44 results about "Baculovirus expression" 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 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 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 discloses akabane disease virus recombinant virus-like particles and a preparation method thereof, and belongs to the technical field of biology. The akabane disease virus recombinant virus-like particles are obtained by recombinant expression of structural protein Gn and 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 as shown in SEQ ID NO. 1; the amino acid sequence of the structural protein Gc is as shown in SEQ ID NO. 2. According to the invention, genes of coding structural proteins Gn and Gc are optimized according to codon preference of insect cells, and AKAV virus-like particles with higher safety are successfully prepared through a baculovirus-insect cell expression system. The AKAV virus-like particle provided by the invention has application potential in various fields of AKAV vaccines, immunotherapy and the like, and provides technical reserve for biological safety monitoring.

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 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 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 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 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 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 application relates to an anti-apoptosis baculovirus expression vector, which realizes a broad-spectrum anti-apoptosis effect by expressing siRNA targeting a Sf-caspase-1 and Tn-caspase-1 common sequence of Spodoptera frugiperda and Trichoplusia ni insect cells through the vector. The baculovirus vector contains a specific DNA sequence, the DNA sequence contains an siRNA sequence targeting the Sf-caspase-1 and Tn-caspase-1 common sequence transcribed by an RNA polymerase III promoter; the recombinant virus of the vector can express double-stranded small RNA in a host cell, silences the caspase-1 encoded by the host cell through an RNA interference pathway, thereby inhibiting the apoptosis of the host cell, and significantly improving the expression level of an exogenous protein. The application can be used for industrialized production of protein preparations and vaccines.

A method for preparing a recombinant Bombyx mori nuclear polyhedrosis virus, and a system and method for preparing a recombinant adeno-associated virus. The system for preparing a recombinant adeno-associated virus comprises: a) BMN cells cultured in low-serum suspension and / or adherent cells of BMN cells cultured in low-serum suspension, and b) a recombinant Bombyx mori nuclear polyhedrosis virus, wherein the recombinant Bombyx mori nuclear polyhedrosis virus infects a host cell or a host which can at least produce a functional protein required for the packaging of a recombinant adeno-associated virus. The method for preparing a recombinant adeno-associated virus comprises infecting the BMN cells cultured in suspension or suspension to adherent culture using the recombinant Bombyx mori nuclear polyhedrosis virus. A recombinant adeno-associated virus prepared on the basis of a Bombyx mori baculovirus expression system has high titer, low empty capsid rate and good infection activity.

The present invention belongs to the technical field of biomedicine, and specifically relates to a recombinant influenza virus protein, a vaccine, and a preparation method therefor and the use thereof. To overcome the defects of existing influenza vaccines with respect to safety and high toxic side effects, a recombinant influenza virus protein is provided, which has an amino acid sequence as shown in at least one of SEQ ID No. 1 to SEQ ID No. 6. Recombinant hemagglutinin proteins of H1N1 and H3N2 subtypes, and Victoria and Yamagata lineages are expressed and purified by means of an insect baculovirus expression system, and are mixed with an adjuvant to prepare a highly immunogenic recombinant bivalent, trivalent or quadrivalent recombinant influenza virus protein vaccine. The prepared vaccine can induce high levels of specific IgG antibodies and hemagglutination-inhibiting neutralizing antibodies, the addition of adjuvant WGa01 improves the immunogenicity of the vaccine, and the provided vaccine is suitable for people allergic to eggs and has high safety.

The invention discloses an orf virus F1L protein monoclonal antibody as well as a blocking ELISA (enzyme-linked immunosorbent assay) detection kit and application thereof. Belongs to the technical field of biological detection. The kit comprises F1L protein expressed by baculovirus and an ORFV F1L protein monoclonal antibody marked by HRP (horse radish peroxidase). The ORFV F1L protein monoclonal antibody disclosed by the invention has the characteristics of strong specificity, high titer, stable property and the like, and is suitable for establishing an ORFV antibody blocking ELISA (Enzyme-Linked Immunosorbent Assay) kit. According to the invention, the HRP-labeled monoclonal antibody is used, so that non-specific reaction possibly introduced due to the use of an enzyme-labeled secondary antibody is reduced. The kit disclosed by the invention can be used for detecting a large batch of samples, has the advantages of low cost, simplicity, convenience, sensitivity, high specificity and the like, and provides an effective technical means for scientific prevention and control of the contagious pustular dermatitis of sheep.

