30 results about "Fusion Protein Expression" patented technology

Fusion proteins containing a targeting sequence, an exosporium protein, or an exosporium protein fragment that targets the fusion protein to the exosporium of a Bacillus cereus family member are provided. Recombinant Bacillus cereus family members expressing such fusion proteins are also provided. Genetically inactivated Bacillus cereus family members and recombinant Bacillus cereus family members that overexpress exosporium proteins are also provided. Seeds coated with the recombinant Bacillus cereus family members and methods for using the recombinant Bacillus cereus family members (e.g., for stimulating plant growth) are also provided. Various modifiations of the recombinant Bacillus cereus family members that express the fusion proteins are further provided. Fusion proteins comprising a spore coat protein and a protein or peptide of interest, recombinant bacteria that express such fusion proteins, seeds coated with such recombinant bacteria, and methods for using such recombinant bacteria (e.g., for stimulating plant growth) are also provided.

The invention relates to the technical field of biological medicines, and discloses a construction method and expression application of an Hcp gene deleted strain of a rahnella aqua KCL-5 strain for fish. The Hcp 0186 gene deletion strain is constructed by using a homologous recombination method, the comparison between the deletion strain and a wild strain is carried out, and the result shows that the deletion of the Hcp 0186 gene has an influence on the growth speed, the pathogenicity of the Hcp 0186 gene to the zebra fish is obviously reduced, the survival rate of the infected zebra fish is increased from 0% to 52%, and the Hcp 0186 gene deletion strain can be used as a fish attenuated live vaccine. A recombinant prokaryotic expression vector pET32a-Hcp and expression and purification of fusion protein of the recombinant prokaryotic expression vector pET32a-Hcp are further constructed, and Hcp 0186 rabbit polyclonal antibody serum is prepared as a primary antibody and can be applied to localization expression analysis of fish tissues infected by the bacterium and detection of WB and IHC. Results prove that the Hcp 0186 gene deletion of the strain provides an attenuated strain vaccine and clinical diagnosis and detection application of infected fish of the attenuated strain vaccine, and a scientific basis can be provided for revealing a molecular pathogenic mechanism of the strain and development and application of a mutant strain of the strain.

Provided are a fusion protein, a preparation method therefor and the use thereof. The fusion protein contains a His tag, a SUMO tag, and natural canine interferon-α, wherein the His tag has an amino acid sequence as represented by SEQ ID NO: 11, the SUMO tag has an amino acid sequence as represented by SEQ ID NO: 12, and the natural canine interferon α has an amino acid sequence as represented by SEQ ID NO: 7. The fusion protein exhibits an extremely significant increase in expression level and yield, has preparation stability and biological activity significantly greater than those of a native canine interferon-α protein, and has good safety.

The invention discloses a fusion protein expression system suitable for expressing long-chain helper protein in yeast, which is a gene expression cassette of fusion protein of helper protein and bovine lactoferrin, and comprises a double KREAEA structural domain + flexible Linker tandem helper protein gene and bovine lactoferrin gene, and the fusion protein assists in folding of bovine lactoferrin in cells, so that the expression of long-chain helper protein is realized. The fusion protein is excised in cells, and the secretion efficiency of correctly folded bovine lactoferrin is remarkably enhanced.

The invention belongs to the technical field of protein engineering, particularly relates to a protein tag with superoxide dismutase activity and application, and provides a protein tag which comprises polypeptide or protein with superoxide dismutase activity. On the second aspect, a fusion protein is provided and comprises the protein tag described in the first aspect and at least one target polypeptide or protein, the target polypeptide or protein is not superoxide dismutase, and the target polypeptide or protein is not superoxide dismutase only by using the protein tag disclosed by the invention. According to the present invention, the technical effects of target polypeptide or protein expression quantity detection (SOD enzyme activity detection), target polypeptide or protein protection from oxidative damage, target polypeptide or protein stability improvement and target polypeptide or protein solubility improvement can be simultaneously achieved without adding any additional step compared with the fusion protein expression operation in the prior art; the operation is convenient.

