25 results about "Perforin" patented technology

The invention relates to an antibody composition for detecting abnormal CTL (cytotoxic T lymphocyte) cells of hemophagocytic syndrome and application of the antibody composition, and the antibody composition comprises a CD2 antibody, a CD3 antibody, a CD4 antibody, a CD5 antibody, a CD7 antibody, a CD8 antibody, a CD56 antibody, a CD57 antibody, a Granzyme B antibody, a Perforin antibody, a TRBC1 antibody and a CD45 antibody. In the technical scheme provided by the invention, key phenotypic characteristics of CTL (Cytotoxic Lymphocyte) can be completely covered by various antibodies aiming at T cell and NK cell surface markers, T large granular lymphocyte markers, cytotoxic granular proteins, T cell receptor constant regions and leukocyte common antigens; wherein the clonality of the CTL cells is judged through the expression level of the TRBC1 antibody, and the cytotoxic function activity of the CTL cells is evaluated through the expression levels of Granzyme B and Perforin.

The present invention relates to a self-cleaving engineered bacterium comprising a metabolic pathway for producing polyhydroxyalkanoate (PHA); the perforin gene and the endolysin gene are expressed under the control of a promoter. According to the method, on the basis of not influencing the dry cell weight and PHA yield of the engineering strain, the self-splitting of the engineering strain after PHA accumulation is completed is realized, the downstream extraction step of PHA biosynthesis is greatly simplified, and the extraction time is shortened.

The invention discloses application of an ATP (adenosine triphosphate) citrate lyase (ACLY) inhibitor in preparation of a medicine for treating leucoderma, and belongs to the technical field of preparation of medicines for treating leucoderma. Through targeted inhibition of ACLY activity or specific knockout of ACLY in CD8 + T cells, abnormal activation of ACLY and secretion of killing effector molecules (Granzyme B, IFN-gamma, Perforin and the like) are inhibited, and infiltration of the CD8 + T cells to skin lesion parts is reduced, so that melanocytes are protected from immune injury, and the progress of vitiligo is blocked. The application is based on the abnormally high expression of ACLY in CD8 + T cells of vitiligo patients and the close association with disease activity, and a brand new targeted treatment thought and a medicine research and development direction are provided for vitiligo.

This invention belongs to the field of biotechnology, specifically relating to a perforin derived from *Staphylococcus warwickiana* temperate bacteriophage and its applications. The perforin has the nucleotide sequence shown in SEQ ID NO:1. This perforin is a key gene for phage lysis and growth, playing a crucial role in the lysis of host bacteria. Therefore, this study aims to verify whether the perforin, whose coding site is located on the genome of *Staphylococcus warwickiana* temperate bacteriophage, as a cell lysis gene, can serve as a potential antibacterial substance, providing a theoretical basis for the prevention and control of Gram-negative pathogens.

The application provides an immune cell culture kit, a culture method and application of gamma delta T cells, and the kit comprises a basic culture medium, a recombinant antibody, a cytokine and a cell proliferation promoting additive, the basic culture medium comprises an RPMI-1640 culture medium, and the cell proliferation promoting additive comprises 1-thioglycerol, taurine, beta-cyclodextrin, thymidine, alpha-lipoic acid, beta-mercaptoethanol and bisphosphonate. The gamma delta T cells obtained by the culture kit have high purity and activity, and have good tumor killing activity. The gamma delta T cells obtained by the method have a high positive rate of 100% of perforin expression.

The invention discloses an engineered dendritic cell as well as a construction method and application thereof, and belongs to the technical field of immunology and medicine. The engineered dendritic cell is a DC cell induced and modified by adopting an engineering means, and an exogenous mRNA sequence is introduced into the DC cell, so that the engineered dendritic cell can efficiently express recombinant IL-18, IL-15Ralpha and TAA. Meanwhile, an exogenous cell factor IL-15 is added into a DC cell culture system after electrotransfection, so that the survival rate and the maturity degree of the cultured engineered DC cells are improved. The IL-15R alpha expressed by the engineered DC and exogenous IL-15 form a membrane anchoring IL-15 / IL-15R alpha compound, the membrane anchoring IL-15 / IL-15R alpha compound and secreted IL-18 play a synergistic effect, T cell activation and proliferation are effectively promoted, meanwhile, generation of cell factors IFN-gamma and release of cytotoxic molecule granzyme B and perforin are expanded, the specific cytotoxicity of T cells to tumors is improved, and the T cells can be used for treating tumor tumors. And the anti-tumor immune effect is sustained and enhanced.

