217 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 present invention discloses a method for producing protein HFBI. The method comprises the following steps: infecting insect cells with a recombinant baculovirus containing an insect cell expression vector expressing a fusion protein, and collecting the supernatant; the fusion protein is composed of a GP64 signal peptide and a protein HFBI; the amino acid sequence of the GP64 signal peptide is shown in SEQ ID NO:2 from the 1st to the 20th position from the N-terminus; then the supernatant is precipitated with ammonium sulfate to obtain a large amount of protein HFBI, and finally the protein is further purified by heating using the thermal stability. The purity of the protein HFBI obtained by the method provided by the present invention is more than 95%, the concentration is 6 mg / ml, and it has biological activity. It can be seen that the present invention can significantly increase the yield of protein HFBI, and the production process is simple, which can be fully applied to actual production. The present invention has important application value.

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 preparation method of influenza D virus-like particles, and belongs to the field of veterinary biological products. Influenza D virus (IDV) is one of important pathogens of bovine respiratory disease syndrome (BRDC), the disease causes a hundreds of billions of dollars economic loss to the global cattle raising industry every year, however, commercialized vaccines for influenza D do not exist in the market at present. The preparation method comprises the following steps: target gene amplification, construction of recombinant shuttle plasmids, construction of recombinant baculoviruses, rescue of recombinant baculoviruses, and preparation and purification of virus-like particles. The D-type influenza virus-like particles (IDV VLPs) are obtained through the steps. After mice are immunized, the IDV VLPs are found to have high immunogenicity and can be used as a candidate vaccine for preventing the D-type influenza.

The invention provides a classical swine fever virus subunit vaccine and a preparation method thereof, the classical swine fever virus subunit vaccine is based on an E2 protein gene and a GP67 signal peptide gene of a CSFV 1.1 strain, a recombinant expression vector is constructed through gene cloning, a classical swine fever virus E2 recombinant protein is obtained through expression of an insect baculovirus system, and the classical swine fever virus subunit vaccine is obtained. And the recombinant protein is combined with a colloidal manganese adjuvant to prepare the swine fever virus subunit vaccine. The hog cholera virus E2 recombinant protein is close to natural E2 protein in structural function, high in reduction degree, high in expression quantity and suitable for vaccine production and application; the vaccine can generate specific humoral immunity and relatively strong cellular immunity, and is beneficial to removal of CSFV in an animal body; meanwhile, the vaccine can be distinguished from attenuated vaccine strain immunization or wild strain infection at the nucleic acid level, can be used for preventing and purifying classical swine fever in an intensive pig farm, and has a considerable application prospect.

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 provides a qPCR (quantitative polymerase chain reaction) method for detecting baculovirus plasmid recombination rate. The qPCR method comprises the following steps: designing a total baculovirus primer; designing a recombinant stem grain primer; the qPCR detection method comprises the following steps: adopting a fluorescent dye qPCR method, taking the extracted stem grain DNA as a template, respectively detecting the same template by using the total stem grain primer pair and the recombinant stem grain primer pair to obtain the Ct total value of the total stem grain and the Ct weight value of the recombinant stem grain, obtaining the difference value delta Ct = Ct total-Ct weight, and calculating the recombination ratio% = 2 delta Ct * 100%. The invention provides a novel method for detecting the proportion of recombinant rods, the total rod primer pairs Bacmid-F and Bacmid-R can specifically amplify non-recombinant rods and recombinant rods, and the recombinant rod primer pairs Tn7L-F and M13-R can only amplify a sample containing the recombinant rods, so that the proportion of the recombinant rods in the sample can be detected by adopting a qPCR (quantitative polymerase chain reaction) method; the recombination proportion can be used for guiding subsequent experiments.

The invention relates to the technical field of biology, and particularly discloses a recombinant vector, a recombinant baculovirus and a preparation method and application of the recombinant vector and the recombinant baculovirus. According to the method for preparing the recombinant vector, pFastBac Dual is adopted as a vector to prepare a recombinant plasmid vector inserted into a VP2 gene of an avian bursal disease virus classic strain, and the recombinant plasmid vector is named as pDual-V1; the method comprises the following steps: preparing a recombinant plasmid vector into which VP2 genes of a classical strain and a variant strain of the avian bursal disease virus are inserted at the same time, and naming the recombinant plasmid vector as pDual-V1-V2; the method comprises the following steps: preparing a recombinant plasmid vector inserted into a group I type 4 fowl adenovirus Fiber-2 gene, and naming the recombinant plasmid vector as pDual-F2; the method comprises the following steps: preparing a recombinant plasmid vector into which a Fiber-2 gene and an avian reovirus sigma C gene sequence are inserted at the same time, wherein the recombinant plasmid vector is named as pDual-F2-sigma C; carrying out double enzyme digestion on the pDual-V1-V2, recovering small fragments, carrying out double enzyme digestion on the pDual-F2-sigma C, recovering large fragments, and then carrying out mixed enzyme ligation. The recombinant baculovirus can express four antigens at the same time, and effective preparation of a multi-combined vaccine is achieved.

