163 results about "Polyhedral virus" patented technology

The invention provides a method for producing porcine circovirus type II capsid protein by utilizing a silkworm bioreactor, and belongs to the field of biotechnology. In the method, by taking a bombyx nuclear polyhedrosis virus as a vector, porcine circovirus type II capsid protein genes are integrated into a polyhedrosis promoter of the bombyx nuclear polyhedrosis virus so as to express target protein by a mode of the homologous recombination of a homologous arm of the nuclear polyhedrosis virus in a transfer vector and bombyx nuclear polyhedrosis virus (BmNPV) genes; a recombinant bombyx nuclear polyhedrosis virus containing target protein genes is obtained by a plaque sieving method, and the target protein is expressed in large scale by using a bombyx bioreactor so as to prepare a subunit vaccine containing recombinant porcine circovirus type II capsid protein; and piglet infection experiments verify that the subunit vaccine has the excellent immune protective effect. The method has the characteristics of high expression efficiency, good activity of the target protein, low production cost and the like, and is suitable for large-scale production.

The present invention discloses beet noctuid nuclear polyhedrosis virus insect killing suspending agent, in which the virus bearer content is 5-20 hundred million PIB/ml., itis composed of chlorbenzuron analogue chlorfluazuron, aromatic amino acid tyrosine, fluorescent whitener, dispersant, glycerin, sterilized water. The present invention is harmless to man and livestock and other higher animals, has no pollution to environment and has no pesticide residue.

The invention belongs to the field of a biology technology, and relates to an expression vector. The expression vector is an expression vector for insect, and contains an AcIE1 promoter of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and a hr5 enhancer of a homologous region 5 and an Opie2 promoter of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). An insect host cell containing the expression vector and a method for preparing a recombinant protein by using the expression vector are provided.

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 preparation method of recombinant nuclear polyhedrosis virus (NPV) which infects ectropis oblique. The preparation method comprises steps that: (1) recombinant vectors pFastBacDual-polh-helicase are prepared; (2) the recombinant vectors pFastBacDual-polh-helicase are transformed into E.coli DH10 BmBacmid competent cells; the cells are cultivated on a cultivating plate; positive plaques are obtained through blue-white selection; and recombinant BmBacmid-polh-helicase DNA is extracted from the positive plaques; (3) the ecombinant BmBacmid-polh-helicase DNA is transfected to domestic silkworm cultivated cells; cultivated cell supernatant is collected; and recombinant virus BmEoNPV is obtained; (4) the recombinant virus BmEoNPV is used for injection; and recombinant virus polyhedron is obtained. The recombinant nuclear polyhedrosis virus is sprayed on tea tree leaves, such that the ectropis oblique can be prevented from harming the tea trees. The preparation method also has advantages of low cost and controllable productions.

The present invention discloses a human interleukin-12 recombinant insect virus strain, the recombinant Autographa california multiple nuclear polyhedrosis virus AcNPV-hIL12 virus strain, CCTCC No.2 V200108 and its preparation. The recombinant virus strain is inserted into human interleukin-12 expressing box, and the expressing box includes double promoter sequence, P35 subunit DNA encoding sequence of human interleukin-12 and P40 subunit DNA encoding sequence. P35 subunit gene locates in the downstream of Polyhederin and P40 subunit gene locates in the downstream of P10 promoter. The present invention can express human interleukin-12, and the cell factor is safe and reliable to human body and suitable for treating tumor bacteria, virus and various infections diseases.

The invention relates to an ELISA diagnosis kit for swine fever virus. In the invention, by gene splicing technology, the swine fever virus E2 is jointed with secretion signal peptide sequence and purification label at two sides and is inserted into the autographa californica nucleopolyhedrosis virus expression vector so as to construct recombinant autographa californica nucleopolyhedrosis virus CGMCC No.2671 and to obtain coated ELISA swine fever virus Shimen Strain E2 gene soluble expression antigen. The kit of the invention is rapid, sensitive, and specific to the diagnosis and the immune evaluation of swine fever virus, and the diagnosis can be completed once within 2 hours.

The invention discloses a method for finding a protein interaction with baculovirus PCNA (Proliferating Cell Nuclear Antigen). The method is characterized by comprising the following steps of cloning full-length DNA of an autographa californica nuclear polyhedrosis virus (AcMNPV) PCNA gene by utilizing the means such as molecular cloning, and DNA sequencing, purifying the full-length DNA, coupling the full-length DNA to an affinity column, and fining the interaction protein by adopting the mass spectrography. The method can be used for finding and functionally studying interaction protein of the baculovirus PCNA.

The invention provides a hyphantria cunea viral insecticide and a preparation method thereof. The preparation method comprises the following steps: feeding hyphantria cunea by virtue of artificial feed, preparing a nuclear polyhedrosis virus precipitate, and adding trace inorganic nano-materials, aromatic amino acids and a small amount of refined fluorescent whitening agent into the virus precipitate. According to the preparation method, the viral insecticide is complete in components and high in suspension property, the quality guarantee period is prolonged, the insecticide has a good protective effect against ultraviolet light during field application, the virus duration is long, and the control effect is greatly improved.

