35 results about "Recombinant Human Interleukin-2" patented technology

The invention discloses a culturing method of an NK (natural killer) cell. The culturing method comprises the following steps of (1) separating a mononuclear cell from peripheral blood or umbilical cord blood of a human body; (2) inoculating the mononuclear cell into a culture medium suitable for culturing lymphocyte, adding a CD3 monoclonal antibody, recombinant human interleukin 2 and autologous plasma, and culturing for 3 to 5 days; (3) adding the recombinant human interleukin 2 and the autologous plasma, and culturing; (4) harvesting the NK cell. According to the culturing method of the NK cell, the culturing cost is reduced, the amplification multiple and cell toxicity of the NK cell are guaranteed, the operation time is saved, meanwhile the probability of error operation is reduced, the obtaining efficiency of the NK cell is higher, and the safety of the NK cell is better.

The invention provides an adult regulatory T cell in-vitro amplification culture medium and an application method thereof. The adult regulatory T cell in-vitro amplification culture medium comprises transform growth factor-beta (1-4 ng/ml), bone morphogenesis protein 4 (8-12 ng/ml), recombinant human interleukin-2 (300-500U/ml), rapamycin (80-100nM/ml), al-trans vitamin A acid (1-4 uM/mL), 4-hydroxyethylpiperazinoethylsulfonic acid (20-30mM/ml), L-glutamine (2mM/ml), 2-mercaptoethanol (40-50uM/ml), 5% human AB type serum, CD3CD28 magnetic bead, penicillin (50U/ml) and streptomycin (50ug/ml). When being used for performing amplification culture and induced differentiation on regulatory T cells, the culture medium can shorten the amplification and differentiation time of the regulatory T cells, and can obtain the high-purity Foxp3CD4+CD25+CD127-regulatory T cells. Therefore, the regulatory T cells can be used in the aspect of clinical treatment of anti-transplantation immunity rejection, autoimmune disease, allergic disease and the like to perform a novel clinical cell therapy.

The present invention provides a method for achieving high-level expression of the therapeutically important lymphokine (human IL-2). The method comprises of identifying the secondary structure in the 5′ region of human IL-2 mRNA, modifying the 5′ region of the human IL-2 DNA sequence to produce a new DNA sequence wherein the mRNA transcribed from the modified human IL-2 DNA sequence has the predicted 5′ secondary structure destabilized with increased free energy compared to that of the secondary structure of the mRNA transcribed from the native DNA sequence without altering the sequence of the encoded amino acids; and using this modified DNA sequence of human IL-2 for high level recombinant expression in a microbial host for large scale production. This method is also applicable to other expression host like yeasts and mammalian cells.

The invention relates to a serum-free cryopreservation medium and method for peripheral blood monouclear cells. The serum-free cryopreservation medium comprises recombinant human interleukin-2, humanserum albumin, polyethylene glycol, trehalose and a basic culture medium or sodium chloride for injection. The serum-free cryopreservation medium has the advantages that the recombinant human interleukin-2 (IL-2) is added into the cryopreservation medium, so that the activity stability of immune cells cultured by thawed cells can be increased greatly, the high activity of original cells can be kept, the immune cells can well keep the physiological function and biological feature of the thawed cells, and the problem of direct large-scale amplification of the thawed cells can be solved effectively; the polyethylene glycol and the trehalose are added into the cryopreservation medium, the cryopreservation medium can replace an existing dimethyl sulfoxide-based (DMSO-based) cryopreservation medium, the toxicity of the cryopreservation medium to the cells is lowered effectively, cell stability is maintained, the direct application safety of the thawed cells is increased, and the thawed cellscan be directly used.

The invention relates to a coupling compound formed by the recombinant human interleukin-2 connecting with the monomethyl polyethylene glycol through the amino acid or the oligopeptide; the coupling compound has rather good plasma stability, and can release and recompose the recombinant human interleukin slowly to actively play the pharmacological action.

The invention relates to an improved expansion culture medium for regulatory T cells of human cord blood origin and an application method of the expansion culture medium. According to the expansion culture medium, heparin anticoagulated autologous cord blood plasma accounting for 10%-12% of the volume of a culture medium, CD3-CD28 antibody co-expressed immunomagnetic beads, recombinant human interleukin 2, 2-mercaptoethanol, rapamycin, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and gentamicin are added into the RPMI (Roswell Park Memorial Institute)1640 culture medium; and then separated cell suspension is inoculated in a 96-well plates with a U-shaped bottom, hole-division expansion can be performed every 1-2 days, and the expansion period is 3-4 weeks. All reagents in the culture system reach the GMP (good manufacturing practice) level or are originated from autologous cord blood, so that risks caused by ingredients of animal origin are avoided, and the regulatory T cells can be used for a third-party unrelated donor and directly applied to clinical disease treatment; and compared with a traditional culture system, Treg cells (the regulatory T cells) expanded by the improved culture medium is excellent in aspects of growth speed, purity, activity, lymphocyte inhibition function and the like, and the Treg cells are expected to be used as the regulatory T cells of the third-party unrelated donor and applied to the clinical disease treatment.

