245 results about "Polerovirus" patented technology

Disclosed and claimed is a new insect cell line, Sf900+, ATCC CRL-12579. The insect cell line was established from Lepidoptera, Noctuidae, Spodoptera frugiperda Sf-9 (ATCC CRL-1711) through multiple rounds of limiting dilution and selection in a serum-free insect medium supplemented with added human insulin. The insect cell line is useful in BEVS or as an adjuvant and has many characteristics and advantages. Also disclosed and claimed are recombinant proteins from recombinant baculovirus expression in insect cells such as Sf900+ cells, for instance, HA, NA, EPO, CD4, CEA, and thrombospondin.

The invention discloses an insect bioreactor capable of expressing multiple exogenous genes, and a construction method and application thereof. The construction method comprises the following steps: (1) introducing multicopy high-efficiency bacteria DNA (deoxyribonucleic acid) replication initiator into chitinase and cysteine proteinase genes of a baculovirus genome to obtain a baculovirus shuttle plasmid; (2) replacing virus duplicated essential gene downstream the polyhedrosis gene of the baculovirus shuttle plasmid with antibiotic gene to obtain a baculovirus plasmid DNA; and (3) replacingother virus duplicated and infected nonessential genes in the baculovirus shuttle plasmid with reverse selection marker gene to obtain the insect bioreactor. The antibiotic gene or reverse selection marker gene in the insect bioreactor which is constructed by replacing the exogenous target genes can express multiple exogenous genes in a host insect or insect cell. The insect bioreactor disclosed by the invention can efficiently expressing one or more exogenous genes in an insect body at the same time, and can produce massive recombinant proteins at low cost.

The present invention relates to production of proteins in insect cells whereby repeated coding sequences are used in baculoviral vectors. In particular the invention relates to the production of parvoviral vectors that may be used in gene therapy and to improvements in expression of the viral rep proteins that increase the productivity of parvoviral vectors.

The invention mainly aims to provide a porcine circovirus type 2 (PCV2) subunit vaccine and a preparation method thereof. Particularly, a baculovirus vector expression system is utilized to express a large amount of recombinant open reading frame type 2 (ORF2) protein in insect cells, so that the subunit vaccine with good immunity effect is developed. A bac-to-bac baculovirus expression system is adopted to perform whole gene amplification on the porcine circovirus type 2 ORF2 gene, and a melittin signal peptide nucleotide sequence is introduced into a terminal 5', so that the recombinant baculovirus of an open reading frame containing the melittin signal peptide nucleotide sequence and a PVC2ORF2 gene sequence is established, wherein the infected insect cell expresses the recombinant ORF2 protein with efficient and high PCV2 antigenicity; and thus the subunit vaccine containing the PCV2 recombinant ORF2 protein is prepared. The inoculation experiments of piglets show that the subunit vaccine has a good immunity protection effect.

The invention provides African swine fever P30 protein recombinant baculovirus expression vector and a preparation method thereof. The method comprises: amplifying in plasmid PCR-4TOPO-P30 of ASFV (African swine fever virus) P30 full-length gene to obtain P39 gene, linking the amplified P30 gene to a baculovirus vector pFastBac 1 to construct recombinant baculovirus vector pFastBac1-ASFV-P30, converting into competent Escherichia coli cells DH10Bac to obtain recombinant shuttle bacmid rBacmid-ASFV-P30, transfecting to insect cells Sf9 after verification is correct to obtain recombinant baculovirus, and passage amplifying the recombinant baculovirus, linking baculovirus high in titer and containing ASFV P30 gene to High Five insect cells for eukaryotic expression of ASFV P30. The African swine fever P30 protein recombinant baculovirus expression vector is constructed by using the method, a recombinant baculovirus expression system is used to express African swine fever P30 protein in insect cells, and basis is laid for African swine fever ELISA (enzyme-linked-immunosorbent serologic assay) detections.