The invention relates to the technical field of gene engineering, in particular to a fusion protein expression cassette for rice multi-gene editing and application of the fusion protein expression cassette. The expression of the fusion protein for multiple gene editing of rice provided by the invention comprises an endonuclease Csy4, a DNA (Deoxyribose Nucleic Acid) endonuclease nickase Cas9, an M-MLV reverse transcriptase and a Cas12a nuclease; in the guide editing fusion protein frame, the ribonuclease Csy4 is located at the N end of the guide editing fusion protein frame, the DNA incision enzyme nickase Cas9 is located between the ribonuclease Csy4 and the M-MLV reverse transcriptase, the M-MLV reverse transcriptase is located between the DNA incision enzyme nickase Cas9 and the Cas12a nuclease, and the Cas12a nuclease is located at the C end of the fusion protein frame. The fusion protein expression cassette not only can ensure the editing efficiency, but also can improve the editing accuracy.

This invention belongs to the field of plant gene editing technology, specifically relating to a bifunctional gene editing system and its application in single-parent maize. The invention provides a bifunctional gene editing system comprising: a fusion protein expression cassette and a guide RNA combined expression cassette; wherein the guide RNA combined expression cassette includes: pegRNA associated with the maize D9 gene; and sgRNA associated with the maize YUC4 and YUC2 genes. Transient expression validation using the bifunctional gene editing system in maize protoplasts revealed that the system achieves a precise editing efficiency of 3.5% at the G571V site of the D9 gene, a knockout efficiency of 40%-65% for the YUC4 gene, and a knockout efficiency of 37%-54% for the YUC2 gene. This demonstrates that the technical solution of this application can simultaneously and efficiently achieve precise point mutation and gene knockout within the same cell, thus enabling its application in the synergistic improvement of lodging resistance in maize.

A derivative of insulin aspart and a method for preparing the same, the derivative comprising a fusion protein of a green fluorescent protein folding unit and an insulin aspart precursor or an active fragment thereof. The fusion protein is expressed in a significantly increased amount, the insulin aspart precursor protein in the fusion protein is folded correctly and has biological activity. Furthermore, the green fluorescent protein folding unit in the fusion protein can be digested by a protease into small fragments, and has a large difference in molecular weight compared to the target protein, and is easy to separate. A method for preparing insulin aspart and an intermediate using the fusion protein is also provided.

The invention provides a plasmid tool for simulating Kras random evolution and application thereof, and the plasmid tool comprises at least two guide RNA expression cassettes driven by a first promoter, which respectively target a Pten gene and a Trp53 gene; the pilot editing guide RNA expression box is driven by a second promoter, targets the 12th codon of the Kras gene, and comprises a repair template sequence for editing the codon into G12D; the fusion protein expression cassette is driven by a third promoter and comprises SpCas9 protein and MMLV reverse transcriptase; and at least one eukaryotic selection marker gene expression cassette. The invention also discloses a method for constructing a simulated Kras random evolution model by adopting the plasmid tool, and application of the plasmid tool in screening targeted drugs aiming at KRAS mutation tumors. The plasmid tool provided by the invention can realize cell multi-gene locus editing, construct a Kras locus random evolution model and simulate a Kras random evolution process in ovarian cancer.

The invention provides a recombinant bovine lactoferrin peptide capable of reducing feedback inhibition on host bacteria and a construction method thereof in order to overcome the practical application problem of a genetic engineering method in the process of producing the bovine lactoferrin peptide at present. According to the invention, bovine lactoferrin peptide, calmodulin and enhanced green fluorescent protein are connected in series to form bovine lactoferrin peptide fusion protein, a pichia pastoris recombinant strain is constructed, and high-level expression of bovine lactoferrin peptide is realized. According to the method, positive charges of bovine lactoferrin are neutralized on the basis of calmodulin, toxicity of antibacterial peptide to a host is weakened, the tandem enhanced green fluorescent protein can emit green fluorescence under 488 nm exciting light, and real-time analysis of the expression level of bovine lactoferrin fusion protein is achieved. According to the method, by adopting a fusion expression technology, the toxicity to a host when the antibacterial peptide is produced by a genetic engineering method is overcome, so that the expression quantity is increased, and a foundation is laid for high-density fermentation production of the bovine lactoferrin peptide.

