95 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 porcine epidemic diarrhea virus antigen fusion protein, a coding gene and a porcine epidemic diarrhea vaccine prepared from the porcine epidemic diarrhea virus antigen fusion protein. The method comprises the following steps: screening to obtain a homosensory sequence of the spike protein of the porcine epidemic diarrhea virus, and fusing the homosensory sequence with a monomeric porcine ferritin heavy chain subunit to obtain an antigen fusion protein; in order to improve the expression quantity or the titer of the antigen fusion protein, the obtained antigen fusion protein is subjected to sequence optimization and then subjected to single-site or multi-site mutation, and the expression quantity and the titer of the obtained mutant are remarkably improved. According to the invention, a recombinant protein is expressed by using a silkworm or AcMNPV-insect cell and other eukaryotic expression systems to obtain a self-assembled ferritin nanoparticle antigen for displaying an S protein antigen protein on the surface of a cage structure of porcine ferritin, and a vaccine prepared from the prepared nanoparticle antigen can induce a widely neutralized anti-porcine epidemic diarrhea virus antibody; the porcine epidemic diarrhea virus vaccine has the potential of becoming a universal porcine epidemic diarrhea virus vaccine with cross immune efficacy.

The invention belongs to the technical field of veterinary medicine, and provides a co-expressed recombinant porcine alpha and gamma interferon. The I-type interferon alpha and the II-type interferon gamma not only exert the anti-virus, anti-cell division and immune regulation effects of the interferon, but also can activate T cells and natural killer cells, and fully exert the multiple activities of the interferon. The two interferons are co-expressed in insect cells, the expression protein can be folded and then processed and modified, the structure of the expression protein is closer to a natural structure, the biological activity is high, the recombinant protein is easy to purify, the production scale is easy to expand, and live viruses or potential dangerous pathogens do not need to be treated, so that the production cost is reduced; by optimizing the production process and fermentation conditions, the quality and stability of the recombinant protein can be further improved.

The invention discloses a preparation method and application of a tetravalent hand-foot-and-mouth disease subunit-new adjuvant vaccine, and relates to the technical field of hand-foot-and-mouth disease vaccines. VP1 proteins of EV-A71, CV-A16, CV-A10 and CV-A6 are expressed through an insect cell-baculovirus system, the VP1 proteins are purified through a nickel affinity chromatography column and mixed with an MF59 adjuvant to prepare a tetravalent vaccine, in-vivo evaluation of mice is conducted, and meanwhile the immunogenicity and protection efficacy of the tetravalent VP1 protein vaccine and the immunogenicity and protection efficacy of a monovalent VP1 protein vaccine are compared. Immune results show that compared with a VP1 monovalent vaccine, all serum of a mouse inoculated with the tetravalent VP1 protein vaccine shows strong reactivity to all captured antigens. Therefore, the results show that the prepared tetravalent subunit-new adjuvant vaccine is a promising broad-spectrum vaccine.

The invention belongs to the technical field of biological medicines, and particularly relates to a PPV-PCV2 nanoparticle vaccine design and a preparation method. According to the invention, PPV1VP2 protein is used as a skeleton, a SpyTag peptide fragment is inserted into a loop 2 region of the VP2 protein, and the fusion protein is expressed by using an insect cell baculovirus system and is marked as PPV-ST; a plurality of B cell epitopes of Cap protein of PCV2 and a plurality of T cell antigen epitopes of Rep protein of PCV2 are connected in series, a SpyCatcher peptide fragment is fused at an N end, and expression is performed by using a prokaryotic expression system and is marked as SC-PCV2. Finally, the PPV-ST protein and the SC-PCV2 protein are subjected to covalent binding assembly, the nanoparticle vaccine is prepared, and the obtained PPV-PCV2 vaccine has good immunogenicity.

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.

Disclosed in the present invention are an expression cassette containing overlapping open reading frames (ORFs), and a use. The overlapping ORFs are overlapping ORFs comprising a first ORF and a second ORF, and the expression cassette sequentially comprises from a 5' end to a 3' end: at least a first promoter used for driving transcription of a gene of the first ORF; a 5' part of the gene of the first ORF; an intron; and a 3'part of a gene of the second ORF, the intron comprising a second promoter only used for driving transcription of the gene of the second ORF. According to the present invention, two promoters are provided in a single expression cassette and are respectively used for driving expression of proteins of overlapping reading frames and adjusting the relative expression time and expression intensity of different proteins, thereby improving the production efficiency of rAAV vectors in insect cells, and also improving the quality of produced rAAV.

In order to solve the problem that an efficient and stable anti-plant-virus compound screening and evaluating technology is lacked, the invention provides an efficient anti-plant-virus compound screening and evaluating method based on insect cells. An insect cell line Sf9JSN106 which is obtained through domestication and can maintain infection and replication of plant viruses (representative species: rice stripe viruses) is used for rapidly screening and evaluating the antiviral effect of a compound. Evaluation is carried out from two aspects: 1) evaluating the inhibition effect of the compound on virus replication, wherein the process comprises cell inoculation, virus cell infection, medicament treatment and virus detection; and 2) evaluating the inhibition effect of the compound on virion infection activity, wherein the process comprises cell inoculation, virus treatment by a medicament, cell infection by the virus and virus detection. The method is completed by using a cell culture plate, various compounds can be evaluated at a time, the whole process does not exceed 60 hours, and the method has the characteristics of simplicity, convenience, rapidness and high efficiency, and can be applied to screening evaluation of plant virus resistant compounds and antiviral mechanism research.