The invention discloses a preparation method and application of a tetravalent hand-foot-and-mouth disease subunit-new adjuvant vaccine, and relates to the technical field of hand-foot-and-mouth disease vaccines. VP1 proteins of EV-A71, CV-A16, CV-A10 and CV-A6 are expressed through an insect cell-baculovirus system, the VP1 proteins are purified through a nickel affinity chromatography column and mixed with an MF59 adjuvant to prepare a tetravalent vaccine, in-vivo evaluation of mice is conducted, and meanwhile the immunogenicity and protection efficacy of the tetravalent VP1 protein vaccine and the immunogenicity and protection efficacy of a monovalent VP1 protein vaccine are compared. Immune results show that compared with a VP1 monovalent vaccine, all serum of a mouse inoculated with the tetravalent VP1 protein vaccine shows strong reactivity to all captured antigens. Therefore, the results show that the prepared tetravalent subunit-new adjuvant vaccine is a promising broad-spectrum vaccine.

The invention belongs to the technical field of biological medicines, and particularly relates to a PPV-PCV2 nanoparticle vaccine design and a preparation method. According to the invention, PPV1VP2 protein is used as a skeleton, a SpyTag peptide fragment is inserted into a loop 2 region of the VP2 protein, and the fusion protein is expressed by using an insect cell baculovirus system and is marked as PPV-ST; a plurality of B cell epitopes of Cap protein of PCV2 and a plurality of T cell antigen epitopes of Rep protein of PCV2 are connected in series, a SpyCatcher peptide fragment is fused at an N end, and expression is performed by using a prokaryotic expression system and is marked as SC-PCV2. Finally, the PPV-ST protein and the SC-PCV2 protein are subjected to covalent binding assembly, the nanoparticle vaccine is prepared, and the obtained PPV-PCV2 vaccine has good immunogenicity.

The invention discloses an H9N2 subtype avian influenza virus virus-like particle. The virus-like particle is formed by assembling M1 protein of an H5N1 subtype avian influenza virus and HA protein and NA protein of the H9N2 subtype avian influenza virus; the M1 protein, the HA protein and the NA protein are respectively coded by genes M1, HA and NA, and the genes M1, HA and NA respectively have base sequences shown as SEQ ID NO.1 to SEQ ID NO.3 in a sequence table. Accordingly, the inventor also establishes a corresponding preparation method, i.e., HA, NA and M1 proteins are expressed by using a Bac-to-Bac baculovirus expression system, and the three proteins are self-assembled into the virus-like particles. Researches show that the expressed HA-M1 and NA recombinant protein can be self-assembled in Sf9 cells to form virus-like particles, the concentrated virus-like particles can agglutinate 1% of chicken erythrocytes, and the hemagglutination titer is 1: 64. Therefore, the invention lays a foundation for further research on the virus-like particle vaccine of the H9N2 subtype avian influenza virus.

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 oral vaccine composition for porcine circovirus-related diseases comprises pupae or cells of baculovirus-infected insects, said pupae or cells being obtained by subjecting a recombinant baculovirus into which DNA encoding a porcine circovirus type 2 protein has been introduced to an infection treatment and a freeze-drying treatment.

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.

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 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 invention belongs to the technical field of insect biological control, and particularly relates to a recombinant insect baculovirus PH-SVT / AC capable of efficiently killing spodoptera frugiperda and application of the recombinant insect baculovirus PH-SVT / AC. The recombinant insect baculovirus PH-SVT / AC is preserved in the China General Microbiological Culture Collection Center on August 7, 2024, the preservation number is CGMCC No.46096, the preservation address is No.3, courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the recombinant insect baculovirus is classified and named as an autographa californica nuclear polyhedrosis virus. The recombinant insect baculovirus PH-SVT / AC has a remarkable prevention and control effect on the spodoptera frugiperda, and has a good application prospect in biological prevention and control of the spodoptera frugiperda.

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 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 a high-efficiency expression and purification method of 5 '-3' exonuclease, which comprises the following steps: based on a Bac-to-Bac system, adding a tag and a TEV restriction enzyme cutting site to optimized 5 '-3' exonuclease Xrn1, then connecting the 5 '-3' exonuclease Xrn1 to a multiple cloning site behind a Polyhedrin promoter of a pFastBac expression vector to prepare expression baculovirus plasmids, and carrying out purification on the expression baculovirus plasmids to obtain the 5 '-3' exonuclease. Then transfecting the recombinant baculovirus particles into insect cells to generate recombinant baculovirus particles; finally, the 5 '-3' exonuclease Xrn1 is obtained through nickel column affinity chromatography, Heparin affinity chromatography and gel filtration chromatography purification, the invention provides a more economical and practical technical route for synthesizing the 5 '-3' exonuclease Xrn1, and a technical foundation is laid for large-scale production of the 5 '-3' exonuclease.

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.

A recombinant baculovirus having a) a nucleotide sequence encoding a fusion protein, wherein said fusion protein includes an antigen fused to a baculovirus capsid peptide operatively linked to a first promoter and b) a nucleotide sequence encoding a lipid viral envelope protein bound to a second promoter.