16 results about "Fusion Protein Expression" patented technology

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 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.

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.

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 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.

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.

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.

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.

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.