relates to a nucleic acid construct for an insect cell-baculovirus expression system and its application, and belongs to the field of genetic engineering. The present application provides a novel nucleic acid construct, a corresponding insect cell-baculovirus expression system and its application. Specifically, the present application constructs a chimeric promoter capable of efficiently driving the transcription of an exogenous gene by creatively combining a specific enhancer and a promoter and designing the order and direction thereof, and further combines an independent polh promoter to construct a double-promoter system for forming two independent transcription units. The double-promoter system can be placed in the same vector to efficiently drive the expression of the same target gene at different stages of baculovirus infection of insect cells, greatly enhancing the expression intensity and persistence of the target gene at the transcription level, and ultimately improving the yield of the target protein.

The invention discloses an insecticidal composition containing entomopathogenic virus and jasmonic acid methyl ester or jasmonic acid, and relates to the technical field of biological insecticide, the active ingredients of the insecticidal composition are entomopathogenic virus and jasmonic acid methyl ester or jasmonic acid, and the entomopathogenic virus is nuclear polyhedrosis virus of baculoviridae. The weight ratio of the virus to the jasmonic acid methyl ester or jasmonic acid is 1: (0.5-1000), and the insecticidal composition can be prepared into dosage forms such as a suspending agent, water dispersible granules, wettable powder and the like. The invention also provides a method for preventing and treating lepidoptera pests, which comprises the step of applying the entomopathogenic virus and methyl jasmonate or jasmonic acid to target pests or a living environment of the target pests in a combined manner, and the combined application is simultaneous application or successive interval application. The insecticidal composition has a synergistic effect on lepidoptera pests such as cotton bollworms and beet armyworms, can obviously reduce the food intake, accelerate the death of pests and improve the field control effect, and has a wide application prospect.

To provide an automated method of producing viral vectors, utilizing engineered viral vector-producing cell lines, or packaging cells, within a fully-enclosed cell engineering system.SOLUTION: A method for automated production of a viral vector, includes: introducing an engineered viral producer cell into a high-temperature chamber of a fully enclosed cell engineering system; transducing the engineered viral producer cell with a vector encoding a gene of interest to produce a transduced viral producer cell; expanding the transduced viral producer cell and producing the viral vector within the cell; transferring the expanded producer cell to a downstream processing module; and isolating the viral vector and purifying the viral vector, the above-described steps being performed in a closed and automated process. Exemplary viral vectors that can be produced include lentivirus vectors, adeno-associated virus vectors, baculovirus vectors and retrovirus vectors.SELECTED DRAWING: Figure 1

The present disclosure is related generally to systems and methods for high level expression of recombinant proteins from baculovirus in insect cells. In particular, the methods and systems described herein allow for high levels of baculovirus production in insect cells and / or high levels of protein production in insect cells using a chemically-defined, yeast lysate-free insect cell medium. The disclosure also relates to compositions and kits for culturing, transfecting, and / or producing recombinant protein in insect cells.

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 the technical field of prevention and control of agricultural and forestry pests, and discloses a synergistic protein ORF76 of insect baculovirus and application of the synergistic protein ORF76. According to the invention, a synergistic protein (ORF76) is obtained from an insect biocontrol resource, i.e., a Pinus fumosa nuclear polyhedrosis virus, and protein function verification shows that the protein has a remarkable synergistic effect on a fall webworm nuclear polyhedrosis virus, an apocheima cinerarius nuclear polyhedrosis virus and a Pinus fumosa nuclear polyhedrosis virus; the compound can be used as a synergistic factor to be added into insect viruses, and has important significance on prevention and control of major forestry pests (fall webworms, spring inchworm and smoke wing pine bees).

The invention relates to a primer pair for size distribution of baculovirus DNA fragments and a detection method. The primer pairs at least comprise any three pairs in the four groups of primer pairs; a forward primer and a reverse primer in each primer pair are respectively and specifically combined with a section shown as SEQ ID NO: 10 on DNA (Deoxyribonucleic Acid) of a baculovirus GP64 protein gene; the lengths of amplification products obtained by amplification of each group of primer pairs are respectively less than 100 bp, 100-299 bp, 300-499 bp and more than 500 bp. The method can be used for quantitatively analyzing the size distribution of baculovirus residual DNA fragments in biological products, and is beneficial to improving the process and improving the product quality. The primer pair can truly and accurately reflect the size and distribution of residual DNA and fragments of baculovirus, and has very high application value for quality control of gene therapy products, vaccines and protein products produced by an insect cell-baculovirus expression system.