The invention provides a fusion protein of flounder netshell virus nucleocapsid protein, bacillus subtilis for expressing the fusion protein and an oral vaccine, and relates to the technical field of biology. According to the present invention, the sequence of the bacillus subtilis anchoring protein CotC is obtained through sequencing, the bacillus subtilis anchoring protein CotC and the flounder nested virus are fused, and after the fusion protein is introduced into the bacillus subtilis, the fusion protein can be anchored on the surface of the bacterial spore, and can be used for preparing the recombinant oral vaccine capable of being stably stored and directly fed, and a new method is developed for preventing the flounder netlike virus CSBV.

The invention provides a nano-antibody-assisted protein or polypeptide expression system as well as preparation and application thereof, and particularly relates to a nano-antibody fusion protein for promoting protein or polypeptide expression and secretion, an expression vector, a host cell and a nano-antibody-assisted protein expression system as well as preparation and application thereof.

Disclosed herein are methods for precise editing of highly homologous regions. The method includes identifying a target region for precise editing of a homologous region. A vector construct for expressing a fusion protein specific for the target region is obtained. The fusion protein is a genetic construct having sequences for expressing an inactivated Cas9 nuclease and an ABE8e base editor for editing at the identified target region. One or more vector constructs for expressing a guide RNA complementary to the target region are created. The gRNA vector construct(s) are transformed into a suitable cell line along with the fusion protein expression vector for precise editing of the highly homologous region. Also disclosed herein is another method for precise editing of highly homologous regions using a truncated guide RNA of 10-15 nucleotides in length for precise editing in highly homologous regions.

Adeno-associated virus (AAV) gene therapy vectors express a therapeutic protein or RNA that treats a genetic defect in a cell. The present disclosure provides AAV gene therapy vectors that additionally express an anti-inflammatory protein or peptide. When the provided AAV gene therapy vectors are used in methods of treatment of. for example. neurodegenerative diseases. the therapeutic protein / RNA treats the genetic defect within the cells directly transduced by the AAV vectors while the anti-inflammatory protein / peptide is secreted by the transduced cells into the intercellular milieu and treats microglial activation associated with the neuroinflammatory component of the neurodegenerative diseases. The therapeutic protein and anti-inflammatory protein / peptide can be expressed as fusion protein in which the two are separated in the fusion protein by a self-cleaving peptide. The provided AAV gene therapy vectors and methods are thus useful in treating neurological and neurodegenerative disorders.

The invention relates to the field of plant genetic engineering, and particularly discloses a recombinant vector construction system for tobacco eIf4e gene editing. The system comprises a recombinant expression vector with pCAMBIA1300 as a basic skeleton, an nCas9-RT fusion protein expression unit driven by a Stubi promoter and a pegRNA expression unit driven by an NtU6-28 promoter are integrated in the recombinant expression vector, and the system further comprises a hygromycin resistance selection marker driven by a 35S promoter; the preparation method comprises the following steps: firstly, synthesizing a specific nucleotide sequence of each element, carrying out directional cloning through BstBI and BlpI restriction endonuclease sites, and sequentially connecting the two core expression units in series according to the direction from 5'to 3 'and assembling the two core expression units into a vector skeleton. The recombinant vector system can be used for specific multi-site precise editing of a VGAD region of the tobacco eIf4e gene, and has the advantages of dual-promoter coordinated regulation and editing element expression coordination.

