54 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 invention discloses a method for improving the efficiency of a baculovirus expression system. According to the invention, actin is overexpressed in insect BmN cells, so that the expression of exogenous genes is improved. The principle is as follows: after overexpression of actin, the recruitment efficiency of virus nucleation and late gene polymerase is improved, so that the replication and transcription of recombinant virus containing exogenous genes are promoted. According to the invention, the problem of low protein expression quantity by using the baculovirus is solved, ultrahigh-level expression of the protein is promoted, and the efficiency of a baculovirus expression system is improved.

To provide a technique for efficient production of a polypeptide usable to induce immunity against Classical Swine Fever Virus (CSFV) in pigs.SOLUTION: A polypeptide having a predetermined amino acid sequence is efficiently produced by using a construct including a polynucleotide encoding the amino acid sequence, the construct being for expressing the polypeptide by means of a baculovirus expression system.SELECTED DRAWING: Figure 1

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.

The invention provides a beet armyworm ovary cell line with high yield of baculovirus, the cell line is named as IOZCAS-Spex18, and the preservation number of the cell line is CGMCC (China General Microbiological Culture Collection Center) No.46039. The invention also discloses a preparation method of the cell line and application of the cell line in large-scale growth of baculovirus. The cell line can be used for replicating baculovirus, large-scale production of baculovirus insecticides, construction of a baculovirus expression vector system and expression of protein with commercial or scientific value.

The invention discloses a method for preparing PRMT2 protein by using a baculovirus expression system and multi-mode chromatography, which comprises the following steps: S1, cloning a base sequence SEQ ID NO.1 of PRMT2 into a plasmid in an artificial gene synthesis mode to form a recombinant expression plasmid; s2, transposing the recombinant expression plasmid obtained in S1 into an escherichia coli competent cell to obtain a recombinant baculovirus, transfecting an insect cell to obtain a baculovirus, and expressing a PRMT2 protein SEQ ID NO.2; and S3, obtaining the purified PRMT2 protein SEQ ID NO.3 through multi-mode chromatography. According to the method disclosed by the invention, the label-free and high-purity PRMT2 protein can be obtained, and a foundation is laid for the biological research of related structures in the future.

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 microorganisms, and particularly discloses a method for preparing self-assembled goose astrovirus vp27 protein-ferritin by using a baculovirus expression system, which comprises the following steps: firstly, constructing a recombinant plasmid pFastBac Dual-VP27 and a recombinant plasmid pFastBac Dual-VP27-ferritin; the preparation method comprises the following steps: respectively transforming a recombinant plasmid pFastBac Dual-VP27 and a recombinant plasmid pFastBac Dual-VP27-Ferritin into a DH10Bac competent cell, so as to obtain a recombinant positive clone bacmid-VP27 and a recombinant positive clone bacmid-VP27-Ferritin, and respectively transforming the recombinant plasmid pFastBac Dual-VP27 and the recombinant plasmid pFastBac Dual-VP27-Ferritin into a DH10Bac competent cell; the method comprises the following steps: respectively transfecting a recombinant positive clone bacmid-VP27 and a recombinant positive clone bacmid-VP27-Ferritin into an insect cell, and carrying out expression of a recombinant baculovirus, so as to obtain a recombinant baculovirus Ac-VP27 and a recombinant baculovirus Ac-VP27-Ferritin; the method comprises the following steps: respectively carrying out subculture on a recombinant baculovirus Ac-VP27 and a recombinant baculovirus Ac-VP27-Ferritin, and carrying out expression of a VP27 protein and a VP27-Ferritin protein; and then separating and purifying the VP27 protein and the VP27-Ferritin protein from the culture supernatant. According to the invention, the VP27 protein and the VP27-Ferritin protein with natural conformations can be conveniently and rapidly obtained, and the VP27 protein and the VP27-Ferritin protein act as candidate vaccines for preventing goose astrovirus infection and antigen substances for detecting antibodies of goose astrovirus.

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 recombinant tachypleus tridentatus C factor based on codon optimization and application of the recombinant tachypleus tridentatus C factor in endotoxin detection, and relates to the technical field of bioengineering. The limulus C factor sequence is used as a starting sequence, the limulus C factor is recoded, and the gene sequence of the recombinant limulus C factor is shown as SEQ ID NO: 1; protein corresponding to the sequence is expressed through an insect cell-baculovirus expression system, and the expression quantity is high, that is, the protein expression quantity of the recombinant horseshoe crab factor C is successfully improved through codon optimization. The production cost of the recombinant tachypleus tridentatus C factor endotoxin detection method kit is reduced, and the recombinant tachypleus tridentatus C factor detection method replaces a tachypleus tridentatus reagent detection method to become a mainstream method for endotoxin detection, so that the problem of raw material supply of tachypleus tridentatus reagent due to the fact that tachypleus tridentatus becomes a protective animal is solved; and the existing recombinant horseshoe crab factor C detection method has the problems of high product price and the like.

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.