The invention belongs to the technical field of pharmaceutical compositions for removing senescent cells, and discloses a pharmaceutical composition for removing senescent cells and a preparation method and application thereof, and the pharmaceutical composition for removing senescent cells contains NK cells and fisetin. According to the pharmaceutical composition for removing senescent cells, the fisetin and the NK cells are combined for use, on one hand, the fisetin can enable the NK cells to easily recognize senescent cells; on the other hand, fisetin can relieve inhibition of SASP factors on NK cell activity, enough amount of cytotoxic particles such as perforin and granzyme are synthesized and released in NK cells, the killing efficiency on senescent cells is high, fisetin can directly trigger an apoptosis pathway of the senescent cells, and the effect of killing the senescent cells is achieved. The NK cells release cytotoxic particles to further accelerate death of the senescent cells, and fisetin and the NK cells have a synergistic effect, so that the senescent cell clearing efficiency is high.

Compositions and methods are provided for isolating, manipulating and using for therapeutic and other purposes, mammalian, MHC Class I restricted, antigen-specific regulatory T cells. The regulatory T cells can be characterized as CD8+ cells that specifically suppress the responses of self-reactive and / or pathogenic CD4+ T cells by cytotoxic mechanisms including, without limitation, perforin, other components of the perforin / granzyme apoptosis pathway, etc. The regulatory T cells are antigen-specific, but are not activated by the same antigen as the self-reactive and / or pathogenic CD4+ T cells. In humans the regulatory T cells express inhibitory KIR proteins, e.g. one or more of KIR2DL2, KIR2DL3, and KIR3DL1.

The present application provides a dendrobium officinale polysaccharide extract, a preparation method and an application, the dendrobium officinale polysaccharide extract is with dendrobium officinale stem as raw material, is extracted by purified water reflux, is precipitated by ethanol, is deproteinized by Sevag, is decolorized and is deashed and is obtained by gel / film separation and purification, the mass percentage content of polysaccharide in the extract is 50-95wt%, the average molecular weight is 10-300 kDa, and the monosaccharide component includes mannose, glucose, galactose, fucose, rhamnose and arabinose.The dendrobium officinale polysaccharide extract provided by the present application has immune activation effect on natural killer cells, can promote the NK cell CD107a degranulation reaction and the expression of IFN-gamma, perforin and granzyme B in a dose-dependent manner, up-regulates the NKG2D related pathway, thereby activating the NK cell function and enhancing the killing activity of NK cells on tumor target cells such as K562.

The application relates to application of arachidonic acid inhibitors in preparation of drugs for improving tumor immune microenvironment. + The arachidonic acid inhibitors improve the ratio and expression amount of perforin and IFN-gamma secreted by T cells and improve the ratio and expression amount of IFN-gamma and IL-17A secreted by Treg cells.

The invention discloses application of a gene modified NK cell in tumor treatment, and belongs to the technical field of biology. The recombinant NK cell for expressing the human perforin-collagenase fusion protein is constructed through a CRISPR-Cas9 technology. The killing activity of a common NK cell to a human breast cancer cell MDA-MB-231 is 59.47%, the killing activity of a recombinant NK cell is 85.71%, and the killing activity is improved by 44.12%. A treatment test on a breast cancer model mouse shows that the average volume of a common NK cell group mouse breast tumor is 117.02 mm, and the NK cell infiltration rate is 15.28%; the mouse breast tumor of the recombinant NK cell group is obviously reduced, the average volume is 86.03 mm, and the NK cell infiltration rate is increased to 22.58%. The infiltration rate and killing activity of the recombinant NK cell on solid tumors are remarkably improved, and the recombinant NK cell can play an effective role in tumor treatment.

The present disclosure relates generally to pharmaceutical formulations (e.g., lyophilized formulations or topical formulations) comprising a perforin inhibitor (e.g., a benzenesulfonamide). In particular embodiments, the pharmaceutical formulations of the present disclosure are useful for treating diseases or disorders associated with undesirable perforin activity.

The invention provides a dendrobium officinale polysaccharide extract as well as a preparation method and application thereof.The dendrobium officinale polysaccharide extract is prepared from dendrobium officinale stems as a raw material through purified water reflux extraction, ethanol precipitation, Sevag deproteinization, decoloration and deliming and gel / membrane separation and purification, the mass percentage content of polysaccharide in the extract is 50-95 wt%, the average molecular weight is 10-300 kDa, and the content of polysaccharide in the extract is 50-95%. The monosaccharide components comprise mannose, glucose, galactose, fucose, rhamnose and arabinose. The dendrobium officinale polysaccharide extract provided by the invention has an immune activation effect on natural killer cells, can dose-dependently promote the degranulation reaction of NK cells CD107a and the expression of IFN-gamma, perforin and granzyme B, and up-regulate NKG2D related pathways, so that the functions of the NK cells are activated, and the killing activity of the NK cells on tumor target cells such as K562 is enhanced.

