60 results about "Insect cell" patented technology

The invention discloses a bovine I-type alpha interferon-ferritin fusion protein, a mutant thereof, a preparation method and an application of the bovine I-type alpha interferon-ferritin fusion protein. The bovine I-type alpha interferon is fused with a ferritin subunit, and interferon molecules are highly repeatedly and orderly displayed on the surface of a ferritin nanocage by utilizing the self-assembly characteristic of ferritin, so that the expression level, the structural stability and the antiviral activity of the interferon are remarkably improved. The fusion protein is further subjected to single-site or multi-site rational design mutation, and a mutant with significantly improved antiviral activity and stability is obtained. According to the invention, a silkworm or insect cell eukaryotic expression system is adopted to express the fusion protein or the mutant thereof, and the expression system is safe to operate, simple and convenient in procedure, low in cost and extremely beneficial to large-scale industrial production; the prepared fusion protein or mutant nanoparticles have application prospects in preparation of drugs or reagents for preventing or treating bovine viral diseases.

The invention discloses a self-assembled ferritin nano antigen particle and application thereof in preparation of a duck hepatitis A vaccine. The VP1 part in the duck hepatitis A virus P1 protein is connected with the N end of a self-assembled ferritin nanoparticle subunit through a connecting sequence to obtain the fusion protein, and the amino acid sequence of the fusion protein is shown as SEQ ID NO.1; in order to improve the expression quantity or expression efficiency of the fusion protein in bombyx mori, a nucleotide sequence of a coding gene of the fusion protein is optimized and modified according to the codon preference of bombyx mori, and an optimized gene sequence with the nucleotide sequence shown as SEQ ID NO.2 is obtained; the fusion protein is subjected to single-site or multi-site mutation, the mutant with obviously improved titer is obtained through screening, and the soluble expression quantity of the mutant is obviously improved. According to the invention, a silkworm or insect cell eukaryotic expression system is used for expressing recombinant protein, and an expression product causes a neutralizing anti-duck hepatitis A virus antibody.

In various embodiments, provided herein are host cells, methods, and pharmaceutical compositions comprising gastrodin, wherein the gastrodin is produced by a genetically modified plant or plant cell, fungal cell, yeast cell, insect cell, or bacterial cell. In certain embodiments, the present disclosure provides methods and compositions for the production of gastrodin. In yet other embodiments, the present disclosure provides enhanced cells and methods for producing gastrodin.

The present application relates to the technical field of molecular biology, and particularly relates to a human parainfluenza virus type I HN recombinant antigen and application thereof. The amino acid sequence of the human parainfluenza virus type I recombinant HN recombinant antigen provided by the present application is shown as SEQ ID NO:1. The amino acid sequence provided by the present application can be efficiently expressed in insect cells, and a stable antigen can be obtained after purification and preparation of the expression product, and the antigen has high immunological activity and can be used for serum specific antibody detection or parainfluenza virus infection diagnosis.

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 bovine I-type tau interferon-ferritin fusion protein as well as a mutant and application thereof. The bovine I-type tau interferon is fused with a ferritin subunit, and interferon molecules are highly repeatedly and orderly displayed on the surface of a ferritin nanocage by utilizing the self-assembly characteristic of ferritin, so that a conformation suitable for the ferritin nanocage to play functions is formed, and the expression level, the structural stability and the antiviral activity of the tau interferon are remarkably improved. The fusion protein is subjected to rational design mutation to obtain a single-site or multi-site mutant with significantly improved antiviral activity and stability. According to the invention, a silkworm or insect cell eukaryotic expression system is adopted to express the fusion protein or the mutant thereof, and the expression system is safe to operate, simple and convenient in procedure, low in cost and extremely beneficial to large-scale industrial production; the prepared fusion protein or mutant nanoparticles can be applied to preparation of a plurality of drugs for preventing or treating bovine viral infection, tumors and immune system diseases and immunologic adjuvants for vaccine compatibility.

