33 results about "Bombyx mori nuclear polyhedrosis virus BmNPV" patented technology

The invention relates to a rapid detection kit and a detection method for bombyx mori nuclear polyhydrosis virus, specifically relates to a quick and convenient detection kit for detecting the bombyx mori nuclear polyhydrosis virus by combining the loop-mediated isothermal amplification technology and the nucleic acid detection lateral chromatography test paper, and a detection method of the detection kit, and belongs to the technical field of viral epidemic disease diagnosis. The detection kit is characterized by comprising a BmNPVLAMP pre-reaction liquid, wherein the desired primer and probe are designed according to the conserved region of the genomic sequence of the bombyx mori nuclear polyhydrosis virus published by GenBank; the kit is capable of effectively and quickly detecting the bombyx mori nuclear polyhydrosis virus; the steps of virus DNA extraction and BmNPVLAMP reaction system amplification are performed, and after the reaction ends, the result is judged by use of the quick nucleic acid detection test paper to obtain that the BmNPV virus DNA is efficiently and specifically amplified; the detection method is simple, convenient and quick, good in specificity, and high in sensitivity, and is suitable for farmers to use daily and advantageous for diagnosing and preventing the bombyx mori nuclear polyhydrosis virus disease.

The invention relates to the effect of small molecule compound prodiginine (PG) for inhibiting BmNPV (bombyx mori nuclear polyhydrosis virus), and application thereof in prevention and control of BmNPV. Cell assay indicates that PG can interfere with proliferation of BmNPV in cells in a concentration range of 0.01-0.8 mu g/L to stop formation of virus polyhedron, and can selectively cause death of silkworm cells infected with BmNPV in a concentration range of 1-300 mu g/L with the death rate being over 97 percent; PG does not show toxicity to normal cells of silkworm in the application ranges. Individual tests indicate that the silkworms infected with BmNPV stop taking food and prematurely die, infected silkworms can be removed easily, and prodiginine does not show toxicity to healthy cells when prodiginine is used for orally feeding silkworm in a concentration range of 1-10mg per silkworm. Proliferation of BmNPV and formation of polynedron can be inhibited by utilizing PG, and PG has the effect of accelerating death of BmNPV, and can prevent and effectively control spread of BmNPV.

The invention discloses a bombyx mori nuclear polyhydrosis virus (BmNPV) 39k inducible promoter and application thereof. A nucleotide sequence of the promoter is shown as SEQ ID No.1; the promoter has obvious BmNPV inducible promotion activity and can make cells start foreign gene expression when the cells are subjected to the BmNPV infection; an RNAi vector using a BmNPV multiplication essential gene as a target can be built by utilizing the promoter; the abundant expression of shRNA can be performed when the bombyx mori is subjected to the BmNPV infection; the shRNA is cut into siRNA in a cell by an enzyme; specific degradation is performed on the BmNPV multiplication essential gene mRNA to initiate the BmNPV multiplication essential gene to be transcribed and then silenced, so the aim of inhibiting the virus multiplication is fulfilled, and the promoter can be used for preventing and controlling the BmNPV infection of the bombyx mori, performing genetic engineering breeding of bombyx mori anti-BmNPV strains and providing a good reference mode for transgenic therapy of other biologic virus diseases.

The invention discloses an application of a bombyx mori nuclear polyhedrosis virus (BmNPV) polygene inverted repeat sequence and a bombyx mori lipase-1 gene and specifically an application of the BmNPV polygene inverted repeat sequence and the bombyx mori lipase-1 gene to the construction of a recombinant vector; the vector can express a hairpin RNA (Ribonucleic Acid) for interfering with BmNPV multiple genes and bombyx mori lipase-1; BmNPV is inactivated by the incremental expression of bmlipase-1 (bombyx mori lipase-1) in the intestinal juice of a bombyx mori, and the number of BmNPV which invades intestinal cells of the bombyx mori is reduced; the expressed hairpin RNA can degrade the mRNA (Messenger Ribonucleic Acid) of a BmNPV gene and inhibit the replication and the proliferation of the virus in the Bombyx mori; and after the vector is used for producing a transgenic bombyx mori, the proliferation of BmNPV in the transgenicbombyx mor can be effectively inhibited, and the virus content is reduced, so that the survival rate of the bombyx mori infected with BmNPV is greatly increased.