The application provides an application of dodartin A1 in activating NK cells. Through in-vitro experiments, it is proved that dodartin A1 can significantly promote the synthesis and release of IFN-gamma and cytotoxic substances (such as granzyme B and perforin) of NK92 cells, and increase the expression of the surface agonistic receptors NKp46 and NKG2D of the NK92 cells; through in-vivo experiments, it is proved that dodartin A1 can promote the expression of the NK cell activation related substances CD107a and NKp46 to play an anti-hepatoma effect; in conclusion, dodartin A1 has the effect of activating NK cells.

The invention relates to the field of metabolic engineering, in particular to an engineered microorganism as well as a construction method and application thereof. A first gene and a second gene are simultaneously expressed in the engineered microorganism, the first gene is a perforin protein coding gene with a nucleotide sequence as shown in SEQ ID NO: 1, and the second gene is a coding gene of hydrolase protein with a nucleotide sequence as shown in SEQ ID NO: 2. The engineered microorganism can complete the extraction of intracellular target products, reduce or completely replace the addition of exogenous enzyme preparations, and significantly reduce the use cost of enzyme.

The invention relates to construction of an engineering strain of high-yield soluble bacteriophage perforin Hol41 protein, a high-yield expression method, application and the engineering strain, fungi are used as host cells, and expression plasmids capable of secreting signal peptides are adopted to carry out free expression on bacteriophage perforin Hol41 genes, so that the engineering strain of high-yield soluble bacteriophage perforin Hol41 protein is obtained. Through the construction, the induced expression quantity of the soluble bacteriophage perforin Hol41 protein is greatly improved.

The invention discloses application of gamma-aminobutyric acid in preparation of a synergist of an immune checkpoint inhibitor, and belongs to the technical field of tumor treatment. Compared with independent treatment, combined administration of the GABA and the immune checkpoint inhibitor can significantly inhibit tumor growth, increase the infiltration proportion of CD8 + T cells in tumor tissues, and increase the proportions of IFN-gamma +, TNF-alpha + and Perforin + CD8 + T cells, so that the cytotoxic function of the T cells is enhanced. The results show that the GABA can promote the anti-tumor immune response of an organism by adjusting immune cell composition and T cell functions in a tumor microenvironment, so that the treatment effect of the immune checkpoint inhibitor is improved, and a new theoretical basis and application prospect are provided for the GABA as an auxiliary strategy for improving the tumor immunotherapy curative effect.

This invention provides a BiTE for targeted immunotherapy of tumors, its preparation method, and its application, relating to the field of biopharmaceutical technology. The BiTE is obtained by conjugating a nanobody to a fusion protein. The nanobody contains the amino acid sequence SEQ ID No. 3 at its C-terminus and has the function of targeting the human epidermal growth factor receptor on tumor cells. The fusion protein also targets the CD3 receptor on T cells and activates T cells, connecting T cells and tumor cells in time and space, activating T cells to secrete perforin and granzyme B to kill tumor cells, thus exerting a targeted immunotherapy effect. This invention utilizes isopeptide bonds to conjugate the nanobody and the fusion protein, completing the conjugation within 15 minutes with a conjugation efficiency of 100%, and extending the in vivo half-life of the nanobody by 6 times.

The application belongs to the technical field of bacteriophages, and discloses a recombinant bacteriophage for displaying bacteriophage perforin as well as a preparation method and application of the recombinant bacteriophage. The preparation method of the recombinant bacteriophage comprises the following steps: S1, using a bacteriophage genome as a template, amplifying a bacteriophage perforin coding gene sequence by using a fusion primer to obtain the bacteriophage perforin coding gene sequence, and performing enzyme digestion and recovery on the amplification product and an expression vector; S2, connecting the bacteriophage perforin coding gene sequence with a homologous arm and the enzyme-digested expression vector skeleton, and transforming the same into Escherichia coli to obtain recombinant Escherichia coli; and S3, performing bacteriophage rescue on the recombinant Escherichia coli to obtain the recombinant bacteriophage for displaying bacteriophage perforin. The lysis spectrum of the recombinant bacteriophage prepared by the method is greatly widened, and the recombinant bacteriophage can cross-species lyse various gram-positive bacteria and mycoplasma, and the lysis efficiency is also improved, so that the recombinant bacteriophage has important significance and value in antibacterial treatment and clinical application.

The invention provides a helicase mutant as well as a preparation method and application thereof in high-throughput sequencing. According to the helicase mutant, at least one cysteine on the surface of a three-dimensional structure of an amino acid sequence as shown in SEQ ID NO: 1 is substituted by alanine. The cysteine is C < 133 >, C < 164 >, C < 292 >, C < 323 > and / or C < 347 >; preferably, the amino acid sequence of the helicase mutant is as shown in SEQ NO: 6. The helicase mutant has good DNA unwinding activity and good protein homogeneity, reduces cross-linking interaction between protein molecules, improves the suitability of the helicase mutant with perforin, and improves the stability of sequencing and the integrity and continuity of an electric signal, so that the helicase mutant has a better sequencing effect, can be used for control and characterization of nucleic acid, and can be applied to the field of biochemistry. And the method is applied to single-molecule nanopore sequencing.