The application discloses a kind of avian influenza and avian infectious bronchitis two-union recombinant subunit vaccine and its preparation method and application.The recombinant protein is by replacing the head domain of AIV HA protein with the RBD domain of IBV S protein, while adding GCN4, Trimer-tag trimer tag in sequence, the recombinant protein obtained, the recombinant protein is expressed in vitro using insect cells and prepared into subunit vaccine.The vaccine prepared by the application can produce high-level neutralizing antibodies against homologous and heterologous H9N2 subtype avian influenza virus after immunizing chicken population, while significantly reducing the virus discharge level and the histopathological changes caused, and can also induce high-level specific antibodies and neutralizing antibodies against QX subtype avian infectious bronchitis virus, effectively inhibit discharge, viral load, and reduce tissue lesions.The application does not depend on chicken embryo, and has low production cost and short cycle, and can simultaneously prevent avian influenza and avian infectious bronchitis.

The invention provides a qPCR (quantitative polymerase chain reaction) primer probe group for detecting various nodaviruses, a detection method and application, and belongs to the technical field of molecular biological detection. Aiming at various nodaviruses capable of infecting insect cell lines, including animal shed virus, Tn5 cell line virus, black beetle virus, borawa virus, nodavirus and macrobrachium rosenbergii nodavirus, a set of primer probe combination is designed. In the combination, part of viruses share the upstream primer and the probe, so that synchronous detection and identification of multiple target viruses in one reaction system are realized. The invention also provides a kit containing the primer probe group and a detection method. The detection method provided by the invention has the advantages of wide coverage range, high detection sensitivity, strong specificity, good matrix interference resistance and the like, and is suitable for safety detection and quality control of nodavirus pollution in insect cell banks, cell cultures and related biological products thereof.

The application describes methods for synthetic synthesis and cell-free synthesis of DNA vectors, particularly closed-ended DNA vectors (e.g., ceDNA vectors) having linear and continuous structure for delivery and expression of a transgene. The present invention relates to an in vitro process for production of closed-ended DNA vectors, corresponding DNA vector products produced by the methods and uses thereof, and oligonucleotides and kits useful in the process of the invention. DNA vectors produced using the methods described herein are free from unwanted side effects due to contaminants introduced during production in cell lines, for example, bacterial or insect cell lines. Further provided herein are methods and cell lines for reliable gene expression in vitro, ex vivo and in vivo using the ceDNA vectors synthesized using the methods herein.

The invention relates to a preparation method and application of 6-methyl-3, 5-dioxocapryloyl coenzyme A. According to the preparation method, 2-methylbutyric acid serves as a starting raw material, the 6-methyl-3, 5-dioxocapryloyl coenzyme A is chemically synthesized through the steps of activation, condensation, coenzyme A coupling and the like, and the structure and purity of the 6-methyl-3, 5-dioxocapryloyl coenzyme A are identified through the technologies of LC-MS / MS, 1HNMR and the like. The nine-carbon coenzyme A is successfully prepared and identified through a chemical synthesis method for the first time, the core problem that an intermediate is unstable and cannot be obtained in vitro is solved, and a stable substrate is provided for in-vitro acyl transfer; the QS-21 in-vitro acyl transfer reaction is realized for the first time, an in-vitro catalytic system of a chemical synthesis intermediate and an insect cell expression enzyme is established, and a foundation is laid for development of a QS-21 total synthesis process.

The invention relates to a spider neurotoxin tail peptide with a cell transmembrane function and application of the spider neurotoxin tail peptide. The amino acid sequence of the spider neurotoxin tail peptide with the cell penetrating function is as shown in SEQ ID NO: 2. The invention also provides application of the cell-penetrating peptide in preparation of a carrier which is used for delivering bioactive molecules and can penetrate cell membranes. The invention further provides a fusion protein composed of the spider neurotoxin tail peptide and spider neurotoxin omega-Pp1b and application of the fusion protein in preparation of insecticides. The invention also provides an amphiphilic peptide and application thereof in preparation of a carrier which is used for delivering bioactive molecules and can penetrate through a cell membrane. The invention also provides a nano-composite and application of the nano-composite in preparation of a preparation for insect cell gene silencing. The invention also provides a pesticide composition. The invention is a novel cell-penetrating peptide derived from an insect system, and has important significance for promoting the development of peptide biological insecticides and RNA pesticides.

The present application relates to the application of enzyme-linked immunoassay for detecting the in vitro relative potency of HPV-VLP antigen stock solution and vaccine finished product (types 6, 11, 16 and 18). The enzyme-linked immunoassay of the present application has universality for detecting antigens produced by different expression systems (Hansenula, Pichia, Escherichia coli and insect cells), and improves the standardization level of HPV vaccine quality evaluation, and lays a foundation for promoting the research and development of high-quality HPV vaccine.