The invention relates to a genetic modification method capable of enhancing disinsection efficiency of baculovirus, belonging to the field of gene engineering. The method comprises the following steps: amplifying EGFP gene by using a pEGFP-N1 vector as a template, carrying out double enzyme digestion, and connecting into a plasmid pFastBacDUAL to obtain a recombinant plasmid pDUAL-EGFP; designing and synthesizing primers according to the Clbi138 gene sequence and open reading frame thereof; carrying out PCR (polymerase chain reaction) amplification by using greenish brown hawk moth karyotype polyhedrosis virus genome DNA (deoxyribonucleic acid) as a template, recovering the target segment, carrying out enzyme digestion, and connecting into the recombinant plasmid pDUAL-EGFP subjected to double enzyme digestion to obtain a recombinant plasmid pDUAL-EGFP-Clbi138; and transforming a Escherichia coli strain containing silkworm karyotype polyhedrosis virus, culturing, purifying, inoculating into an LB (Langmuir-Blodgett) liquid culture medium, carrying out shake culture, and extracting DNA of the recombinant BmNPV. The experiment proves for the first time that the disinsection efficiency of the recombinant BmNPV is obviously enhanced, the median lethal concentration is reduced by 11 times as compared with the control group, the median lethal time is shortened by 42.9% as compared with the control group, and the liquefaction in the sick polypide is more severe, thereby achieving the effect of enhancing control effects on pests. The recombinant BmNPV has obvious economic and ecological benefits, and has wide application prospects.

The invention discloses an LAMP primer combination for detecting Bombyx mori nuclear polyhedrosis virus and a kit. The primer combination comprises external primers, internal primers and a loop primer, wherein the external primers F3 and B3, the internal primers FIP and BIP and the loop primer LF have base sequences of a sequence list from SEQ.ID.No.1 to SEQ.ID.No.5. The primer combination is contained in the kit. By means of the LAMP primer combination for detecting Bombyx mori nuclear polyhedrosis virus and the kit, Bombyx mori nuclear polyhedrosis virus can be detected only by performing DNA extraction and loop-mediated isothermal amplification on a tissue sample, thus early diagnosis of Bombyx mori nuclear polyhedrosis virus in a production field can be carried out quickly, accurately and easily. Compared with the prior art, the LAMP primer combination for detecting Bombyx mori nuclear polyhedrosis virus and the kit have the advantages of high specificity, high sensitivity, quick result obtaining and no pollution.

The invention discloses a Cas9 system for efficiently editing a bombyx mori genome and an application of the Cas9 system. The system comprises a bombyx mori transgenic expression vector for expressing a Cas9 gene and bombyx mori nuclear polyhedrosis virus packaging virion containing an expression cassette U6-sgRNA expressed by a U6 promoter regulating sgRNA. A bombyx mori nuclear polyhedrosis virus (BmNPV) genome is used as a vector, the U6-sgRNA expression cassette is recombined to the virus genome through Bac-to-Bac transposition, a recombinant virion is constructed, a Cas9 cell line or Cas9 bombyx mori is infected with the recombinant virus, and a target gene can be effectively edited. The established Cas9 system does not need a transfection process, is high in transfection efficiency and good in safety, can stably and efficiently edit a target gene, and can be suitable for research on functions of viral genes and related host genes, a baculovirus expression system and the like.

The invention discloses an electrochemical immunosensor for detecting a bombyx mori nuclear polyhedrosis virus and a detection method thereof. The preparation method comprises the following steps: modifying a gold electrode by using an undecylmercaptoundecanoic acid/beta-mercaptoethanol mixed self-assembled membrane, wherein the hybrid self-assembly membrane is used for immobilizing a BmNPV polyhedrosis protein antibody; completing the capturing of BmNPV polyhedrosis protein through specific binding of an antigen and the antibody, and achieving the ultra-sensitive detection of the bombyx morinuclear polyhedrosis virus based on the electro-catalysis effect of the hybrid self-assembly membrane. The detection method has the characteristics of simplicity, convenience, quickness, good specificity and high sensitivity, the linear range is 0.0001-100ng/mL, the detection limit is 14.54 fg/mL, and the detection method is suitable for early-stage quick diagnosis and prevention of the bombyx mori nuclear polyhedrosis.