The invention provides a universal baculovirus transfer vector and application thereof. The universal baculovirus transfer vector comprises the following elements: (a) a 3CD protease coding gene containing Q184L mutation; (b) a baculovirus homologous repeat region enhancer hr1; (c) a baculovirus late expression factor lev5 gene and an expression regulatory element thereof; (d) a polh-pSel promoter used for driving the expression of the P1 protein; (e) a target serotype enterovirus P1 protein; wherein the elements (a)-(d) constitute a general core skeleton, and the element (e) allows insertion or replacement of a specific P1 protein gene. The 3CD stability is remarkably enhanced, the VLP yield and the assembling efficiency are greatly improved, the method has outstanding universality, and an efficient technical platform is provided for development of broad-spectrum enterovirus vaccines.

The present invention provides a kind of transfer vector, for Bac-to-Bac baculovirus expression system, relate to biotechnology and virology technology field, described transport vector includes 3 expression cassettes that are independently regulated and expressed by 3 promoters, can express multiple foreign proteins independently simultaneously, wherein promoter OP166 is early and late promoter, VP39 and P6.9 are late promoters, relative to commonly used promoters P10 and PH, expression time is advanced by 8 hours. Through experimental verification, transport vector p-BT has diversified selection for foreign protein expression mode, and the foreign protein expressed has biological activity, and virus-like particles (VLP) can be formed. Therefore, the expression of transport vector p-BT is more diversified, sequential, multi-quantity, large capacity, is conducive to being applied to multiple fields such as genetic engineering, drug development, vaccine production, expression of immunocompetent molecules and some oncogenic virus proteins and gene expression regulation research.

The use of queuine, queuosine or a salt or solvate thereof either: in cell-based or non-cell-based protein synthesis; as an additive to cell fermentation media; or as an additive to serum-free media for cell-based protein synthesis. The queuine, queuosine or salt or solvate thereof may be present in a concentration range from 0.01-200 micromolar. The cells may be selected from bacteria, yeast, baculovirus, insect, mammal or filamentous fungi cells. The cells may be proliferating. The use of queuine, queuosine or a salt or solvate thereof may increase the yield or quality of protein produced. The protein to be produced may be poorly expressed from cells in serum-free media. The protein to be produced may have a high AT content. The process may take 1-7 days. The cells may have been optimised to have elevated levels of the enzyme tRNA guanine transglycosylase (TGT).

This invention discloses a SARS-CoV-2 virus-like particle, its preparation method, and its application. The genes encoding the structural proteins of the novel coronavirus include the S gene, M gene, or E gene. The nucleotide sequence of the S gene is shown in SEQ ID NO.1; the nucleotide sequence of the M gene is shown in SEQ ID NO.2; and the nucleotide sequence of the E gene is shown in SEQ ID NO.3. This invention provides, for the first time, a virus-like particle (VLP), which is self-assembled after expression of the three structural proteins S, M, and E of the SARS-CoV-2 virus via a baculovirus-insect cell expression system. This invention solves the problem of the current lack of a safe and effective VLP vaccine for the prevention and control of COVID-19. The VLP is suitable for mucosal immunization, improving the immunization effect while reducing vaccination and labor costs, and has potential economic benefits.

The invention provides a gene with an antiviral effect and application thereof, the nucleotide sequence of an open reading frame of the gene is SEQ ID NO: 2, and the amino acid sequence of a coding protein of the gene is SEQ ID NO: 1. The invention also provides a method for increasing the sensitivity of the beet armyworm to the beet armyworm nuclear polyhedrosis virus, which is used for reducing the expression quantity of the gene in the beet armyworm. According to the invention, a gene with an antiviral effect is screened from an important agricultural pest beet armyworm. The sequence structure and tissue expression mode of the gene are analyzed, incremental expression and RNA interference technologies are utilized to prove that the gene has an anti-SeMNPV effect, a new target is provided for better utilizing baculovirus to prevent and control beet armyworms in production, and the gene has a good application prospect.

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.