The present disclosure provides insect cell lines for expression of viral vectors, and methods of making and using the same. Provided methods employ use of identifiers that are capable of being packaged into a viral vector to select and / or identify insect cell lines with engineered sequences associated with beneficial characteristics for viral vectors production. Exemplary viral vectors include AAV vectors.

This invention discloses a SARS-CoV-2 virus-like particle, its preparation method, and its application. The genes encoding the structural proteins of the novel coronavirus include the S gene, M gene, or E gene. The nucleotide sequence of the S gene is shown in SEQ ID NO.1; the nucleotide sequence of the M gene is shown in SEQ ID NO.2; and the nucleotide sequence of the E gene is shown in SEQ ID NO.3. This invention provides, for the first time, a virus-like particle (VLP), which is self-assembled after expression of the three structural proteins S, M, and E of the SARS-CoV-2 virus via a baculovirus-insect cell expression system. This invention solves the problem of the current lack of a safe and effective VLP vaccine for the prevention and control of COVID-19. The VLP is suitable for mucosal immunization, improving the immunization effect while reducing vaccination and labor costs, and has potential economic benefits.

This invention relates to the field of biomedical technology, providing a rabbit hemorrhagic disease virus (RHDV) type 2 fusion antigen protein and its applications. The fusion antigen protein comprises the RHDV2 VP60 protein sequence and an immunomodulatory CRM197 protein functional fragment, fused together via a linker peptide, and expressed and purified in an insect cell expression system. This invention improves the immunogenicity of the antigen protein by fusing the major capsid protein VP60 of RHDV2 with the immunomodulatory fragment of CRM197. Rabbit immunization experiments verified that the fusion antigen protein can induce high-titer specific antibodies, with serum antibody titers reaching 1:12800 and above, demonstrating good immunization effects. The fusion antigen protein of this invention can be used to prepare a vaccine for the prevention of RHDV2 infection, providing a new technical solution for the prevention and control of RHDV2 disease.

The application discloses a bovine type I alpha interferon-ferritin fusion protein, a mutant thereof, and a preparation method and application thereof. The bovine type I alpha interferon is fused with a ferritin subunit, the self-assembly characteristics of the ferritin are utilized, the interferon molecules are highly repeated and orderly displayed on the surface of the ferritin nanocage, and the expression level, structural stability and antiviral activity of the interferon are significantly improved. The fusion protein is further subjected to rational design mutation at a unit point or multiple points, and a mutant with significantly improved antiviral activity and stability is obtained. The fusion protein or the mutant thereof is expressed by using a silkworm or insect cell eukaryotic expression system, the expression system is safe in operation, simple in procedure, low in cost, and extremely beneficial to large-scale industrial production, and the prepared fusion protein or mutant nanometer particle has application prospects in the preparation of medicines or reagents for preventing or treating bovine viral diseases.

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 present invention relates to means and methods for producing adeno-associated virus capsid libraries using insect host cells. In particular, the present invention relates to novel DNA constructs and methods of using these constructs to produce libraries of adeno-associated virus capsids in insect host cells, which minimize the occurrence of cross-packaging and chimeric phenomena in these libraries produced. The present invention further relates to methods for identifying AAV capsid variants having one or more desired characteristics such as, for example, a combination of CNS tropism and peripheral organ de-targeting.

The invention discloses a method for prolonging the half-life period of polypeptide by introducing composite N-glycosylation modification, which comprises the following steps: expressing trypanosoma buchneri-derived oligosaccharyl transferase TbSTT3A in insect cells; the method comprises the following steps: synthesizing a mutant GLP-1M containing asparagine by using GLP-1; oligosaccharide in the humanized compound fatty alcohol oligosaccharide is transferred to GLP-1M by utilizing the catalysis of oligosaccharyl transferase TbSTT3A, so that the humanized compound N-glycopeptide with prolonged half-life period is obtained. According to the invention, a conventional complex in-vivo human N-carbohydrate chain biosynthesis pathway is broken, fatty alcohol oligosaccharide containing a human compound N-carbohydrate chain is synthesized in vitro, oligosaccharyl transferase TbSTT3A with high substrate adaptability is used for catalysis, and the compound N-carbohydrate chain is transferred from the fatty alcohol oligosaccharide to GLP-1M polypeptide, so that the stability of GLP-1M is remarkably enhanced, and the human N-carbohydrate chain-containing fatty alcohol oligosaccharide-GLP-1M can be used for preparing the GLP-1M-GLP-1M-GLP-1M-GLP-1M-GLP-1M-GLP-1M-GLP-1M. And a new strategy for prolonging the half-life period of the GLP-1 is provided.