The invention relates to the technical field of veterinary biological products, and discloses a porcine reproductive and respiratory syndrome virus (PRRSV) genetic engineering subunit vaccine and a preparation method and application thereof, the vaccine comprises a PRRSV ZJ266 GP5-M protein complex and an adjuvant, and the adjuvant is a mannose modified chitosan solution. The PRRSV ZJ266 GP5-M protein compound is formed by GP5 protein and M protein of a PRRSV ZJ266 strain expressed and purified by a Bac-to-Bac baculovirus expression system, the nucleotide sequence of the PRRSV ZJ266 GP5-M protein compound is shown as SEQ ID NO: 1, and the amino acid sequence of the PRRSV ZJ266 GP5-M protein compound is shown as SEQ ID NO: 2. According to the invention, a high-level neutralizing antibody can be induced to generate, 100% protection is provided for attack of PRRSV NADC30-like virulent strains, the virus load can be significantly reduced, and detoxification can be prevented. The vaccine provided by the invention is safe and effective, the preparation process is stable, and a new high-quality product choice is provided for preventing the PRRSV.

The invention belongs to the technical field of oligosaccharide preparation, and particularly relates to a novel method for preparing oligosaccharide containing Lex epitope through catalytic synthesis. According to the method, an insect cell-baculovirus expression system is adopted for secretory expression of alpha 1, 3-fucosyltransferase F (FucT-F delta TM) from truncated schistosome; carrying out nickel affinity chromatography purification to obtain a target protein, and catalyzing a substrate GalGn [beta] AsnFmoc or Gal2Gn2Man3Gn2 [beta] AsnFmoc under optimized reaction conditions; and after the product is purified by HPLC (High Performance Liquid Chromatography), ODS (Octa3) reversed-phase chromatography purification and desalination are performed to obtain a high-purity oligosaccharide product containing the Lex epitope (Galbeta4 (Fucalpha3) GlcNAc-R). According to the invention, the glycosyl transferase FucT-F [delta] TM is obtained by means of secretory expression of insect cells, and milligram-scale preparation of a Lex epitope-containing target oligosaccharide product is realized. The product preparation process is low in cost and high in purity, and is obviously superior to a chemical synthesis or natural extraction method.

The invention discloses a neutralizing monoclonal antibody aiming at a pig delta coronavirus S1 protein conserved linear epitope and application of the neutralizing monoclonal antibody, and belongs to the technical field of biology. The amino acid sequence of a light chain of the neutralizing monoclonal antibody is as shown in SEQ ID NO.9, and the amino acid sequence of a heavy chain of the neutralizing monoclonal antibody is as shown in SEQ ID NO.13. According to the invention, an insect baculovirus expression system is utilized to express the S1 protein of the porcine delta coronavirus and screen out a linear epitope with a neutralizing effect, and a neutralizing monoclonal antibody is harvested. The monoclonal antibody can be subjected to neutralization reaction with multiple strains of porcine delta coronaviruses, and is strong in neutralization activity and high in titer. The neutralizing monoclonal antibody provided by the invention can be used for efficiently preventing and treating the porcine delta coronavirus. The invention provides a new way for diagnosing, preventing and treating the porcine delta coronavirus, and provides a theoretical basis for development of related medicines and vaccines.

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 application discloses application of a silkworm silk gland BmSPI45 protein in preparation of a product resisting red hair moss, and relates to the technical field of bioengineering, and has the technical scheme as follows: the application of the silkworm silk gland BmSPI45 protein in preparation of the product resisting red hair moss, and the amino acid sequence of the silkworm silk gland BmSPI45 protein is SEQ ID NO:1.The application uses an insect baculovirus expression system to perform in-vitro expression on a BmSPI45 recombinant protein, and obtains a recombinant pFastBac-BmSPI45 eukaryotic expression vector.The constructed pFastBac-BmSPI45 eukaryotic expression vector is transformed into a DH10Bac competent cell, and a recombinant Bacmid is extracted and obtained, the recombinant Bacmid is transfected, and the BmSPI45 protein is massively expressed.The BmSPI45 recombinant protein is subjected to antibacterial function research, and the result shows that the protein has the effect of resisting red hair moss, thereby providing a new protein alternative for development of an antibacterial preparation.

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 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 concerns a baculovirus expression vector for recombinantly expressing a Foot-and-mouth disease virus (FMDV) capsid precursor protein under control of a promoter, the expression vector comprising a nucleic acid sequence encoding the FMDV capsid precursor protein, wherein the ATG start codon of an open reading frame encoding the FMDV capsid precursor protein is preceded at position −4 to −1 by the nucleic acid sequence 5′-AAAT-3′. The invention further relates to a host cell comprising the baculovirus expression vector, a method of producing FMDV virus-like particles (VLPs), and a method of producing a vaccine.