The application discloses an expression plasmid system of antigen-loaded PHA nanoparticles and preparation and application thereof. The expression plasmid system comprises pACYC-PhaA-PhaB plasmid and pET28a-PhaC-Target plasmid, nucleotide sequences of the plasmid and the pET28a-PhaC-Target plasmid are respectively shown as SEQ ID NO: 4 and SEQ ID NO: 5; the pACYC-PhaA-PhaB plasmid is a PhaA and PhaB protein expression frame inserted into a multiple cloning site of a vector pACYCDuet-1 and driven by two T7 promoters respectively; the pET28a-Phac-Target plasmid is a PhaC-vaccine antigen fusion protein expression frame inserted into a multiple cloning site of a vector pET-28a. The application lays a good foundation for preparation of PHA nanoparticle vaccines and related research.

This invention relates to the field of biomedical technology, and in particular to a feline allergen fusion protein, an mRNA immunomodulator, and their applications. The amino acid sequence of the feline allergen fusion protein of this invention is shown in SEQ ID NO. 11. The feline allergen fusion protein provided by this invention is a fusion protein obtained by fusing key sensitizing segments of Fel d2 and Fel d4 proteins with the full-length Fel d1 protein. This fusion protein can simultaneously cover the major sensitizing epitopes of Fel d1, Fel d2, and Fel d4, and can more comprehensively block the sensitizing effect of feline allergens. The feline allergen fusion protein provided by this invention enables the rapid design and synthesis of mRNA immunomodulators, allows for precise control of feline allergen fusion protein expression in poultry, results in a more durable immune response, and the prepared IgY antibody can effectively block allergen proteins in feline oral saliva.

The invention discloses an aeromonas salmonicida and infectious hematopoietic necrosis bivalent subunit vaccine and application thereof, and belongs to the technical field of aquatic vaccines. The vaccine is characterized in that it comprises a fusion protein and an immunologic adjuvant. The fusion protein comprises a VapA protein antigen of aeromonas salmonicida and a G protein antigen of infectious haematopoietic necrosis virus, the VapA protein antigen and the G protein antigen are connected through a flexible Linker, and the preferable structure of the fusion protein is VapA-Linker-IHNV-G-6 * His. The invention also discloses a preparation method of the vaccine. The preparation method comprises the following steps: constructing a fusion protein expression vector, transforming a host, inducing expression, purifying the fusion protein and emulsifying with an adjuvant. The bivalent vaccine disclosed by the invention can simultaneously stimulate dual specific immune response aiming at bacteria and viruses, has a remarkable immune protection effect on aeromonas salmonicida and infectious hematopoietic necrosis viruses, is high in safety as a subunit vaccine, and provides an effective new way for prevention and control of multi-pathogen cross infection in aquaculture.

The invention discloses a fusion protein with an anti-transplant rejection function and an engineering cell for expressing the fusion protein, and further relates to a method for resisting transplant immunological rejection, in particular to a method for resisting NK (Natural Killer) cell immunological rejection.

A recombinant humanized collagen type III alpha-1 (rhCol III α1), and an expression vector and use thereof are provided, relating to the technical field of bioengineering. A sequence on positions 154 to 1,221 of a human type III collagen α chain is ligated to an optimized vector pcDNA3.1, a resulting optimized expression vector is transfected into a human embryonic kidney cell Expi293F, and then rhCol III α1 is secreted to form a fusion protein with a soluble green fluorescent protein (GFP). The expressed rhCol III α1 that forms the fusion protein with the GFP shows an activity of promoting the migration of BALB / c 3T3 cells, thus exhibiting a great application potential in the field of skin care.

Relates to the field of immunology and tumor treatment. Specifically, the invention relates to a TP53R282W mutant epitope peptide and a pMHC complex, an antigen presenting cell expressing the epitope peptide or the pMHC complex, a tumor vaccine containing the epitope peptide or the pMHC complex, and an application of the antigen presenting cell in prevention or treatment of tumors with TP53R282W mutation. The invention also relates to a T cell receptor (TCR) capable of specifically recognizing the TP53R282W mutant, a conjugate and a fusion protein containing the TCR, an immune cell expressing the TCR, a T cell medicine containing the TCR, and application of the T cell receptor and the fusion protein to prevention or treatment of tumors with the TP53R282W mutant.