The invention provides a preparation method for poly(ethylene glycol) modified recombinant human interleukin-2, belonging to the field of biological medicine. The method comprises the following steps: activating mPEG with an activator so as to obtain an activated mPEG molecule; and reacting the activated mPEG molecule with recombinant human interleukin-2, adding a glycine solution to terminate a reaction and separating and purifying an obtained product; wherein the activator is one selected from the group consisting of succinic anhydride and N-hydroxysuccinimide, p-toluenesulfonic acid-chlorine, N,N'-carbonyl diimidazole, N,N'-disuccinimidocarbonate, p-nitrophenyl carbonate, benzotriazole carbonate, phenylsuccinimide carbonate and N-acetoxysuccinimide. The preparation method provided by the invention is simple and convenient to operate and is easy for quality control and enlarged production.

The invention discloses a frozen stock solution of immune cells. The frozen stock solution of the immune cells comprises the following components: 25 to 100 U/mL of recombinant human interleukin-2, 0.5 to 2.5 mg/mL of alpha-1,4-glucan, 0.05 to 0.2 mg/mL of trehalose, 0.1 to 0.5 mL/mL of polyethylene glycol, 0.05 to 0.15 mL/mL of propylene glycol and 0.7 to 0.9 mL/mL of a basic medium. The frozen stock solution of the immune cells disclosed by the invention has the beneficial effects of being high in safety, small in damage to the cells and capable of guaranteeing normal biological functions after the cells are recovered.

The invention provides a culture method for monkshood polysaccharide-induced nature killer T cell (Nature Killer T cell, NKT) proliferation. The method comprises the characteristics as follows: human peripheral blood, marrow or a mononuclear cell with an umbilical cord blood source are continuously cultivated by traditional Chinese medicine monkshood polysaccharide and recombinant human interleukin 2, a lot of nature kill T cells are obtained, NK<1.1+> and CD<8+> are expressed, interferon gamma is secreted, the ratio in lymphocyte at the 14th day is more than 16.15%, the order of magnitude can be up to over 5*10<9>, and the nature kill T cell have effective anti-tumor and anti-virus functions. A clinical application of the NKT cell obtained by the method is also provided by the invention.

The invention discloses immune cell preserving fluid. The immune cell preserving fluid comprises 0-50 U/mL of recombinant human interleukin-2, 0-10 mg/mL of human albumin, 0-1.5 mg/mL of alpha-1,4-glucan, 0.5-2.0 mg/mL of glucose, 3.5-5.0 mg/mL of sodium citrate, 2.0-3.5 mg/mL of sodium gluconate, 0.2-0.5 mg/mL of potassium chloride and 0.2-0.5 mg/mL of magnesium chloride. The immune cell preserving fluid has the advantages of being suitable for cell preservation within a wide temperature range, and capable of guaranteeing high cell viability of immune cells and facilitating follow-up cell experiment or clinical treatment.

The invention belongs to the field of biotechnology, and particularly relates to an improved cryopreservation solution for peripheral blood mononuclear cells, which comprises the following components:fetal bovine serum, dextran, DMSO, trehalose, polyethylene glycol and recombinant human interleukin-2. Fetal bovine serum and dextran are selected as nutritional support components of the cells to ensure cell nutrients and maintain a good osmotic pressure environment. The combination of permeable and non-permeable protective agents DMSO, trehalose and polyethylene glycol is used to effectively reduce cytotoxicity, maintain cell stability and improve the survival rate of recovery. By adding recombinant human interleukin-2, the activity and stability of cultured immune cells after recovery aresignificantly improved. The experimental results show that the PBMC cells cryopreserved with the improved cryopreservation solution provided by the invention have higher PBMC recovery rate, cell survival rate, stronger cell proliferation ability and high activity to be induced cultured into other immune cells after resuscitation. The cryopreservation solution provided by the invention can be directly frozen at minus 80 DEG C during cryopreservation.

