65 results about "Thymosin α1" patented technology

The invention relates to a solid-phase synthesis process of a thymosin alpha 1, belonging to the polypeptide solid-phase synthesis technical field. The invention comprises the following steps: a. a Fmoc-Rink Amide AM resin or a Fmoc-Rink Amide MBHA resin is used as carrier, an H2N-Rink Amide AM resin or an H2N-Rink Amide MBHA resin is obtained after deprotection of the Fmoc; b. side chain carboxyl group of Fmoc-Asp-X is connected with resin amino by the method of solid-phase synthesis to obtain the Fmoc-Asp (resin)-X; c. the left amino acid in the sequence is synthesized in solid-phase with the Fmoc strategy; d. after the amino protection group Fmoc of N terminal amino acid is removed, the N terminal amino acid is acetylated by acetic anhydride and pyridine; e. then the acetylated N terminal amino acid is cut by a cracking agent (tri fluoroacetic acid / benzoylate sulfide / 1, 2- dithioglycol / Anisole) to obtain the thymosin alpha 1; f. crude product of the thymosin alpha 1 is prepared and separated by HPLC to obtain the pure thymosin alpha 1. The invention can increase significantly the yield of the thymosin alpha 1 and decrease the production cost, which is helpful for scale production and has better industrialization prospect.

The invention relates to a solid phase synthesis process of thymosin alpha1. The process comprises the following steps of: (1) preparation of NH2-Asn(Trt)-CTC resin; (2) synthesis of AA(1-27)-Asn(Trt)28-CTC resin; (3) preparation of Ac-AA(1-27)-Asn28(Trt)-CTC resin; (4) preparation of crude thymosin alpha1 by using a cracking agent; and (5) purification of the crude thymosin alpha1.

A method for overcoming immune suppression includes the steps of inducing production of naïve T cells and restoring T cell immunity. A method of vaccine immunotherapy includes the steps of inducing production of naïve T cells and exposing the naïve T cells to endogenous or exogenous antigens at an appropriate site. Additionally, a method for unblocking immunization at a regional lymph node includes the steps of promoting differentiation and maturation of immature dendritic cells at a regional lymph node and allowing presentation of processed peptides by resulting mature dendritic cells, thus, for example, exposing tumor peptides to T cells to gain immunization of the T cells. Further, a method of treating cancer and other persistent lesions includes the steps of administering an effective amount of a natural cytokine mixture as an adjuvant to endogenous or exogenous administered antigen to the cancer or other persistent lesions; preferably the natural cytokine mixture is administered in combination with thymosin α1.

The present invention relates to polyethylene glycol derivatives of thymosin alpha-1, their preparation process, the medicine composition containing them, and their application in the medicine for preventing and treating diseases related with immune deficiency and hypoimmunity, including hepatitis B, hepatitis C, malignant melanoma, non-small cell lung carcinoma, SARS, etc.

A method for overcoming immune suppression includes the steps of inducing production of naïve T cells and restoring T cell immunity. A method of vaccine immunotherapy includes the steps of inducing production of naïve T cells and exposing the naïve T cells to endogenous or exogenous antigens at an appropriate site. Additionally, a method for unblocking immunization at a regional lymph node includes the steps of promoting differentiation and maturation of immature dendritic cells at a regional lymph node and allowing presentation of processed peptides by resulting mature dendritic cells, thus, for example, exposing tumor peptides to T cells to gain immunization of the T cells. Further, a method of treating cancer and other persistent lesions includes the steps of administering an effective amount of a natural cytokine mixture as an adjuvant to endogenous or exogenous administered antigen to the cancer or other persistent lesions; preferably the natural cytokine mixture is administered in combination with thymosin α1.

The invention discloses a method and special engineering bacteria for producing N-terminal acetyl protein or polypeptide. The recombinant engineering bacteria capable of expressing transferase of archaebacteria on chromosomes are obtained by integrating the expression box of the transferase of archaebacteria onto the chromosomes of a host; the expression box for expressing the transferase of archaebacteria comprises a promoter and an archaebacteria transferase gene connected with the downstream of the promoter; and the nucleotide sequence of the archaebacteria transferase gene is represented by the sequence No.1 in a sequence list, and the amino acid sequence of the archaebacteria transferase gene is represented by a sequence No.2 in a sequence table. The engineering bacteria of the invention can directly produce complete N-terminal acetyl protein or polypeptide, such as thymosin extrasin alpha 1 and thymosin extrasin beta 4, the drawback of incompetence of acetylation or partial acetylation in a conventional genetic engineering technique is overcome, the production of N-terminal acetyl thymosin extrasin by the genetic engineering technique is realized completely, and the method and the special engineering bacteria are very practical.