The invention provides a broad-spectrum rod-shaped viral pesticide produced by substitute hosts, the components and the weight proportion of which are: 20 billion PIB / ml virus suspension 2.5 to 15 percent, 60nm silicon dioxide 0.1 to 0.5 percent, aromatic amino acid 0.1 to 0.15 percent, refined fluorescent whitening agent 0.5 to 1 percent, sodium lignin sulfonate 0.1 to 1 percent, chlorfluazuron 0.01 to 0.1 percent, glycerol 1.0 to 2.0 percent, and remaining no sterilized water; the virus suspension is received by the infection of nuclear polyhedrosis virus of cabbage armymoth and the substation for the host---cotton bollworm or beet armyworm. The pesticide not only maintains the biological pesticide properties of viral pesticides and has no harms to human beings and higher animals, but also is suitable for the the massive employments on vegetables, and has the wide insect disinfestation spectrum. Therefore, a broad-spectrum viral pesticide can prevent and control various harmful insects while with only a rod-shaped virus. The invention has the advantages of good insecticidal effect, fast insecticidal speed, and very good application prospect.

The invention relates to a coxsackie virus A16 type virus-like particle vaccine. The inventor fortuitously finds that proteins which have better spatial configurations and are suitably cut can be obtained by expressing P1 protein of CVA16 and 3CD protein of CVA16 by infecting insect cells with rhabdovirus. The proteins can be automatically assembled into a virus-like particle which has high immunogenicity.

The invention discloses a SARS vaccine and preparing method, which comprises the following steps: 1) constructing external baculoviral surface display carrier of S protein of SARS coronary virus; 2) transmitting the carrier into susceptive cell with baculoviral genome plasmid Bacmid; obtaining recombinant baculoviral genome plasmid; 3) using the plasmid to infect insect cell; purifying the recombinant plasmid; obtaining the product.

Disclosed and claimed is apparatus and methods for the growth of cells to high density, products therefrom and uses thereof. Also disclosed and claimed is the use of this method for the growth to high-density insect cells, such as the Spodoptera frugiperda Sf900+ cell line (ATCC: CRL 12579). Further disclosed is the infection of Sf900+ cells at high cell density with wild type and recombinant baculoviruses to produce baculovirus and DNA or gene or expression products.

The invention discloses a method for producing a porcine parvovirus antigen and its product. The method comprises the following steps: porcine parvovirus capsid protein VP2 gene or optimized VP2 gene is cloned in a baculovirus carrier so as to obtain a transfer expression carrier; the constructed transfer expression carrier and baculovirus DNA are carried out cotransfection to obtain recombined baculovirus; the recombined baculovirus is used to infect insect host and cell; the infected insect host is cultured to express corresponding porcine parvovirus capsid protein; and the expressed antigen is ingathered and purified so as to obtain the porcine parvovirus antigen. The method adopts a baculovirus expression system to make safe and efficient porcine parvovirus antigen capsid particles in a domestic silkworm bioreactor; the prepared purified antigen by the method has high safety, and can be directly produced to vaccines for animal immunity. The method for producing porcine parvovirus antigen has the advantages of high expression efficiency, high immunization activity of the expressed antigen, low production cost, large scale production realization and the like.

The invention discloses a method for building a domestic silkworm silk gland biologic plant. A recombinant transgene vector which is provided with domestic silkworm silk protein specificity promoter controlled extraneous gene expression cassettes, an enhancer activity component, antibiotic resistant genes and fluorescent protein reporter genes and based on a piggyBAC transposon is established through gene engineering operations. The transgene vector is introduced into primarily laid eggs, special antibiotics are fed to hatched newly exuviated silkworms continuously until the fourth instar. In this way, silkworms which can resist the specific antibiotics and are provided with fluorescent protein reporter genes are obtained; target genes are detected; and transgene silkworms are manufactured by breeding. By adopting the silk gland tissues of the transgene silkworms, protein drugs can be obtained. With the method disclosed by the invention, transgene domestic silkworm silk gland plants can prepare protein drugs at low manufacture cost without potentially hazardous rhabdovirus. The method is safe and is practically valuable.

