171 results about "Sf9" patented technology

The invention relates to a preparation method of virus-like particles (VLPs) of Chikungunya virus (CHIKV). The method comprises the steps of: modifying genetic elements of a structural protein encoding gene C-E3-E2-6K-E1 of CHIKV, cloning the modified genetic elements into the expression vector of an insect cell, then transfecting the obtained recombined expression vector and baculovirus linear DNA respectively to an SF9 insect cell and making the cell secrete and express CHIKV VLPs. Additionally, the invention also makes preliminary studies on the immune effects of CHIKV VLPs and applicationof CHIKV VLPs in virus specific antibody detection, thus laying a foundation for research and preparation of immunological detection reagents and even vaccines based on CHIKV VLPs.

The invention discloses porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as a preparation method and application thereof. Sequences of VP0, VP1 and VP3 genes are artificially synthesized by referring to an FMDV (Foot And Mouth Disease Virus) O-type epidemic strain gene sequence; the VP0, VP1 and VP3 genes are connected to pFBDPHmHNM1P10eGFP plasmid by adopting the plasmid as a framework vector, so that a baculovirus transfer vector pFBDPHmVP013 is obtained. The baculovirus transfer vector pFBDPHmVP013 is mixed with DH10Bac escherichia coli competent cells, and the positive bacterial colony is selected to obtain a recombinant shuttle vector Bacmid; the shuttle vetcor Bacmid is transferred with a sf9 cell, and the recombinant baculovirus QP-Ac-FVLP is obtained by collecting the cell supernatant. The recombinant baculovirus can be used for efficiently expressing FMDVVP0, Vp1 and Vp3 proteins and forming virus-like particles. And the virus-like particles are used for preparing subunit vaccine, so that the organism is induced to generate specific immunity response after the mouse is immunized.

The invention relates to virus-like particle recombinant protein of a virus variation strain VP2 gene of an infectious bursal disease, belonging to the field of biologic pharmacy. The IBDV variation strain (AH1) VP2 gene is cloned, converted and transfected to obtain a recombinant baculovirus vBac-VP2; an infected Sf9 insect cell has specific fluorescence, and the antigen valence of the infected Sf9 insect cell is above 1.6*10<3>; the molecular weight of a recombinant VP2 protein is 53kDa, and the recombinant VP2 protein is in the state of virus-like particles; an indirect ELISA detection method established for an envelope antigen by the purified recombinant VP2 protein has good specificity and sensibility; an immune chicken can resist IBDV virulent attack, and the protection ratio achieves 100 percent. The novel virus-like particle recombinant VP2 protein prepared by the IBDV variation strain VP2 gene has high pertinence on the immune prevention of a current prevalent IBDV virulent strain and good practical value and popularization prospects.

The invention relates to recombinant baculoviral of capsid protein of rabbit viral hemorrhagic disease viruses and gene engineering vaccines, which belong to the bio-pharmaceutical field. A capsid protein VP60 whole gene of the rabbit viral hemorrhagic disease viruses is amplified through RT-PCR, a baculoviral recombinant vector is constructed through the molecular biotechnology, Sf9 cells are transfected, which enables the VP60 gene to obtain expression, and then gene engineering vaccines of the rabbit viral hemorrhagic disease are prepared through taking the capsid protein VP60 of the rabbit viral hemorrhagic disease viruses of the recombinant adenovirus expression as antigen and adding corresponding adjuvant. The invention can provide the capsid protein gene engineering vaccines of the rabbit viral hemorrhagic disease with good immune effects and simple technology, which is used for preventing and controlling the rabbit viral hemorrhagic disease.

The invention discloses recombinant porcine reproductive and respiratory syndrome virus (PRRSV) virus-like particles (VLP) and a preparation method and an application thereof. Based on comparative analysis of GP5 of a PRRSV epidemic strain and an M gene sequence, a GP5 and M tandem sequence GP5M is synthesized artificially, the synthesized GP5M gene sequence is cloned into a vector with a pBAC5 plasmid as a skeleton, the baculovirus transfer vector pBAC-PRRSVGP5M is obtained, the recombinant bacmid rBacmid-GP5M is obtained, sf9 cells are transfected with the bacmid, and the recombinant baculovirus Ac-PRRSVGP5M is obtained. The PRRSV GP5 and M protein are expressed efficiently by the recombinant baculovirus, and the virus-like particles are formed. A subunit vaccine prepared by the proteinexpressed by the recombinant baculovirus can induce a body to produce a specific immune response after immunizing animals and can protect the pig body against the strong poison attacking of porcine reproductive and respiratory syndrome virus.