The invention discloses an extraction method of a recombinant human interleukin-2 fermentation inclusion body. The extraction method comprises the following steps: (1) culturing an engineering strain of an escherichia coli expression recombinant interleukin-2; (2) breaking a thallus obtained by fermentation and culturing to collect an inclusion body; (3) extracting and washing the inclusion body to obtain a refined inclusion body, wherein the engineering strain is E.Coli K802(ply-4), the culturing method comprises multiplication culture and fermentation tank culture, which both adopt a yeast and peptone-containing antibiotic-free culture medium, and the thallus is broken by adopting a high-pressure homogenizer, and is degenerated to obtain a high-purity ril-2 stock solution. The extraction method disclosed by the invention is short in production period, high in production efficiency, large in production scale, and especially suitable for industrialized production; the production cost is lowered.

The invention discloses a fusion protein of interleukin 2 and human serum albumin. Plasmids with a fusion gene of interleukin 2 and human serum albumin are electrically transferred into CHO cells to obtain a CHO monoclonal cell strain capable of stably and efficiently expressing human recombinant protein. The monoclonal cell strain of the invention can secrete and express the fusion protein of the interleukin 2 and the human serum albumin, and the fusion protein can prolong the plasma half-life period of the human interleukin 2 and can be used for preparing human interleukin 2 drugs or drugs for treating various diseases, such as tumors and immunodeficiency diseases.

The invention discloses a method for improving the yield of recombinant human interleukin-2 (rhIL-2). The method comprises the steps of purification, eluent recovery after the purification, reductionof rhIL-2 dimer and wrong ligand by using 2-mercaptoethanol, reoxidation renaturation, and purification. The method performs reduction of rhIL-2 dimer and wrong ligand by using the 2-mercaptoethanol,opens disulfide bonds, and performs reoxidation renaturation and purification, so that correctly-paired disulfide bonds with biological activity can be formed so as to obtain the rhIL-2 with the biological activity, and the yield is improved by about 20 percent.

The invention relates to the field of proteins, in particular to application of recombinant human calcineurin B subunit, and mainly provides application of rhCNB to preparation of a medicine for killing and/or inhibiting a gastric cancer cell MGC-803. When rhCNB for injection plays a role in treatment of human gastric cancer cell MGC-803 carcinoma in situ transplantation tumors in BALB/c nude mice, the effective tumor treatment (compared with that of a solvent control group, p is smaller than 0.05) time starts after continuous medication for 6 times; in the whole medication process, the tumors can be effectively controlled; one week after medicine withdrawal, rhCNB for injection still has a continuous and effective treating effect (compared with that of the solvent control group, p is smaller than 0.001) on the tumors; the performance of rhCNB for injection is superior to those of cisplatin for positive control injection and hydroxycamptothecine for injection, and equivalent to recombinant human interleukin-2 for injection.

The present invention discloses an application of the interleukin-2 (IL-2) and the derivatives in the preparation of the drugs for treating rhinitis and pertains to the field of biomedicine. The present invention has the advantages that: the present invention firstly uses the recombinant human interleukin-2 in the treatment of rhinitis, the results show that the interleukin-2 can obviously alleviate the symptoms and physical signs of the rhinitis, significantly alleviate the nasal lesions and have the accurate efficacy.

The invention discloses an optimized nucleotide sequence of recombinant human interleukin-2. The nucleotide sequence is optimized according to preferred codons of Escherichia coli under the circumstance of not changing amino acid coding sequence. The nucleotide sequence is as shown in SEQ ID NO:1, or is complementary nucleotide sequence. Furthermore, expression vector and engineering bacteria of the nucleotide sequence are constructed, and recombinant human interleukin-2 is expressed in the engineering bacteria. The expressed recombinant human interleukin-2 accounts for 10-30% of total mycoprotein, and soluble expression amount accounts for 3-15% of total expression amount. The amount of recombinant human interleukin-2 inclusion body is reduced, expression amount of soluble protein is raised, and conditions are created for mass production of soluble recombinant human interleukin-2.

The invention belongs to the field of medicine preparation, and particularly discloses application of low-dose interleukin 2 in medicine for treating chronic spontaneous urticaria. The low-dose recombinant human interleukin 2 is innovatively injected to the subcutaneous or intramuscular part of a patient suffering from chronic spontaneous urticaria, the severity and frequency of erythema and whealattack of the patient suffering from chronic spontaneous urticaria with poor treatment control of conventional antihistamine can be reduced, the pruritus degree can be reduced, or the medicine takingdose and medicine taking frequency of the antihistamine can be reduced, and the antihistamine can even be completely stopped from being taken for part of patients.