The invention discloses a genetic engineering subunit vaccine for a porcine circovirus as well as a preparation method and application of the genetic engineering subunit vaccine. By cloning the nucleocapsid protein of the novel porcine circovirus 3 (PCV3), a PCV-Cap protein with higher purity is successfully expressed by using an escherichia coli or baculovirus expression system. The subunit vaccine for the PCV3 is successfully developed for the first time by using the PCV-Cap protein; the prepared vaccine is high in antigen purity, good in safety and strong in immunogenicity, and has no pathogenicity to pigs and other animals; the antigen has good solubility in a neutral PH buffer solution; furthermore, the preparation method is simple and low in cost, thus being suitable for large-scaleindustrial production; an effective and powerful means is provided for the prevention and control of novel PCV, and the genetic engineering subunit vaccine has a wide application prospect in the fieldof the prevention and control of the PCV3.

The invention relates to a method of increasing protein expression in baculo vector virus expression systems. The invention provides a method to produce a recombinant protein in insect cell culture which comprises selecting a recombinant baculovirus expressing said protein, growing insect cells in growth medium in a culture vessel and infecting the cells with an inoculum of at least one baculovirus at a cell density of 1×105 to 5×106 cells / ml with an m.o.i of <0.01. The invention also provides a method to produce recombinant pestivirus E2 or Em9 protein or fragments thereof in insect cell culture characterized by a final concentration of the protein fragments in the growth medium at harvest of at least 100 μg / ml. The invention also provides a method of producing recombinant FSH, α-units and / or β-units, and complexes and fragments thereof, at a concentration in the growth medium at harvest of at least 15 μ / ml.

The invention provides rabbit hemorrhagic disease virus-like particles. The rabbit hemorrhagic disease virus-like particles are prepared by optimizing rabbit hemorrhagic disease virus VP60 gene according to insect cell biased codons, connecting the optimized VP60 gene with an insect cell efficient expression vector, and then producing the rabbit hemorrhagic disease virus-like particles through a rhabdovirus / insect cell expression system. The rabbit hemorrhagic disease virus-like particles can be mixed with a preservative and adjuvants to prepare a rabbit hemorrhagic disease vaccine. The rabbit hemorrhagic disease virus-like particles can greatly increase the expression quantity of the rabbit hemorrhagic disease virus-like particles, and have the advantages of high safety, high immunogenicity, low cost and the like, and is easy to ferment.

The recombination, expression and application of Chinese prawn's antibacterial peptide gene are disclosed. Its recombination expression method includes configuring recombinant carrier (pET series, pGEX-4T series, pPIC series, pAO815, and baculovirus expression carrier), transferring the carrier to colibacillus and yeast and transfecting insect cells, screening engineered bacterial strain or cell, and inducing expression. The expressed product can be used to prepare feed additive, antistaling agent, cosmetics and medical products.

The present invention provides an insect infection method for use in the production of a protein with a baculovirus expression vector in the insect, the method comprising the steps of:(g) providing a plurality of insect larvae or pupae;(h) providing a solution comprising a wild type baculovirus or a baculovirus expression vector having a desired gene encoding a protein;(i) stressing the insect larvae or pupae;(j) soaking the insect larvae or pupae in the solution for an appropriate time so that they are infected with the wild type baculovirus or the baculovirus expression vector; and(k) incubating the infected larvae or pupae for production of the protein; and(l) harvesting the protein.The method can treat a great quantity of larvae simultaneously, achieve batch infection of larvae or pupae, save manpower and effectively infect larvae or pupae at a high infection rate.

The invention discloses a recombinant baculovirus vector, a virus like particle, a preparation method and use and aims at providing tandem expression and accurate cutting expression of a plurality of genes with a single promoter and successfully packaging the virus like particle. The recombinant baculovirus vector is simple to operate, high in expression quantity and high in cutting efficiency. The obtained virus like particle is good in immunogenicity and does not have an exogenous sequence. The technical essential is that picornaviridae 3C / 3CD gene and P1 / P1op gene are expressed under the single promoter, the 3C / 3CD gene and the P1 / P1op gene are connected by a 2A sequence to build a 3C / 3CD-2A-P1 / P1op expression frame, then the 3C / 3CD gene is placed under the promoter of baculovirus vector plasmid to obtain a recombinant shuttle vector, a host cell is transfected to obtain recombinant baculovirus, and the host cell is infected to obtain the virus like particle.

It is intended to provide virus vector vaccine preparations containing as the main component a virus vector with the use of a virus showing no pathogenicity on humans and being free from a risk of the re-acquisition of pathogenicity. Namely, vaccine preparations containing as the main component a baculovirus having a gene encoding an antigen integrated thereinto are provided.