The invention discloses a snake venom metal protease inhibitor BJ46a capable of resisting attack and transference of tumor and application thereof, and belongs to the field of medical organism. In the invention, according to the BJ46a gene sequence (AF294836) in GenBank, the BJ46a full gene is designed and synthesized. An expression vector cloned to baculovirus can generate the recombined BJ46a protein capable of inhibiting the activity of the substrate metal protease in Sf9 insect cells, and in vivo and in vitro experiments show that the BJ46a protein has the functions of resisting the attack and transference of melanoma cells B16. The inhibitor adopts the genetic transfection technique, and can establish B16/pcDNA3.1HisC-BJ46a cell strains with stable transfection, and the in vivo and in vitro experiments prove that the BJ46a can inhibit the attack and transference of B16 cells at the gene level. The inhibitor is applied to preventing and treating the attack and transference of tumor, and has great application prospect.

The invention relates to a rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida bivalent inactivated vaccine and a preparation method thereof, and belongs to the field of immune technology. Recombinant rabbit hemorrhagic disease virus VP60 baculovirus is inoculated into Sf9 insect cells and cultured at 27-28 DEG C. When cell lesion reaches 85% or more, a cell culture is harvested and inactivated, and the inactivated cell culture is used as a rabbit hemorrhagic disease virus antigen. Rabbit Pasteurella multocida capsular serotype A C51-17 strain is amplified and cultured, a bacterial solution is inactivated, and the inactivated bacterial solution is used as a Pasteurella multocida antigen. The rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida bivalent inactivated vaccine can be prepared by mixing the rabbit hemorrhagic disease virus antigen and the Pasteurella multocida antigen with adjuvants in proportion. The rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida bivalent inactivated vaccine has high safety, good immune effect and simple process, and can be used for preventing and controlling Rabbit Hemorrhagic Disease (Rabbit Plague) and Rabbit Pasteurella multocida.

The invention relates to an insect cell line and discloses a Sf9 cell line free from Sf-RV pollution as well as a screening method and application of the Sf9 cell line. The cell line is screened fromcommercial Sf9 cells, named as an Sf9-ZY cell line and has the biological collection number of CCTCC NO:C201952. Detection of the proliferation level and protein expression quantity of baculovirus inthe Sf9-ZY cell line indicates that the Sf9-ZY cell line can replace Sf9 cells to serve as host cells of an insect cell-baculovirus expression vector system (BICS) to be applied to expression of recombinant protein for preparing biological products. The Sf9-ZY cell line provided by the invention solves the problem that host cells of the insect cell-baculovirus expression vector system (BICS) are polluted by Sf-RV and guarantees safety of the biological products.

This invention provides a kind of preparation method of gene vector which made chitosan as matrix, and made haluronic acid as target molecule. The method is: at 37DEG C, use hydoscine N butyl bromide degrade natural haluronic acid to the desired length, after dislysis and purification then freezing and drying, then according to the specific quality proportion, at 37DEG C coupled with four kinds of chitosans with different molecular weight catalysed by the EDC, and formed different target chitosan gene vector with four kind of different molecular weight. Dissolve the obtained gene vector with 5 mmol/l pH 5.5 buffer solution of acid acetic and sodium acetate to get 0.1 - 10 mg/ml solution, after the 0.22 mm membrane filter, composite with the reporter gene with FP to form a series of composition with quality proportion of vector and plasmid DNA. Using it to transfect Insect cell lines Sf9, can achieve the same or better transfection efficiency with the commercial transfection agent liposome. The effect of using this vector transfect silkworm is excellent. The best transfection effeciency is over 50%. This vector is safe and innocuity, cheap and easy to use.