The present invention provides a method of inactivating enveloped viruses during the production of a biologic product without harming the biologic product. The method comprises contacting cells containing the biologic product with at least one purified non-ionic detergent, wherein the detergent(s) lyses the cells to release the biologic product, thereby producing a lysate, and wherein the detergent(s) inactivates any enveloped viruses within the lysate. The detergent can be an ethoxylated alcohol, sorbiton ethoxylate, triblock copolymer and combinations thereof. Examples of biologic products include non-enveloped viruses and therapeutic recombinant proteins produced via lysis of mammalian or insect cells.

The present application relates to the technical field of molecular biology, in particular to a truncated form of recombinant human glutamate decarboxylase 65 and application thereof. The present application screens and retains a main immunogenic fragment of GAD65, and constructs the truncated form of recombinant human glutamate decarboxylase 65. The truncated form provided by the present application can be efficiently expressed in insect cells, and a stable antigen can be obtained after purification and preparation of the expression product, and the stable antigen can be used for glutamate decarboxylase antibody detection.

The invention discloses a recombinant expression and purification method of SLFN14 protein. The invention provides a method for improving the expression quantity and / or the stability of SLFN14 protein, which comprises the following steps of: A1) mutating cysteine at 365th, 775th and 808th sites of an amino acid sequence of the SLFN14 protein into serine, and mutating glutamic acid at 211th site into alanine to obtain an SLFN14 protein mutant; a2) expressing the SLFN14 protein mutant through a baculovirus-insect cell system, so as to obtain an insect cell for expressing the SLFN14 protein mutant; a3, the insect cells expressing the SLFN14 protein mutant are subjected to splitting decomposition, nickel column purification, ion exchange purification and molecular sieve purification in sequence, and the target protein.The purity, yield and stability of SLFN14 are remarkably improved through the method.

Nucleic acid delivery vehicles for delivering nucleic acid, e.g., for RNAi to cells that are typically refractory to RNAi by using extracellular vesicles (EVs) from cultured beetle cells as delivery vehicles. Instead of using high levels of long dsRNA and transfection reagents to accomplish suppression of an mRNA target in cells that don't respond to treatment with naked dsRNA, this approach applies the dsRNA to cultured beetle cells, collects nucleic-acid loaded EVs from the culture, then treats our target cells with the intracellularly loaded EVs, which results in significant enhancement of the RNAi response and greater suppression of transcript levels.

This invention provides a primer-probe set, detection method, and application for qPCR detection of various nodaviruses, belonging to the field of molecular biology detection technology. This invention designs a primer-probe combination for various nodaviruses that can infect insect cell lines, including farm virus, Tn5 cell line virus, black beetle virus, Boralwa virus, Nodamura virus, and Macrobrachium rosenbergii nodavirus. In this combination, some viruses share upstream primers and probes, enabling simultaneous detection and identification of multiple target viruses in a single reaction system. This invention also provides a kit and detection method containing this primer-probe set. The detection method of this invention has advantages such as wide coverage, high detection sensitivity, strong specificity, and good resistance to matrix interference, and is suitable for the safety detection and quality control of nodavirus contamination in insect cell banks, cell cultures, and related biological products.

The present invention relates to the production of adeno-associated viral vectors in insect cells. The insect cells therefore comprise a first nucleotide sequence encoding the adeno-associated virus (AAV) capsid proteins, whereby the initiation codon for translation of the AAV VP1 capsid protein is a non-ATG, suboptimal initiation codon and wherein the coding sequence for one or more amino acid residues have been inserted between the suboptimal translation initiation codon and the codon encoding the amino acid residue that corresponds to the amino acid residue at position 2 of the wild type capsid amino acid sequence of which the first amino acid residue is alanine, glycine, valine, aspartic acid or glutamic acid. The insect cell further comprises a second nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) nucleotide sequence; a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to expression control sequences for expression in an insect cell; and, a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to expression control sequences for expression in an insect cell. The invention further relates to adeno-associated viral vectors with an altered ratio of the viral capsid proteins.