The invention discloses a bombyx mori actin 6A gene and application in detection of methylation level of the bombyx mori actin 6A gene. The CG methylation level of the gene in a 1884-2071bp nucleotideregion is obviously higher than that of normal cells in silkworm cells infected with nuclear polyhedrosis viruses, and the nucleotide sequence of the region is as shown in SEQ ID NO. 1. The inventionalso provides a primer pair, a kit and a detection method for specifically detecting the CG methylation level in the region. The forward sequence of the primers is shown as SEQ ID NO.2, and the reverse sequence is shown as SEQ ID NO.3. The invention provides a new research direction for researching the interaction mechanism of bombyx mori DNA methylation and the nuclear polyhedrosis virus. The primers and the detection kit for detecting the methylation level of the gene disclosed by the invention have important practical application value for detecting the bombyx mori nuclear polyhedrosis virus.

The invention discloses a probe for rapidly detecting a bombyx mori nuclear polyhedrosis virus. The bombyx mori nuclear polyhedrosis virus can be rapidly detected through an RAA technology. The invention further discloses a kit for rapidly detecting the bombyx mori nuclear polyhedrosis virus, and the kit is rapid in detection and high in sensitivity. The invention further discloses a detection method of the kit for rapidly detecting the bombyx mori nuclear polyhedrosis virus, the steps of reagent mixing and sample adding are reduced, operation is convenient, and time is saved.

The invention discloses a method for preparing analgesic polypeptide from silkworms and belongs to the field of gene engineering. The preparation method comprises steps as follows: a conotoxin omega-MVIIA gene sequence is optimized according to BmNPV (Bombyx mori Nucleopolyhedrovirus) codon usage frequency, genes obtained after chemical synthesis are linked to a donor plasmid pFastBacDual and introduced to EGFP (enhanced green fluorescent protein) genes, and a recombinant donor plasmid pFBD-MVIIA/EGFP is established. MVIIA/EGFP is transposed to BmNPV with a Bac-to-Bac method, and a recombinant virus vBmMVIIA/EGFP is formed. DNA of the recombinant virus is transfected to cells of the silkworms, BV particles are obtained and injected into the silkworms at 200 pfu after the titer of the BV particles is tested by the aid of green fluorescence, and blood of the silkworms is collected after five days of transfection. Acetic-acid-induced mouse writhing experiments show that the blood of the silkworms infected with expressed conotoxin omega-MVIIA has remarkable analgesic activity, and the number of the average writhing times is only 3.55 and is remarkably higher than 37.30 in a control group of mice injected with normal saline. A new method is provided for expression of conotoxin omega-MVIIA with analgesic activity, and has potential application value.

The invention discloses application of 5-Pyridoxolactone in preparation of a medicine for treating bombyx mori nuclear polyhedrosis virus (BmNPV). Micromolecule 5-Pyridoxolactone in an anti-BmNPV host cell is reasonably utilized to inhibit the replication of BmNPV in the cell, that is, after the cell is infected by the BmNPV, the 5-Pyridoxolactone is firstly added to inhibit the replication of the BmNPV, so that a new thought is provided for treating the BmNPV infection of the cell, and a foundation is laid for virus infection prevention and treatment in sericulture production.

The invention discloses application of a bombyx mori BmUAP56 gene, and particularly discloses application of the bombyx mori BmUAP56 gene in cultivation of bombyx mori varieties resisting bombyx mori nuclear polyhedrosis viruses and preparation of drugs for inhibiting bombyx mori nuclear polyhedrosis virus proliferation. The nucleotide sequence of the BmUAP56 gene is shown in SEQ ID NO.3. The expression quantity of the BmUAP56 gene in bombyx mori is reduced through RNA interference or/and gene editing, or a reagent for inhibiting the binding of the bombyx mori UAP56 protein and the BmNPV virus protein 25K is used, and the binding force of the bombyx mori UAP56 protein and the BmNPV virus protein 25K can be weakened through BmUAP56 gene mutation, so that the capability of resisting bombyx mori nuclear polyhedrosis virus is obtained, and the bombyx mori UAP56 protein has a very good application value.

The invention discloses a method for screening bombyx mori nuclear polyhedrosis virus disease resistance with a BmIML-2 gene. According to the method, nuclear polyhedrosis virus is added during the last mulberry leaf feeding before mounting, and the transcription level of the BmIML-2 gene is adopted as the detection index to judge the resistance strength of bombyx mori to the virus. Compared withthe prior art, the method disclosed by the invention has the advantages that: (1) the virus is added before 5-instar mounting, so that the test period can be shortened, the workload is small, and theprobability of cross infection among bombyx mori is reduced; (2) the resistance of the bombyx mori variety screened by the method to the nuclear polyhedrosis virus is improved by 10-100 times; and (3)the bombyx mori variety screened by the method is stable in resistance and vigorous in physique after multi-generation feeding.