The invention is an application of cnidiadin as insect virus synergist to the biologic prevention and cure. Cnidiadin derives from common cnidium fruit extract, when used singly, has not pesticidal activity, but when associated with insect nuclear polyhedrosis virus (AcNPV, SINPV, MbNPV, etc.) and granulosis virus (XcGV, etc.), can remarkably enhance the infection capability of insect virus to pest. The dosage is low (1-228mg/L), and the concentration lower, the synergistic action stronger, safe to entironment.

The present invention discloses a method for preventing vegetable noctuid through propagating virus by attractant. According to the method, a noctuid attractant is used for inducing the imago to carry and propagate the nuclear polyhedrosis virus so that the virus propagates vertically and horizontally in the host group. The progeny larvae falls ill and dies after the female which copulates with the virus-taking male or the female carrying the virus spawns. The method of the invention can promote the natural propagation of virus in the field, reduces the dosage of virus insecticide and increases the effect of insect disinfestation with virus. Compared with the currently submerged virus releasing, the method of the invention has evident advantage and has great market development potential.

The invention mainly aims to provide an ectropis oblique warren nuclear polyhedrosis virus insecticidal suspending agent and a production method thereof. The insecticide consists of virus suspension, silicon dioxide, aromatic amino acids, a refined fluorescent whitening agent, sodium lignin sulfonate, polyvinyl alcohol, chlorfluazuron, glycerin and sterile water. The production method comprises the following steps: paired spawning on pupas by virtue of emerged adults, sterilizing the eggs, hatching larvae, feeding the larvae by using artificial feed, inoculating ectropis oblique warren virus on the feed surface to feed, grinding and filtering the larvae infected with the virus, centrifuging filtrate, collecting precipitates, and preparing the corresponding insecticide. By utilizing the insecticide, plant diseases and insect pests of ectropis oblique warren can be effectively controlled, the control cost is low, the environment friendliness is promoted, and the problems that the drug resistance of injurious insects is enhanced, the natural enemy number is reduced and pesticide residues in tea leaves are increased due to use of lots of chemical pesticides are solved.

The invention relates to a rabbit hemorrhagic disease virus recombinant subunit vaccine and a preparation method thereof. A vaccine production strain is a recombinant autographa californica nuclear polyhedrosis virus CGMCC No.8052 (China General Microbiological Culture Collection Center Number 8052), an expression vector adopted by the strain is a secretory expression vector and is provided with six histidine tags easy for protein detection and analysis; the strain is used as the production strain for producing the rabbit hemorrhagic disease virus recombinant subunit vaccine; the vaccine is free from viral genome, animal safety hazard and potential dispersity, is high in immunity pertinence, and can be used for exciting immunized animals to generate sufficient immune antibodies and long protective efficacy. RHDV-like (rabbit hemorrhagic disease virus-like) particles are produced according to the technology provided by the invention, the hemagglutination titer of virus liquid obtained from a 1000L bioreactor is (1 to 32768) to (1 to 262144), the protein expression quantity of the RHDV particles is about 150mg/L, 1L of semi-finished virus liquid can be prepared into 5,000-100,000 standard vaccines, and the preparation efficiency is higher than the domestic technical level by 8-20 times; the recombinant subunit vaccine is added with an adjuvant, so that the immune effect of the vaccine is improved; and the vaccine is a novel vaccine remarkably superior to an inactivated tissue vaccine produced in the prior art.

A fast doner plasmid with singly embedded polyhedronvirus gene of bollworm and P10 gene promoter sequence is configured through inserting exogenous gene in polycloning site under control of P10, transfecting it to bollworm cell by inserting the exogenous gene and polyhydrous gene in the shuttle expression carrier, collecting recombinant virus particles, transfecting again, and detecting the exogenous gene expression level. Its advantages is short period (7-14 days).

A single embedded polyhedronvirus shuttle expression carrier of bollworm is configured through inserting the low-copy bacterium F replicon, kanamycine resistant gene and beta galactosidase gene to the polyhedrom gene site of the virus genom. The doner plasmid is used to transfer the exogenous gene to the insetion site of transposon in the said shuttle expression carrier. After bollworm cell is transfected by said DNA, the infections recombinant virus particles are formed to express exogenous gene under control of polyhedron promoter and P10 promoter.

The invention discloses a virus insecticidal suspension agent containing an insect hormone insecticide. The virus insecticidal suspension agent comprises the following components in percentage by weight: 0.1-30% of the insect hormone insecticide, 1-10% of a dispersant, 1-7% of an antifreezing agent, 0.2-10% of a stabilizer, 0.1-5% of a synergist, 0.5-2% of an ultraviolet protecting agent, 0.1-2% of a defoaming agent, 0.1-3% of a pH regulator and the balance of water, wherein the prepared suspension agent also contains an insect virus which is one of or a combination of a nuclear polyhedrosis virus (NPV) and granulosis virus, and the content of an insect virus inclusion body in the suspension agent is 0.5-20 billion PIB/ml. The insect hormone insecticide and a virus insecticide of the virus insecticidal suspension agent disclosed by the invention are subjected to complex pairing, so that the problem of insect resistance can be solved, and the virus insecticidal suspension agent is safe and environmentally friendly.