The present invention discloses the preparation process of thynosin-alpha with acetylation modified N-end by using recombinant colibacillus. The preparation process includes obtaining thynosin-alpha gene, constructing recombinant expression vector containing thynosin-alpha, transforming prokaryotic cell, culturing prokaryotic cell and expressing thynosin-alpha, and separating and purifying thynosin-alpha with acetylation modified N-end. The present invention also discloses the process of utilizing recombinant colibacillus in preparing thynosin-alpha antigen with acetylation modified N-end and then cutting and separating to prepare thynosin-alpha-1 antigen with acetylation modified N-end, thynosin-alpha-2 antigen with acetylation modified N-end and similar matter. The thynosin-alpha with acetylation modified N-end has the functions of regulating immunity, cell proliferation, etc. and has wide application foreground in antivirus and antitumor.

It is described the use of thymosin alpha in combination with dacarbazine and optionally with Interferon alpha, for preparing a medicament for the treatment of malignant melanoma on stage IV characterized by distant unresectable metastases.

The invention discloses a method for preparing N-terminal acetylation extrasin alpha1, and special engineering bacteria thereof. The engineering bacteria is obtained in such a way that encoding genes of the extrasin alpha1 and encoding genes of N-terminal acetyl transferase are introduced to host strains. The N-terminal acetyl transferase is the protein of the following a or b: a) protein is composed of amino acid sequences shown by a sequence 7 in a sequence table; and b) protein is derived from the a) and is provided with the N-terminal acetyl transferase through the replacement and / or deletion and / or adding of one or a plurality of amino acid residue of the amino acid sequences shown by the sequence 7 in the sequence table. Talpha1 obtained through the preparation of the engineering bacteria is all N-terminal acetylized. The invention overcomes the defects of no acetylization or only partial N-terminal acetylization in a traditional gene project technology, thoroughly realizes N-terminal acetylization Talpha1 through a gene engineering technology, and has strong practical purposes.

The invention relates to a recombined extrasin alpha 1 two-strand body protein and a preparation method thereof. Glycin and serine are connected in series with bimolecular extrasin alpha 1 (T alpha 1), a connection mode is as follows: T alpha 1-Gly-Ser=T alpha 1, and an extrasin alpha 1 two-strand body gene expression vector is constructed by synthesized extrasin alpha 1 two-strand body genes so that extrasin alpha 1 which can not be directly expressed in colon bacillus can be efficiently expressed in the colon bacillus in a two-strand body way. The purified extrasin alpha 1 two-strand body protein has biological activity and can promote lymphocytes of small mice to proliferate, restrain tumor cell strains from proliferating and obviously restrain the tumor growth of tumor-bearing mice, thereby establishing a base for the further research and the extensive application of the extrasin alpha 1.

The present invention belongs to the field of biosynthesis and chemical modification technology. By means of bioengineering process, several thymosin genes are recombined and the recombinant gene is expressed in host cell to obtain high-expression thymosin fusion body. The fusion body is cracked and modified chemically to form thymosin alpha 1 monomer finally. The production process can produce thymosin alpha 1 identical with that produced chemically and is simple, low in cost and suitable for production in large scale. The present invention also provides the production process of several thymosin alpha 1 derivatives.

The present invention relates to active fragment of natural or artificial amino acid substituted thymosin alpha-1 and its polyethylene glycol derivatives, their preparation process, the medicine composition containing them, and their application in the medicine for preventing and treating diseases related with immune deficiency and hypoimmunity, including hepatitis B, hepatitis C, malignant melanoma, non-small cell lung carcinoma, SARS, etc.

Polyethylene glycol modifications of thymosin alpha 1 (T&agr; 1-PEGs), their preparation process, the medicine composition containing them, and their application in the medicine for preventing and treating diseases related with immune deficiency and hypoimmunity, including hepatitis B, hepatitis C, hepatoma, malignant melanoma, non-small cell lung cancer, SARS, and AIDS etc.

An orally taken medicinal composition of thymosin alpha 1 carried by microspheres features that it can be located in intestinal tract to release the thymosin alpha 1. It is prepared from thymosin alpha 1, acrylic resin as enteric material, polyoxyvinyl laurinol ether and laury azazole ketone.

The invention discloses a solid phase synthesis and purification process for thymosin [alpha]1 and analogues thereof, and belongs to the technical field of peptide synthesis. The process comprises the following steps of 1) taking a tricyclic amide linker resin as a carrier; 2) connecting side chain carboxyl groups of Fmoc-Asp-Y to amino groups of the resin after the resin is subjected to deprotection of Fmoc; 3) synthesizing residual amino groups in order in a solid phase by using a Fmoc strategy; 4) removing Fmoc protection groups from N-terminal amino protection groups, acetylating the N-terminal amino protection groups by using a DMF solution of Ac2O and DIPEA; 5) cutting by using a lysis reagent to obtain a crude peptide and precipitating the crude peptide with ether; and 6) separating the crude peptide by HPLC to obtain a pure product.