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.

Described herein are methods for making edible fat product using insect cells. The methods include isolating a population of insect cells, which may be precursor cells, seeding the population of insect cells onto a scaffold, and contacting the population of insect cells with a free fatty acid composition or a lipid composition. The cells seeded on the scaffold accumulate lipids, resulting in an edible fat tissue. Further disclosed are a composition of matter made from the method for making edible fat cells, a food product that includes the composition of matter, and an in vitro edible fat tissue comprising a population of insect precursor cells seeded on the scaffold.

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.

The invention provides a separated Actin5C promoter as well as a construction method and an expression vector thereof, the separated Actin5C promoter is a partial region of a wild Actin5C promoter, and the partial region comprises a region from the 94th site in the downstream direction of a transcription start site to 400bp-600bp in the upstream direction. The separated Actin5C promoter is short in length, so that the expression vector containing the Actin5C promoter is small in size and high in transfection efficiency. By utilizing the expression vector provided by the invention, the target protein yield of in-vitro insect S2 cells can be remarkably improved, and the scientific research and clinical application values are high.

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.

The invention relates to the technical field of microbial cells, and discloses a method for transfecting a symbiotic bacterium Wolbachia into a lepidoptera insect cell line for culture, which comprises the following steps: collecting insect eggs infected with a Wolbachia host; culturing a lepidoptera insect cell line; adding eggs of the insect infected with the Wolbachia host into a centrifugal tube containing PBS (Phosphate Buffer Solution); washing the eggs; adding a cell culture medium into the centrifugal tube after the eggs are washed, and fully grinding the eggs by using a sterilization grinding rod to obtain an egg homogenate; according to the method, bacteria and insect cells are tightly combined together by utilizing weak centripetal force generated by low-speed centrifugation, and the bacteria are absorbed into the cells by combining the endocytosis and exocytosis characteristics of the cells, so that the cross-host transfection of the symbiotic bacteria is realized. The technology is of great significance to establishment of new insect strains infected with specific symbiotic bacteria.

The invention discloses a preparation method and application of a bivalent feline calicivirus VLP vaccine, an insect cell endogenous molecular chaperone protein HSP60 is creatively utilized to construct a recombinant plasmid co-expressing HSP60 and FCV VP1, and the expression quantity of VP1 is successfully improved, the expression quantity of GII VP1 is improved by 9.2 times, and the expression quantity of GI VP1 is improved by 17 times. By screening assembly buffer solutions with different salt ion concentrations, pH values and components, the assembly conditions of GI VLP and GII VLP are optimized, and the yield and yield of VLP are improved. Animal model evaluation shows that the bivalent VLP vaccine has an immune synergistic effect, the immune effect of the bivalent VLP vaccine is remarkably superior to that of a monovalent VLP vaccine and a commercially available inactivated vaccine, the same immune effect of a high dose can be achieved by a low dose of the bivalent VLP vaccine, the antigen dose demand is reduced by 4-8 times, meanwhile, protection on two FCV types is provided, and a new direction is provided for research and development of FCV vaccines.

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 invention belongs to the technical field of biology, and relates to a preparation method and application of a chemokine functional active body. Specifically, based on the earlier stage of finding that insect cells sf9 can specifically recognize and cut the human-derived secretory protein signal peptide MRRLLIPLALWLGAVGVGVA under the condition of the method in scientific research, the inventor researches and develops a specific signal peptide cleavage method which can specifically recognize and cut the human-derived secretory protein signal peptide MRRLLIPLALWLGAVGVGVA through an insect cell secretory pathway. The technical scheme of preparing a chemokine functional active body with the first amino acid at the N end being non-initiation codon methionine is adopted, soluble expression is realized, and an optimized ultrafiltration purification technical system is provided. The method is simple and efficient, column chromatography is not needed, later-stage enzyme digestion processing on protein is not needed, the prepared chemokine functional active body does not contain redundant amino acid or tag sequences which interfere normal folding and functions, and good conditions are provided for large-scale preparation of the chemokine functional active body and research and development of anti-inflammatory lipolytic research and development of the chemokine functional active body.

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 present invention relates to recombinant proteins, encoding nucleic acids, a vector, transformed microorganism and host cell, a vaccine composition intended for salmon farming, and use for preventing and treating infections caused by Piscine orthoreovirus (PRV). In particular, the present invention relates to a vaccine based on recombinant proteins expressed in a baculovirus / insect cell system and corresponding to the structural proteins in the first and second layer of the viral capsid. This vaccine can be administered intraperitoneally, reducing the viral load of Piscine orthoreovirus in salmon infected with this virus.