The invention discloses a construction method for carriers for prokaryotic expression and eukaryotic expression of candidate gene of porcine rotavirus vaccines VP4 and VP7, comprising: cloning and prokaryotic expression of genes of porcine rotavirus VP4, construction and transfection of the recombinant vector of genes of rotavirus VP4 and VP7 and the expression carriers of insect cell sf9, and the construction of a recombinant vector of genes of rotavirus VP4 and VP7 and the expression vector of yeast cells GS115.

The invention discloses a main cis-acting element of a shrimp white spot syndrome virus (WSSV) iel promoter and a transcription factor combined with the same and application. In the invention, by starting from transcriptional regulation of WSSV iel and carrying out structural and functional analysis on the promoter of the WSSV iel through deletion and mutation, a 12-bp DNA is found to be the maincis-acting element of the WSSV iel and is a crucial factor for the high expression of the iel. In the invention, a DNA affinity chromatography method is used for purifying a protein combined with a DNA segment from a nucleus protein Sf9, the protein is identified to be PHB2 (Poly-Beta-Hydroxybutyrate 2) through biological mass spectrometry, and the interaction between the protein and the DNA segment is proved to be specific by an electrophoretic mobility shift assay. Experiment results prove that PHB2 serves as a transcription factor and is specifically combined with a 12-bp DNA sequence of the iel promoter to start WSSV immediate early gene transcription so as to further regulate the replication of the WSSV, and can be used as an effective action target of medicaments for screening medicaments for resisting the shrimp WSSV.

The invention relates to the biotechnical field, and in particular relates to a recombination cell line and a method for determining the titer of baculovirus. A construction method of the recombination cell line for determining the titer of the baculovirus comprises the following steps: constructing reporter plasmids; transfecting Sf9 insect cells through the reporter plasmids; screening the transfected Sf9 insect cells through a culture medium containing bleomycin and fetal bovine serum to obtain single cell clones; continuously screening the single cell clones through the culture medium containing the bleomycin to obtain the recombination cell line. According to the recombination cell line and the method for determining the titer of the baculovirus, the titer of the baculovirus can be quickly determined, and a determination result is accurate.

The invention discloses a harmaline oxazoline compound and a preparation method and application thereof .The structural formula of the harmaline oxazoline compound is shown as the formula (I) in the description, wherein in the formula (I), R1 is methyl or phenyl or p-chlorophenyl or o-hydroxybenzyl, and R2 is o-hydroxybenzyl or 3-methoxy-4-hydroxyphenyl or 3,5-dimethyl-4-hydroxyphenyl or 2-furyl .The compound has significant cytotoxic activity on spodoptera frugiperda cultured cell lines Sf9, has good insecticidal activity on culex fatigans and lipaphis erysimi, has good growth and development regulating activity on prodenia litura, and also has good inhibitory activity on rhizoctonia solani, colletotrichum gloeosporioides, phytophoria melonis, fusarium oxysporum f .sp .niveum, pseudoperonospora sp, botrytis cinerea and fusarium oxysporum f.sp.cubense .Besides, the compound is simple in structure, easy to synthesize, simple in synthetic process, high in product purity and suitable for large-scale industrial application and popularization.

The invention discloses an optically-controlled expression vector for insect cells and application. An applicant successfully constructs a positive plasmid pMIB-C120-mCherry-GFP-EL222 and a control plasmid pMIB-C120-mCherry-GFP on an insect cell expression vector pMIB/V5-HisA based on a VP-EL222 optically-controlled transcription activating system; after the positive plasmid pMIB-C120-mCherry-GFP-EL222 is transfected with Sf9 cells, a green fluorescent protein expresses while a red fluorescent protein does not express; after the positive plasmid pMIB-C120-mCherry-GFP-EL222 is irradiated and stimulated by blue light, the red fluorescent protein expresses; after the control plasmid pMIB-C120-mCherry-GF is transfected with the Sf9 cells, the green fluorescent protein expresses while the red fluorescent protein does not express; after the control plasmid pMIB-C120-mCherry-GF is irradiated and stimulated by the blue light, the red fluorescent protein still does not express due to no EL222 transcription factor. An optically-controlled inducible expression system is successfully applied to the insect cells for the first time, and the application range of the technology is widened. According to a verification method constructed by the invention, the plasmids also provide a flexible and easy-to-use method for using an optically-controlled induced expression technology.