A method of treatment of hepatitis B virus (HBV) infection in a patient by administering to the patient a drug regimen including an antiviral-effective amount of thymosin alpha 1 (Talpha1), an antiviral-effective amount of lamivudine, and optionally an antiviral-effective amount of famciclovir is disclosed.

The invention relates to a porcine circovirus type 2 Cap protein, thymosin alpha1 fusion protein and an application, and an amino acid sequence of the fusion protein is shown as SEQ ID NO:1. The fusion protein can be used for preparing porcine circovirus type 2 subunit vaccine. The screened porcine circovirus type 2 Cap protein and a thymosin alpha1 gene having immunological enhancement effect are connected through a Lingker sequence to obtain the fusion protein. The genetic engineering subunit vaccine of the invention has good immunogenicity, and the immune response of piglet can be caused with high efficiency.

Pharmaceutical compositions that include thymosin alpha 1 peptide derivatives modified at the C-terminal of the peptide chain with polyethylene glycol, and their pharmaceutical acceptable salts, are generally disclosed. Also, new methods used to prepare these thymosin alpha 1 peptide derivatives modified at the C-terminal of the peptide chain with polyethylene glycol are generally provided. The presently disclosed compounds and their salts can be prepared administered to humans to treat immune disease and can also be used in adjuvant treatment.

The invention discloses a 4*thymosin alpha1 gene sequence and a method for preparing transgenic tomato, which belongs to the field of gene engineering. The method comprises the following steps: a nucleotide sequence with bioactive thymosin alpha1 polypeptide is encoded according to a plant preferential codon, and has at least 75 percent of homology with a nucleotide sequence containing 16th to 408th nucleotides in SEQ ID NO.2; or the nucleotide sequence can be hybridized with the nucleotide sequence containing 16th to 408th nucleotides in the SEQ ID NO.2 under moderate stringent conditions. The invention also provides a method for expressing thymosin alpha1 functional protein by utilizing a plant system. A 4*T alpha1 gene expresses the important effect and application value of a product in improving the immunity of human bodies and preventing and treating human major diseases such as hepatitis, HIV, cancer and diabetes in eukaryotic cells.

The invention discloses a recombined Talpha 1-BP5 fusion peptide, a gene, an engineering bacteria and an application. The recombined fusion peptide is prepared by fusing thymosin alpha 1 with Fabricius bursa pentapeptide BP5 through a soft Linker. The recombined Talpha 1-BP5 fusion peptide gene is inserted into an expression vector to transfer the escherichia coli so as to obtain the gene engineering bacteria for effectively express the Talpha 1-BP5 fusion peptide; the Talpha 1-BP5 fusion peptide is prepared through liquid cultivation and purification; and the thioredoxin of the fusion peptide is eliminated by using enterokinase with His tag at the N-end, and the fusion peptide is further subjected to affinity chromatography purification so as to obtain the single recombined Talpha 1-BP5 fusion peptide. The recombined Talpha 1-BP5 fusion peptide disclosed by the invention can be used as a novel polypeptide immunologic adjuvant which is used in match with vaccines, can effectively enhance the organism cell immune level and the body liquid immune level, and has wide application prospects.

The invention relates to a method to produce human thymosin alpha 1 that uses transgene tomato as bio-reactor. It optimizes the nucleotide sequence of human thymosin alpha 1 according to plant preference codon. Compounding the gene and making it connect end to end to form tetrad, then, the plant expression transforming tomato expressed by thymosin alpha 1 cascading gene that is driven by cauliflower 35s promoter would be constructed. The thymosin alpha 1 would have important effect in vivo that could promote T cell function and the producing of special antibody and cell element. It could cure autoimmune diseases like lupus erythematosus, chronicity hepatitis B, etc. It could be also used as assistant medicine for curing cancer and tumor. The fruit containing thymosin alpha 1 could enhance human body immunity.

The invention discloses a method and special engineering bacteria for producing N-terminal acetyl protein or polypeptide. The recombinant engineering bacteria capable of expressing transferase of archaebacteria on chromosomes are obtained by integrating the expression box of the transferase of archaebacteria onto the chromosomes of a host; the expression box for expressing the transferase of archaebacteria comprises a promoter and an archaebacteria transferase gene connected with the downstream of the promoter; and the nucleotide sequence of the archaebacteria transferase gene is represented by the sequence No.1 in a sequence list, and the amino acid sequence of the archaebacteria transferase gene is represented by a sequence No.2 in a sequence table. The engineering bacteria of the invention can directly produce complete N-terminal acetyl protein or polypeptide, such as thymosin extrasin alpha 1 and thymosin extrasin beta 4, the drawback of incompetence of acetylation or partial acetylation in a conventional genetic engineering technique is overcome, the production of N-terminal acetyl thymosin extrasin by the genetic engineering technique is realized completely, and the method and the special engineering bacteria are very practical.