This invention discloses a method for the bioenzymatic synthesis of ascorbate phosphate succinyl tripeptide-1, belonging to the field of bioengineering technology. This method involves constructing a fusion protein expression system through genetic engineering, releasing the target tripeptide GHK (glycylhistyllysine) through specific enzymatic cleavage, and then conjugating it to the succinyl group of ascorbate phosphate via an enzymatic catalytic esterification reaction, ultimately obtaining high-purity ascorbate phosphate succinyl tripeptide-1. Compared with traditional chemical synthesis methods, this method has advantages such as shorter synthesis steps, milder conditions, environmental friendliness, and high yield (>85%), making it suitable for large-scale industrial production and showing broad application prospects in the cosmetics and pharmaceutical fields.

Aspects of the disclosure relate to Botulinum toxin X (BoNT X) protein variants (e.g., BoNT X protease variants). The variants provided herein have been evolved to cleave gasdermin D. Some of the variants provided herein do not cleave the native substrate of BoNT X protease, VAMP1 protein. Further aspects of the disclosure relate to nucleic acids encoding the gasdermin D cleaving polypeptides described herein and expression vectors comprising the nucleic acids, as well as host cells and fusion proteins comprising the gasdermin D cleaving polypeptides described herein and kits comprising the gasdermin D cleaving polypeptides, nucleic acids, fusion proteins, expression vectors, or host cells described herein. Further aspects of the disclosure relate to methods of producing BoNT X protein variants (e.g., BoNT X protease variants) and methods of using the BoNT X protein variants (e.g., BoNT X protease variants), for example, to induce cell death.

The present invention relates to the fields of immunology and tumor treatment. Specifically, the present invention relates to a TP53 R282W mutant epitope peptide and a pMHC complex, an antigen-presenting cell expressing the epitope peptide or the pMHC complex, a tumor vaccine comprising same, and a use thereof in preventing or treating a tumor harboring a TP53 R282W mutation. The present invention further relates to a T-cell receptor (TCR) that specifically recognizes a TP53 R282W mutant, a conjugate and a fusion protein comprising the TCR, an immune cell expressing the TCR, a T-cell drug comprising same, and a use thereof in preventing or treating a tumor harboring the TP53 R282W mutation.

The invention discloses a probiotic EcN lpp-A5-alpha TN strain as well as a construction method and application thereof, Escherichia coli Nissle 1917 is taken as a chassis, CRISPR (clustered regularly interspaced short palindromic repeats) is used for knocking out an outer membrane lipoprotein gene lpp, and an Lpp-OmpA-Annexin V fusion protein expression cassette is integrated in situ, so that the probiotic EcN lpp-A5-alpha TN strain which is stable in heredity and free of exogenous resistance is obtained. The strain retains the beneficial characteristics of EcN, and meanwhile, realizes targeted colonization, long-time retention and continuous secretion of anti-TNF-alpha nano antibody alpha TN in intestinal tracts, inhibits immune cell infiltration and rebuilds the immune microenvironment of the intestinal tracts. Animal experiments prove that the traditional Chinese medicine composition has a remarkable curative effect on acute and chronic ulcerative colitis, can be used in combination with 5-aminosalicylic acid, glucocorticoid or a biological preparation, and is safe to orally take, mature in process, low in cost and suitable for large-scale production.

This invention belongs to the field of agricultural biotechnology, specifically relating to a laccase fusion protein and its fermentation preparation method, and its application in fungal toxin detoxification. The fusion protein is derived from laccase of *Auricularia ochracea*. So Lac, linker peptides, and xylanase from Aspergillus niger An XynB composition. This invention constructs xylanase. An XynB and Laccase So The Lac fusion protein expression vector was transformed into Pichia pastoris for heterologous expression of the fusion protein, which significantly improved laccase expression. So Lac expression level. The recombinant laccase obtained in this invention. So Lac and xylanase An XynB fusion protein can effectively detoxify mycotoxins such as aflatoxin B1 and zearalenone, and can be widely used in the field of mycotoxin detoxification in food and feed.