This invention discloses self-assembled ferritin nanoparticles and their application in the preparation of duck hepatitis A vaccine. The invention connects the VP1 portion of the duck hepatitis A virus P1 protein to the N-terminus of a self-assembled ferritin nanoparticle subunit via a linker sequence to obtain a fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1. To improve the expression level or efficiency of the fusion protein in silkworms, the nucleotide sequence of the encoding gene of the fusion protein is optimized and modified according to the codon preference of silkworms, resulting in the optimized gene sequence shown in SEQ ID NO.2. The invention also involves single-site or multi-site mutations of the fusion protein, screening for mutants with significantly improved titers and significantly increased soluble expression levels. This invention utilizes a silkworm or insect cell eukaryotic expression system to express the recombinant protein, and the expression product induces neutralizing anti-duck hepatitis A virus antibodies.

The application discloses a multiplex qPCR primer probe set, a kit, a detection method and application for detecting alphaviruses, and belongs to the technical field of molecular biological detection. The application designs a set of primer probe combination for a plurality of alphaviruses capable of infecting insect cell lines, including western equine encephalitis virus, eastern equine encephalitis virus, sindbis virus, chikungunya virus, AURA virus and Venezuelan equine encephalitis virus. The combination realizes synchronous detection and identification of a plurality of target viruses in one reaction system. The application also provides a kit containing the primer probe set and a corresponding detection method. The detection method has the advantages of wide coverage, high detection sensitivity, strong specificity, good anti-matrix interference capability and the like, and is suitable for safety detection and quality control of alphavirus contamination in insect cell banks, cell cultures and related biological products.

The application discloses a bovine type II gamma-interferon-ferritin fusion protein, a mutant thereof, and a preparation method and application thereof. The bovine type II gamma-interferon and a ferritin subunit are connected through a flexible peptide to realize surface display of the interferon protein, form a dimer conformation suitable for the bovine type II gamma-interferon to play a function, effectively improve the expression amount and activity of the bovine type II gamma-interferon, improve the stability of the bovine type II gamma-interferon in the body, and prolong the half-life period. The fusion protein is further rationally designed to obtain a mutant with significantly improved antiviral activity and stability. The application adopts a silkworm or insect cell eukaryotic expression system to express the fusion protein or the mutant, the expression system is safe to operate, simple in procedure, low in cost, and extremely beneficial to large-scale industrial production; and the prepared fusion protein or mutant can be applied to the preparation of a medicine for preventing or treating bovine viral infection, tumor, and immune system diseases, and an immune adjuvant for vaccine compounding.

The application discloses a method for determining baculovirus titer, which adopts a mouse IgG2a type monoclonal antibody specifically recognizing envelope protein gp64, and comprises the following steps: step 1, cell plating: cells are plated in a cell culture plate; step 2, baculovirus inoculation: a baculovirus sample to be detected is inoculated into the cell culture plate in step 1; step 3, virus infection; and step 4, flow cytometry detection: infected cells are collected, excessive baculovirus is removed, the cells are washed, fluorescently labeled antibodies are added, and machine detection is carried out. The method of the application is more simple in operation process by using the to-be-detected baculovirus to directly infect insect cells and flow cytometry analysis based on the gp64 antibody, and does not depend on subjective judgment of an experimenter on cytopathic effect, so that the result is more representative.

The invention discloses saccharomyces cerevisiae for synthesizing active cow milk peroxidase, and belongs to the technical field of bioengineering. According to the invention, the pGal1 promoter is adopted to regulate the cow milk peroxidase, and co-expression is carried out on the pGal1 promoter and ERp57 regulated by the pTEF1 promoter, so that active synthesis of the cow milk peroxidase is realized, and the yield reaches the level of 85U / L fermentation liquor (based on ABTS). Compared with methods for extracting from milk or synthesizing from mammalian cell lines or insect cell lines and the like in the prior art, the method disclosed by the invention is low in cost and simple in process, and has industrial application potential.