An improved baculovirus expression system for enhanced heterologous protein expression in hosts selected from Bombyx mori larvae, pupae, primary cells, or established insect cell lines is disclosed. The expression system is derived from a wild-type strain of Bombyx mori nucleopolyhedrovirus (BmNPV) by deletion or inactivation of non-essential genes, of the BmNPV genome. The system includes novel transfer vectors designed for targeted gene modification and incorporation of a bacterial origin of replication and an insertion site for a gene of interest (GOI). This engineered bacmid maintains infectivity while offering enhanced genetic stability and significantly improved heterologous protein expression in insect cells, larvae, or pupae. Methods for constructing this bacmid via homologous recombination and using it for high-level protein production are also described.

This invention discloses a fusion multi-epitope antigen of the extracellular region of the rabies virus G protein and its application, belonging to the field of vaccines. This invention uses an insect cell-baculovirus expression system to express a recombinant fusion protein containing the extracellular region of the rabies virus G protein, the dendritic cell targeting peptide DCpep, and the universal T cell epitope PADRE, and displays it on the surface of bacterial-like particles via the peptidoglycan binding domain PA to obtain a recombinant subunit vaccine. Comparative experiments with this vaccine and commercial inactivated vaccines and adjuvanted recombinant subunit vaccines show that the recombinant subunit vaccine provided by this invention has good immunogenicity, can induce specific immune responses in mice and increase neutralizing antibody levels, achieves 100% protection in lethal rabies virus standard strain challenge experiments, significantly reduces viral load in the brain of infected mice, and provides effective challenge protection.

The invention discloses a baculovirus expression vector as well as a construction method and application thereof. The vector takes pFastBacDual as a skeleton, 2-9 repetitive BS sequence modification promoters are connected in series to the downstream of a polh promoter, an extremely advanced transcription factor VLF-1 gene is introduced, and the two components synergistically enhance the transcriptional activity. According to the invention, three types of recombinant vectors pBSX-eGFP, pBSX-eV and pBSX-GV are constructed, and the optimal combination is determined as overexpression of two repeated BS sequences and VLF-1, so that the expression quantity of the target protein can be increased. The vector construction process is standardized, enzyme cutting sites such as BamHI and EcoRI are reserved, and high-expression protein can be obtained after Sf9 cells are transfected and cultured for 4-5 days. The recombinant protein expression efficiency of a baculovirus expression system is improved, the advantages of correct folding and post-translational modification of the recombinant protein are reserved, and the method is suitable for large-scale industrial recombinant protein production scenes such as biopharmacy and vaccine research and development and has important application value.

The invention belongs to the technical field of biological medicines, and particularly relates to a recombinant influenza virus protein, a vaccine and a preparation method and application thereof. In order to overcome the defects of high safety and toxic and side effects of the existing influenza vaccine, the invention provides a recombinant influenza virus protein, the amino acid sequence of which is as shown in at least one of SEQ ID No.1-SEQ ID No.6. According to the invention, H1N1 subtype, H3N2 subtype, Victoria line and Yamagata line recombinant hemagglutinin proteins are expressed and purified through an insect baculovirus expression system, and are mixed with an adjuvant to prepare a recombinant divalent, trivalent or tetravalent recombinant influenza virus protein vaccine with high immunogenicity. The prepared vaccine can induce high-level specific IgG antibodies and hemagglutination inhibition neutralizing antibodies, the immunogenicity of the vaccine is improved by adding the adjuvant WGa01, and the vaccine is suitable for egg allergic people and high in safety.

This invention utilizes the recombinant porcine deltacoronavirus protein PDCoV-S constructed in an insect-baculovirus expression system, and uses it as an immunogen to prepare egg yolk antibodies. The egg yolk antibodies are further processed into oral formulations. Through optimization of emulsifiers and synergists, microcapsule formulations with high encapsulation efficiency, high gastric acid throughput, and good intestinal release performance are obtained. Furthermore, the addition of synergists enhances the therapeutic efficacy of the formulation. When the mass ratio of baicalin, Polygonatum odoratum polysaccharide, and nano-selenium is 2:1:2, the encapsulation efficiency of the microcapsules can be effectively improved, thus enhancing the drug's effect. This microcapsule formulation can be used as an active ingredient in the production of drugs or feed additives for the treatment or prevention of PDCoV infection.