The subject of this invention is a construct of an expression vector for expressing recombinant µ-conotoxin TIIIA or TIIIAlaMut, characterized in that it comprises the nucleotide sequence of µ-conotoxin TIIIA SEQ ID NO:1 or the nucleotide sequence of µ-conotoxin TIIIAlaMut SEQ ID NO:4, both sequences containing a sequence encoding six histidine residues (6His) at the 5' end, which is linked via a serine-glycine-serine linker (SGS) to a construct encoding a TRX::TIIIA fusion protein or a TRX::TIIIAlaMut fusion protein, wherein the TRX::TIIIA fusion protein comprises the µ-conotoxin TIIIA gene and a gene encoding a leader protein, and the TRX::TIIIAlaMut fusion protein comprises the µ-conotoxin TIIIAlaMut gene and a leader protein, wherein the leader protein is a thioredoxin (TRX) modified by site-directed mutagenesis, wherein the amino acid methionine at position 37 is replaced by lysine. Another subject of the invention is an expression vector comprising a construct according to the invention under the control of a constitutive promoter. Another subject of the invention is isolated *E. coli* cells comprising an expression vector according to the invention. Another subject of the invention is a method for producing µ-conotoxin TIIIA or TIIIAlaMut in *E. coli* using an expression vector comprising a construct according to the invention, characterized in that the method comprises the steps of: a) transforming *E. coli* cells with an expression vector comprising a construct according to the invention under the control of a constitutive promoter, said expression vector encoding a TRX::TIIIA fusion protein having the amino acid sequence SEQ ID NO:2 or having SEQ ID NO:2. a) TRX::TIIIAlaMut fusion protein NO:5; b) Expression of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; c) Isolation and purification of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; d) Formation of disulfide bonds by glutathione treatment of the purified TRX::TIIIA or TRX::TIIIAlaMut fusion protein in GSH / GSSG and dialyzing in buffer; e) Cleavage of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein with the formed disulfide bonds by cyanogen bromide; f) Purification of the cleaved TIIIA or TIIIAlaMut peptide.

Aspects of the disclosure relate to Botulinum toxin X (BoNT X) protein variants (e.g., BoNT X protease variants). The variants provided herein have been evolved to cleave gasdermin D. Some of the variants provided herein do not cleave the native substrate of BoNT X protease, VAMP1 protein. Further aspects of the disclosure relate to nucleic acids encoding the gasdermin D cleaving polypeptides described herein and expression vectors comprising the nucleic acids, as well as host cells and fusion proteins comprising the gasdermin D cleaving polypeptides described herein and kits comprising the gasdermin D cleaving polypeptides, nucleic acids, fusion proteins, expression vectors, or host cells described herein. Further aspects of the disclosure relate to methods of producing BoNT X protein variants (e.g., BoNT X protease variants) and methods of using the BoNT X protein variants (e.g., BoNT X protease variants), for example, to induce cell death.

The invention relates to the field of plant genetic engineering, and particularly discloses an agrobacterium tumefaciens-mediated NtU6-27 driving type tobacco pilot editing transformation identification method. Wherein the nCas9-RT fusion protein expression unit is driven by a 35S promoter, and the pegRNA expression unit is driven by a 35S promoter-Cmylc enhancer-NtU6-27 promoter. The preparation method comprises the following steps: preparing a tobacco explant, constructing a recombinant expression vector, introducing the vector into agrobacterium tumefaciens, carrying out enlarged culture, carrying out agrobacterium tumefaciens-mediated transformation on the tobacco explant, carrying out screening culture to obtain a regenerated plant, and identifying an editing effect through PCR (Polymerase Chain Reaction) and sequencing. The composition can be used for tobacco precise genetic improvement and gene function research, and has the advantages that pegRNA is driven by NtU6-27 to efficiently express, and the editing efficiency is high; in addition, the preparation method has the advantages of being clear in step, high in repeatability and accurate in identification.