The invention provides application of strepsterol in preparation of a liver cancer radiotherapy sensitizing drug, and belongs to the technical field of biological medicine. The invention discloses a technical scheme for reducing the generation of macrophage chain sterol in radiation-resistant liver cancer tissues and promoting the sensitization of liver cancer radiotherapy by using chain sterol treatment for the first time. Wherein the CD8 < + > T cells serve as anti-tumor immune core effector cells, the killing function of the CD8 < + > T cells directly affects the curative effect, and the strepsterol can promote infiltration of the cells into tumor tissues and improve secretion of killing molecules such as perforin and granzyme, so that radiation-resistant hepatoma carcinoma cells are effectively eliminated. Streptosterol treatment not only reduces radiotherapy resistance factor expression, but also amplifies the radiotherapy killing effect, finally realizes efficient radiotherapy sensitization and significantly improves the tumor recession rate, provides a brand new efficient scheme for radiation-resistant liver cancer treatment, and has great clinical and social values.

The present application relates to the technical field of NK cell culture, and discloses a method for improving the killing function of NK cells and application thereof.The method comprises the following steps: (1) adding a haem oxygenase 1 antibody to an NK cell culture medium to prepare an antibody culture medium; (2) performing sterile filtration treatment on the antibody culture medium; and (3) separating NK cells and culturing the NK cells in the antibody culture medium.The present application adds a haem oxygenase 1 antibody to treat NK cells, blocks the expression of haem oxygenase 1 in the NK cells, or inhibits the activity of an effective substance of haem oxygenase 1.After being treated with the haem oxygenase 1 antibody, the secretion of granzyme B and perforin of the NK cells can be promoted, and the killing effect of the NK cells on target cells Yac-1 and K562 can be improved, so that the anti-tumor function of the NK cells is improved.

The invention discloses a Perforin CaMACPF6 and an application of the Perforin CaMACPF6 in construction of a novel flood-resistant vegetable germplasm. According to the invention, an Agrobacterium tumefaciens-mediated inflorescence dip dyeing method is utilized to construct an arabidopsis thaliana plant of the overexpressed perforation protein CaMACPF6 gene, and the obtained transgenic plant is cultured under a hypoxia stress condition to find that compared with a wild type control, the overexpressed perforation protein CaMACPF6 gene in the plant can significantly improve the hypoxia stress resistance of the plant. On the basis, the perforation protein CaMACPF6 can be used for constructing a crop variety resistant to hypoxia stress, so that the crop variety can resist waterlogging and hypoxia stress caused by other conditions, and the adverse effect of hypoxia stress on crop growth is relieved. The method is beneficial to the construction of hypoxia stress-resistant germplasm, and further reduces the loss of crops caused by flood disasters and the like.

This invention discloses an NK cell exosome capable of targeting and killing lung cancer and its preparation method. Specifically, it is based on NK cells derived from umbilical cord blood stem cells, and involves constructing CRISPR-Cas9-PD-1 knockout recombinant plasmids to transfect cells, and rapidly inducing umbilical cord blood stem cells to transform into CD3+ cells using IL-21. ‑ CD56 + NK cell differentiation and maturation, tandem ultracentrifugation, and size exclusion chromatography were used for high-efficiency purification to obtain PD-1 knockout NK cell exosomes containing granzyme, perforin, and FasL. L NK-EXO). The PD-1 constructed in this invention L NK-EXO treatment significantly prolonged the survival of a mouse model of lung cancer and reduced tumor size, while also demonstrating clear lung cancer targeting and specific killing functions. More importantly, this invention reveals that PD-1 knockout of NK cell exosomes is crucial for tumor targeting and killing.

The invention relates to a perforin Hol41 protein, an engineering strain, a soluble expression method and application. The soluble perforin Hol41 protein comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 3, and the soluble perforin Hol41 protein is obtained by encoding a codon optimized bacteriophage SP4 perforin Hol41 gene and an optimized SUMO soluble tag. Wherein the nucleotide sequence of the codon optimized bacteriophage SP4 perforin Hol41 gene and the nucleotide sequence shown as SEQ ID NO: 1 have at least 85% of identity; the nucleotide sequence of the optimized SUMO soluble tag has at least 85% of identity with the nucleotide sequence as shown in SEQ ID NO: 2. Through the design, the perforin protein with high antibacterial activity is obtained, the soluble expression of the perforin protein is also realized, and the stability and the induced expression quantity of the protein are remarkably improved.