The invention supplies a recombination thymosin alpha 1 gene, expression particle pKAT, engineering bacterium YKAT containing pKAT and filtered engineering strain YKAT-8, and the manufacture method for recombination thymosin alpha 1. It uses colibacillus preference codon, designing and combining recombination thymosin alpha 1 gene, recombining the upstream sequence, constructing pKAT and constructing and filtering YKAT-8. After fermenting and cultivating, directly excreting rT alpha-1 into culture medium, and the excreting quality could reach 500mg / L, and the purity could be over 95% after separating and purifying. It could sharply decrease producing cost and production cycle.

A GP-thymin alpha 1 (GP-T alpha 1) is prepared through chemically synthesizing thymin alpha 1 gene, cloning it to karyogamy expression carrier pTRX, configuring fusion expression plasmid pTRX-GP-T alpha 1, adding proteinase ReScission Protease (3C) recognition site between pTRX and Talpha 1, expressing thioredoxin-T alpha 1 fusion protein, purifying by chromatography, enzyme severing of Pre Scission Protease (3C) to release GP-Talpha 1, affinity chromatography, and HPLC purifying. It can be used to prepare the medicines for treating viral hepatitis, tumor and AIDS.

The invention provides a method for preparing thymosin alpha 1 by liquid phase fragment condensation, belonging to the technical field of biochemistry. According to the method, high capacity value (not less than 0.8mmol / g) resin is used as a starting material, firstly, a standard solid phase polypeptide synthesis (SPPS) technology is adopted for synthesizing a high-purity peptide fragments with selected structures, then, a liquid phase condensation technology is adopted for connecting the peptide fragments, and thus, a high-purity (more than 99%) target peptide is obtained. Compared with the solid phase thymosin alpha 1 synthesis technology, the method provided by the invention avoids the problem of low coupling ratio of amino acids behind the 12th position, and greatly improves yield (up to 25-30%) of the thymosin alpha 1; meanwhile, the peptide fragment formed by solid phase synthesis is free from purification, post-treatment technology is simplified, finally, the thymosin alpha 1 is purified by means of high performance liquid chromatography, preparation difficulty is reduced, preparation times is decreased, synthesis cost of the thymosin alpha 1 is lowered, and implementation of large-scale and industrialized production is facilitated.

The invention discloses a thymosin [alpha]1-porcine interferon [alpha] fusion protein gene and a preparation method of recombinant protein of the fusion protein gene. A nucleotide sequence of the reconstructed thymosin [alpha]1-porcine interferon [alpha] fusion protein gene provided by the invention is shown as SEQ ID NO.1. Meanwhile, the invention discloses a method for preparing the recombinant protein expressed by the gene and for conducting activity detection, wherein specifically, the method comprises such steps as reconstructing the thymosin [alpha]1-porcine interferon [alpha] fusion protein gene, cloning the gene and promoting secretory expression of the gene in pichia pastoris, preparing the recombinant protein and controlling fermentation culture conditions, improving such operating methods as the activity detection method and the like. The method provided by the invention is simple and easy to implement and is relatively low in cost; the efficient and stable secretory expression of the thymosin [alpha]1-porcine interferon [alpha] fusion protein in the pichia pastoris is achieved; and the expressed recombinant fusion protein is high in anti-viral activity and immune cell regulating activity levels; therefore, the invention lays a foundation for preparing green and no-residue biological products having functions of treating porcine viral diseases and immune regulation.

The invention discloses a new application of thymosin Alpha 1, which relates to the thymosin Alpha 1 and provides a new application of the thymosin Alpha 1. The thymosin Alpha 1 is used for preparing diabetes medicines. By the verification of animal experiments, the thymosin Alpha 1 can not only slow down the weight increase of a mouse, lower the normal fasting blood sugar of the mouse and obviously enhance the sugar tolerance of the mouse, but also prevent STZ from inducing the occurrence of diabetes of the mouse; besides, the thymosin Alpha 1 also plays an important role in protecting the structure of islet cells and the secretion level of insulin. The thymosin Alpha 1 can be used for maintaining normal glycometabolism and protecting the structure and functions of islet cells; by adopting the method of taking the thymosin Alpha 1 orally, the effect of obviously slowing down weight increase can be realized; therefore, the thymosin Alpha 1 can be applied to preparing drugs for preventing diabetes from occurring and treating diabetes and clinical practice.

Thymosin-α1 is used as an adjuvant in combination with carmustine (BCNU) as an effective treatment for malignant glioblastoma.