The invention discloses a foot and mouth disease virus recombinant virus sample particle as well as a preparation method and application thereof. The recombinant virus sample particle is commonly assembled and expressed by components in a DNA molecule composition. The DNA molecule composition contains an O-type foot and mouth disease VP0 gene, an O-type foot and mouth disease VP1 gene and an O-type foot and mouth disease VP3 gene and further contains a green fluorescent protein gene. By virtue of the property that FMDV VLPs is self-assembled through VP0, VP1 and VP3, a construction method of a baculovirus recombinant vector is improved, a green fluorescent protein label is added into the vector, and FMDV VLPs is successfully prepared by virtue of a pFBDM Bac-to-Bac system, so that a theoretical foundation is laid for the further development of safe and efficient O-type FMDV genetic engineering vaccines.

An insect larva aerosol infection method for producing recombinant proteins and baculovirus bio-insecticides is disclosed. A liquid spray of budded form baculoviruses are employed to infect insect larvae in order to reduce the manpower for manual injections and the energy wasted in feeding infection technologies. The invention may be applied to the production of recombinant proteins and baculoviruses as the production platform for bio-insecticides.

The present invention discloses a recombinant adeno-associated virus preparation method and recombinant baculovirus. The recombinant adeno-associated virus preparation method specially comprises: (1) infecting a corresponding host packaging cell line with recombinant baculovirus constructed with a recombinant adeno-associated virus ITR core expression element and a Cap gene or Rep gene; (2) carrying out amplification culture on the host packaging cell line infected with the recombinant baculovirus in the step (1) to produce recombinant adeno-associated virus; and (3) separating and purifying the recombinant adeno-associated virus obtained in the step (2), wherein the recombinant adeno-associated virus has the genome constructed with the adeno-associated virus ITR core expression element and the corresponding serotype Cap gene or has the genome constructed with the adeno-associated virus ITR core expression element and the Rep gene. According to the present invention, the flexibility and versatility of the recombinant adeno-associated virus preparation method using the baculovirus system are substantially enhanced.

The invention discloses a method for improving expression of extraneous genes by insect transgene cells. In the method, active promoter controlled neomycin resistance gene expression cassettes, enhancer elements and extraneous gene expression cassettes inside the cells of insects are cloned into a vector with reporter genes based on transposon piggyBAC factors; subsequently, the vector is mixed with subsidiary plasmids expressing transposase to transfect insect cell lines; transgene insect cells are acquired through sectionalized screening of G418. Engineering cells expressing extraneous genes at high level are acquired by cell clone technology in combination practical examination of expression level of extraneous genes. With the method, transgene insect cells can express extraneous genes at high level continuously; the expression products are free of rhabdoviruses with good bio-safety, and are processed perfectly as well as natural.

The invention relates to a porcin circovirus type 2 (PCV2) recombinant autographa californica multicapsid nucleopolyhedrovirus construction method and a subunit vaccine preparation method thereof. The novel recombinant lucerne fork vein noctuid nuclear polyhedrosis virus construction method comprises the steps that: the main surface antigen which expresses the PCV2 is used to prepare the subunit vaccine with better immunity. Specifically, in the carrier expression system of the PCV2 recombinant autographa californica multicapsid nucleopolyhedrovirus, a hepatitis E virus open reading frame (ORF2) gene of the PCV2 is directionally inserted, so the gene efficiently expresses in insect cells and has good immunogenicity, the prepared corresponding subunit vaccine can stimulate pigs to generate the protective immunoreaction against the attack of the strong virus of the PCV2.

This invention relates to the methods of detecting Penaeus monodon baculovirus (MBV). Two methods are established: the first one is a polymerase chain reaction (PCR) and the second one is an ELISA. For the PCR method, two sets of primers are designed. The first set of primers is designed from the conserved sequences of nuclear polyhedrosis viruses (NPVs) DNA polymerase genes. The second set of primers is designed from the genomic DNA of MBV. The antibody for ELISA is an antiserum against the occlusion bodies of MBV.