The invention discloses a II-set alphabaculovirus shuttle expression vector MbBacmid capable of expressing foreign protein in various insect cells and application thereof. The Bacmid contains whole genome sequences of mamestra brassicae nuclear polyhedrosis viruses, and contains genetic elements capable of being copied in escherichia coli. Foreign protein genes can be quickly inserted into the Bacmid provided by the invention through a donor plasmid pFast-Bac Mbph, and are efficiently expressed inside various commercial insect cells including Sf9, Tn368, high five and the like. The vector and a donor plasmid pFast-Bac Mbph+piel-cath can be subjected to homologous recombination to obtain a recombinant virus MbMNPV-ph+ piel-cath capable of increasing insect killing speed. The expression system provided by the invention is the only II-set alphabaculovirus shuttle expression vector system capable of efficiently expressing foreign protein in various commercial insect cells.

The invention provides a large-scale preparation method of new H5N1 avian influenza virus-like particle vaccines, which sequentially comprises the following steps: (1) constructing and extracting the recombinant plasmid rbacmid/HANAM1; (2) transfecting the recombinant plasmid rbacmid/HANAM1 to Sf9 insect cells to revive the recombinant baculovirus, and assembling virus-like particles in the Sf9 cells; and (3) culturing and recombining the Sf9 cells in a large scale, and purifying the virus-infected particles and the virus-like particles. The method is suitable for large-scale preparation of human anti-highly pathogenic H5N1 avian influenza VLPs vaccines, and has the advantages of safety, high efficiency and good development and application prospects.

The invention discloses a method for realizing efficient covering of a foreign protein by using an insect virus polyhedron, wherein the polyhedrin of a bombyx mori cytoplasmic polyhedrosis virus (BmCPV) and a target protein gene are constructed under the PH and P10 promoters on a pFastBac Dual carrier, and a baculovirus capable of expressing the polyhedron and the target protein simultaneously can be obtained through recombination. The method is characterized in that the cytoplasmic polyhedrosis protein gene and the target protein gene are simultaneously constructed under the PH and P10 promoters on the pFastBac TMDual carrier through a baculovirus expression system, respectively, a flexible link peptide is introduced between the target protein and a signal peptide, and the recombinant baculovirus is obtained through recombination with a virus skeleton. Once Sf9 or Sf21 insect cells are infected by the single virtus, the desired covered protein can be directly generated in cells; the recombinant virtus also can be amplified.

The invention discloses a method for efficiently producing recombinant avian adeno-associated virus. The method comprises the steps of first respectively constructing recombinant baculovirus rBacRep for expressing functional protein genes Rep78 and Rep52 of avian adeno-associated virus (AAAV), recombinant baculovirus rBacVP for expressing three structural protein genes VP1, VP2 and VP3 of the AAAV and recombinant baculovirus rBacGFP for expressing green fluorescence protein reporter genes; infecting insect cells Sf9 with the rBacRep, the rBacVP and the rBacGFP simultaneously, and obtaining rAAAV-GFP. According to fluorescent quantitative polymerase chain reaction (PCR) detection, each Sf9 cell can generate about 28000 VG of rAAAV-GFP, and the yield is improved by about 20 times compared with a conventional three-plasmid method. The method not only is simple and convenient in operation procedure and greatly improves virus titer, but also is easy to produce in a large scale, and the bottleneck of using rAAAV as a novel virus gene transfer vector is overcome.

A process for preparing the recombinant chicken gamma-interferon (rCHIFN-gamma) with high activirus activity includes such steps as cloning ChIFN-gamma gene from eukaryotic plasmid to transfer carrier by dual enzyme servenings to obtain recombinant transfer plasmid pFASTBACI-ChIFN-gamma, taking DH10Bac competent cell, adding IngpFASTBAC1-ChIFN-gamma, transposition, diluting to culture object by SOD culture medium, coating on Luria plate, culturing, choosing white clony, purifying, naming positive plasmid as Bacmid-ChIFN-gamma, extracting its DNA, and transfecting Sf9 cells. It can be used to suppress the pathogenic action of both MDV GA to CEF and AIV(H5NI) virus to cell, and the reproduction of NDV F48E8 on cells.