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

The invention relates to a method for 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 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 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 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.

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.

The invention relates to a cat hepatitis virus core antigen virus-like particle as well as a preparation method and application thereof. The virus-like particle is prepared by expressing a wild type gene DCHcWT of a cat hepatitis virus core antigen or a 1-149 amino acid truncated gene DCHcN149 of an amino terminal of the wild type gene DCHcWT in an insect baculovirus expression system, and purifying and self-assembling the wild type gene DCHcWT or the 1-149 amino acid truncated gene DCHcN149 in the insect baculovirus expression system. The prepared virus-like particles are regular in structure and good in immunogenicity, and antigen characteristics of natural virus particles can be efficiently simulated. The virus-like particle can be used as a diagnosis antigen of cat hepatitis B virus infection, and is used for preparing a serum detection kit for cat hepatitis. The vaccine can also be used as an immunogen to prepare a subunit vaccine for preventing cat hepatitis B; the compound can also be used for vaccine nano-delivery carriers.

The present invention discloses a SmartBac baculovirus expression system and application thereof. The system can comprise a acceptor plasmid (containing fragment A or fragments B and C) and a donor plasmid (containing fragment D); the fragment A contains a promoter, a sequence encoding a protease, a protease cleavage site, an insertion region of a gene encoding a target object to be expressed and a termination sequence; the fragment B contains a promoter, a sequence encoding a protease and a termination sequence; the fragment C contains a promoter, an insertion region of a gene encoding a target object to be expressed and a termination sequence; the fragment D contains a promoter, an insertion region of a gene encoding a target object to be expressed and a termination sequence. The present invention also provides three cloning strategies to achieve the expression of protein complexes with molecular weights of less than 600 kDa and the expression of protein complexes with molecular weights of no less than 600 kDa and efficient screening of a subunit most suitable for adding a purification tag. The present invention is of great significance for recombinantly expressing protein complexes with complex components and large molecular weights in insect cells.

The present application relates to the technical field of molecular markers, and particularly relates to a porcine epidemic diarrhea virus (PEDV) bacterium-like particle and a preparation method thereof, which takes S1 genes and COE genes of Guangxi PEDV epidemic strains as target genes, and successfully expresses fusion proteins S1-PA and COE-PA fused with anchor hook proteins PA by using an insect cell-baculovirus expression system; meanwhile, lactic acid lactobacillus MG1363 is treated by heat and acid to remove bacterial nucleic acids and proteins, and GEM particles are successfully prepared, and the fusion proteins S1-PA and COE-PA are successfully displayed on the surfaces of the GEM particles to obtain S1-GEM and COE-GEM particles, the two kinds of particles are mixed with GEL-01 adjuvant to prepare two kinds of bacterium-like particle vaccines, and the vaccines are used for immunization of mice, and it is confirmed through immunization routes, immunogens and immunization doses that 24 mu g COE-GEM can achieve the optimal immunization effect for the mice immunized by nose drops.

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.

The invention discloses a BLV p24 recombinant antigen protein for detecting a bovine leukemia antibody, a test strip and application of the BLV p24 recombinant antigen protein. The amino acid sequence of the BLV p24 recombinant antigen protein is as shown in SEQ ID NO. 2. The eukaryotic recombinant p24 protein is prepared by adopting a baculovirus expression system, and the recombinant p24 protein expressed by the system can be correctly folded and modified after translation, better simulates native conformation and has higher sensitivity. And the His tag on the recombinant protein is beneficial to one-step purification to achieve higher purity, and better activity can be shown without enzyme digestion. The formed colloidal gold test strip is applied to detection of bovine leukemia, and the accuracy, the sensitivity and the detection are well improved.

The invention relates to a primer, a probe, a method and a kit for detecting Sf-RVN cell DNA residual quantity, and belongs to the technical field of gene detection. The invention discloses primers and a probe for detecting the residual quantity of Sf-RVN cell DNA (deoxyribonucleic acid). The primers comprise an upstream primer and a downstream primer; the sequence of the upstream primer is as shown in SEQ ID NO: 1; the sequence of the downstream primer is as shown in SEQ ID NO: 2; the sequence of the probe is as shown in SEQ ID NO: 3. The primer and the probe can stably, accurately, quickly, sensitively and specifically detect the residual quantity of the Sf-RVN cell DNA, and can be applied to quantitative detection of the residual quantity of the host insect cell Sf-RVN cell DNA in an intermediate process sample, a semi-finished product and a finished product of an influenza virus recombinant HA protein product produced by an insect cell-baculovirus expression system.

The invention relates to the technical field of biological medicines, in particular to an optimized SARS-CoV-2 chimeric virus-like particle based on an influenza virus skeleton and an application of the optimized SARS-CoV-2 chimeric virus-like particle. The amino acid sequence of the spike protein of the virus-like particle comprises an extracellular domain amino acid sequence; the amino acid sequence of the extracellular domain has S-6P mutation (F817P, A892P, A899P, A942P, K986P and V987P) and Frelin restriction enzyme cutting site substitution (RRAR-GSAS). The optimization mode can maintain natural trimer conformation before S protein fusion and effectively improve the stability of the S protein, so that the dissociation phenomenon of the protein is effectively improved, and the influence of the dissociation phenomenon on effective epitopes is avoided. In order to improve the secretory expression efficiency of the VLPs, the protein expression is optimized by using a signal peptide derived from an insect cell-baculovirus expression system. The immunogenicity and immune protection efficacy of the vaccine can be effectively improved.

This invention utilizes an insect-baculovirus expression system to prepare the recombinant protein SADS-CoV-S of porcine acute diarrhea syndrome coronavirus, which is then used to prepare egg yolk antibodies. By optimizing the composition of the subunit vaccine composition for immunizing laying hens, IL-2 and ZnCl2 solution are added as immunostimulants to the conventional adjuvant, increasing the antibody concentration in the egg yolk antibody extract and improving the neutralizing antibody titer and protective efficacy. When IL-2 and zinc chloride aqueous solution are added to the immunization composition simultaneously, the resulting egg yolk antibody aqueous solution exhibits the highest protein concentration, and at the same protein concentration, the egg yolk antibody has the highest neutralizing titer. Compared to the immunization composition using Freund's complete adjuvant alone, the neutralizing titer is increased by more than 60 times.

The invention discloses a large yellow croaker iridovirus double-antigen subunit vaccine and a preparation method thereof. By optimizing the sequences of the large yellow croaker iridovirus MCP protein and ATPase protein, the immunogenicity and stability of the large yellow croaker iridovirus MCP protein and ATPase protein can be remarkably improved, the expression quantity of the large yellow croaker iridovirus MCP protein and ATPase protein in baculovirus can be remarkably improved, and the two recombinant proteins are expressed by using a baculovirus expression system, so that large-scale production of the large yellow croaker iridovirus MCP protein and ATPase protein can be realized through large-scale serum- the production batch is stable, the protein expression quantity is high, and the production cost is low. According to the large yellow croaker iridovirus double-antigen subunit vaccine prepared by mixing the two recombinant proteins, the level of antibodies generated by immunizing large yellow croakers is high and is far superior to that of existing single-antigen vaccines and inactivated vaccines, the protection effect is strong, the protection effect of 78.1% or above can be obtained only through one-time immunization with a very small amount, and the large yellow croaker iridovirus double-antigen subunit vaccine is free of pathogenicity to the large yellow croakers and high in safety.