The invention discloses a method for producing human collagen type II, and belongs to the technical field of gene engineering. The method is characterized in that a baculovirus multi-gene expression system is adopted, insect cells are infected by the bacteria converting the baculovirus multi-gene expression system to produce baculovirus with multiple genes expressed, the virus liquid is infected in the insect cells or injected into silkworm larva bodies to produce recombinant human collagen type II full-length protein, the protein structure is that 3 peptide chains with (G-X-Y) n repetitive sequences form a three-strand helical structure, to both keep the conformation of the natural collagen type II and have the activity of the natural collagen type II. By adopting the method disclosed by the invention, multiple genes can be expressed to facilitate the production of then collagen type II full-length protein; the method disclosed by the invention is simple in process method, simple and convenient to operate and short in process route.

A method for constructing a recombinant double-expression silkworm polyhedral baculiform virus which contains the SOD gene comprises that the general RNA of the silkworm is extracted and an MnSOD primer is designed; a single-stranded cDNA is synthesized and an MnSOD gene is synthesized; the MnSOD gene is connected with a p FastBacHTa to transform XL-Blue cells; and the recombinant donor plasmid p FastBacHTa / MnSOD can be extracted; the baculiform virus DNA of the silkworm is taken as the template to synthesize the silkworm polyhedral gene and the gene is cloned to the p FastBac Dual plasmid; the MnSOD gene is inserted under the P10 promoter of the double-expression plasmid to construct the double-expression vector: Bm polyhedrin pFB dual / MnSOD; after the transinfection of the Bm polyhedrin pFB dual / MnSOD, the double-expression virus DNA which contains the MnSOD gene and the polyhedral gene is distilled; the DNA is transduced into the silkworm culture cell of transinfection with the mediation of transinfection reagent lipidosome to obtain the recombinant virus. Virus particles which can feed and infect the silkworm is provided and the object gene MnSOD is expressed by the silkworm and large-scale and industrial production is realized.

The invention discloses PRRSV (porcine reproductive and respiratory syndrome virus) virus-like particles with immunogenicity as well as preparation and application thereof, and belongs to the technical field of agricultural veterinary biology. The preparation comprises the following steps: cloning coding sequences of N protein and GP5 protein of PRRSV into rod granules to obtain recombinant rod granules; infecting the recombined rod granules on insect cells, culturing the transfected insect cells to obtain recombinant baculovirus. The N protein and GP5 protein are expressed by adopting the recombinant baculovirus infected insect cells, protein with better steric configuration can be obtained, and PRRSV virus-like particles can be automatically assembled. The virus-like particles with high immunogenicity can be used for preparing vaccine for preventing infection of PRRSV. The PRRSV virus-like particles have high immune protective rate, can produce attacking protection on PRRSV virulent strain, can be safer and more effective, and do not have the risk of virulence return.

A recombinant baculovirus displaying on its envelop a fusion protein is disclosed. The fusion protein comprises a heterologous antigen, and a C-terminal region of baculovirus envelope GP64 protein, which has at least 100 amino acid residues in length and lacks a B12D5 binding epitope located within the central basic region of the GP64 protein. The genome of the recombinant baculovirus comprises a transgene encoding a fusion protein that comprises a signal peptide, the heterologous antigen, and the C-terminal region of the baculovirus envelope GP64 protein, in which the antigen is located between the signal peptide and the C-terminal region of the GP64 protein. Methods for eliciting an antigen-specific immunogenic response in a subject in need thereof are also disclosed.

The invention discloses a preparation method and system of recombinant adeno-associated virus (rAAV), and recombinant bacmid. The method comprises: (1) separately preparing shuttle plasmids and a corresponding recombinant bacmid containing baculovirus genome; (2) integrating a rAAV core expression element which has a heterologous functional gene fragment with other expression cassettes which produce functional protein components necessary for rAAV so as to obtain a recombinant bacmid containing recombinant baculovirus genome producing the rAAV; and (3) transfecting the obtained recombinant bacmid into a host cell line for culturing. The system comprises the shuttle plasmids and the corresponding recombinant bacmid containing the baculovirus genome. The recombinant bacmid comprises at leastone expression cassette that produces functional protein components necessary for rAAV. The system has flexibility, compatibility, and higher passage stability.