The invention discloses a microbial pesticide for preventing and controlling prodenia litura. In the microbial pesticide, the prodenia litura larva, prodenia litura nuclear polyhedrosis virus production strain stock solution and fresh tobacco are used as major raw materials, and virus reproduction is performed. The microbial pesticide is prepared mainly by the following steps of: breeding the larva, inoculating the viruses, collecting the larva died of illness and performing crude extraction and filtration to obtain the virus stock solution; mixing the virus stock solution, glycerin and water at a weight ratio of 1:(1.2-4):(2.3-6) to prepare a virus stabilizer; preparing a virus mixture from the virus stock solution and 5% emamectin benzoate at a weight ratio of 1:(1.1-1.25); mixing and dissolving the virus stabilizer and the virus mixture; and adjusting the pH value to 6.8 with dilute sulphuric acid solution to obtain the microbial pesticide. The microbial pesticide disclosed by the invention is used for preventing and controlling the prodenia litura pests on crops such as vegetables, tobacco and the like, has high efficiency and environmental protection effect, and improves the quality and yield of the tobacco.

The present invention discloses one kind of compound pesticide prepared through mixing plant extract liquid and nuclear polyhedron virus liquid in the volume ratio of 1/9-9 at normal temperature. The plant extract liquid may be capsaicim or rotenone. The compound pesticide has high toxicity, high pesticidal effect, obvious synergistic effect, relatively low cost and wide latent market.

The technique to modify NPV and construct high-virulence NPV of Lepidoptera pest with modern dsRNA interference molecular biological technique comprises: (1) cloning CS conservative sequence of geometrid; (2) constructing the dsRNA intermediate carrier for the lepidopterid CS gene; (3) constructing the NPV transfer carrier contained CS dsRNA interference sequence; (4) recombining NPV, validating the NPV with PCR or other methods; finally, contrast with wild NPV, evaluating the insect disinfectation capacity of recombined NPV to screen the virus strain with higher virulence and faster diffusion and secondary infection speed and better prevention effect.

The invention discloses a manufacturing method and application of an insect in-vitro protein interaction detecting system. At the positions of amino acid residues at the 159th bit and the 160th bit, mCherry is divided into a segment NmC and a segment CmC through a PCR method, and then the segment NmC and the segment CmC are each fused with a connecting zone sequence, a c-Myc label and a poly(A) sequence to form four fused segments; the four fused segments are each connected with a pIE-MCS plasmid with an autographa californica multiple nuclear polyhedrosis virus ie1 gene promoter and a gp64 gene poly(A) sequence to establish expression plasmids; the expression plasmids are combined to form an insect cell dimolecular fluorescent complementary detecting system. The method is simple and easy to implement, and the established dimolecular fluorescent complementary detecting system expands the application of an insect cell expression system and provides a convenient and effective technological means for insect proteomics and research on protein interaction between insects and pathogenic microorganisms of the insects.

The invention discloses a method for using silkworm cultured cell to express antibacterial peptide Cecropin B. The method comprises the following steps: (1) using the total RNA extracted from the fat body cells of wild silkworm chrysalis as template, adopting RT-PCR amplification to obtain wild silkworm antibacterial peptide Cecropin B gene; using 1% agarose gel electrophoresis to perform PCR product analysis to the antibacterial peptide Cecropin B gene, using a PCR purification kit of Qiagene for purification, then cloning the purified PCR product to TA vector pCR2.1 to obtain pCR2.1-Cecropin B, utilizing the dideoxynucleotide chain termination to confirm the correctness of cloned gene order; using restriction endonuclease BamHI and HindIII to digest pCR2.1-Cecropin B and obtain Cecropin B genetic fragments, then cloning rhabdovirus transfer vector pBlueBacHisa in the genetic fragments to obtain reconstituted transfer vector; performing cotransfection of the reconstituted transfer vector and silkworm wild-type nuclear polyhedrosis virus BmNPV DNA in silkworm cultured cell, performing homologous recombination to generate recombinant virus; and (3) inoculating the recombinant virus containing wild silkworm antibacterial peptide Cecropin B gene in the silkworm cultured cell, infecting at 27 DEG C for three days to express the infection, and centrifuging to collect silkworm cultured cell.

The invention relates to a qualitative detection method of a tea geometrid nuclear polyhedrosis virus, which belongs to the technical field of the detection of biological preparations and is characterized by comprising the following steps of: firstly, preparing a polyhedrin monoclonal antibody of the tea geometrid nuclear polyhedrosis virus, then coating a sample to be detected and detecting by using an indirect Elisa method. The invention breaks through a traditional biological assay technology by means of an immunological means and solves the problem of accurate qualification of the tea geometrid nuclear polyhedrosis virus. Compared with a traditional biological assay method, the virulence index detection of the method is finer, the virulence evaluation period is greatly shortened, the method can be widely used for estimating the quality of a virus preparation and specification of the production and the application of a tea geometrid virus.