39 results about "TRAIL Protein" patented technology

The present invention relates to the construction of a TRAIL DNA cassette for the production of a secretable trimeric rTRAIL, the development of pCMVdw vectors and pAAVdw vectors harboring a feed-forward amplification loop type Tet-On system that can be packaged into AAV particles, the preparation of a recombinant vectors by the combination of the TRAIL DNA cassette and the two vectors, and the treatment of diseases including cancer using such vectors. The present invention provides a TRAIL DNA cassette comprising a secretion signal (SS) sequence, a trimer-forming domain (TFD) and a TRAIL(114-281) coding cDNA. The TRAIL cassette thus constructed can be cloned into an appropriate expression vector, and subsequently used in the mass production of a secretable recombinant trimeric TRAIL protein or administered to a patient for a gene therapy.

The present invention discloses recombinant soluble human tumore necrosis factor related death inducing ligand and its preparation process and application in preparing antitumor medicine. The present invention adopts human tonsil tissue mRNA as template to amplify TRAIL coding sequence, constitute engineering colibacillus for expressing rhsTRAIL and engineering saccharomycete, establish the rhsTRAIL engineering colibacillus fermentation, occlusion body washing and destination protein purification process and the engineering saccharomycete and destination protein purification process separately, and obtain pure rhsTRAIL product, which has molecular weight of 19.6-24 KD and exists in both monomer and dimmer. The rhsTRAIL can induce death of several kinds of cancer cells outside body, inhibit amplification of liver cancer cell in tumor loading mouse obviously and kill tumor cell selectively, so that it may be used in preparing effective antitumor medicine.

The invention belongs to the technical field of genetic engineering drugs, and discloses a double-target-spot fusion protein capable of enhancing the TRAIL antitumor activity. The cDNA sequence of the overall-length fusion gene sequence 1 is shown in SEQ ID NO1; the coding amino acid sequence of the overall-length fusion gene sequence 1 is shown in SEQ ID NO2; the coding cDNA sequence of the overall-length fusion gene sequence 2 is shown in SEQ ID NO4; and the coding amino acid sequence of the overall-length fusion gene sequence 2 is shown in SEQ ID NO5. In comparison with the prokaryotic expression carrier of separate TRAIL protein soluble fragment coding cDNA sequence, the fusion protein of the prokaryotic expression carrier has higher soluble expression, and the recycling and purifying efficiency of the fusion protein is higher; and the in-vitro biological activity analysis indicates that the fusion protein can activate death receptors on the cell membrane and inhibit expression of an XIAP gene in the cell plasma, enhance the activity of inducing tumor cell apoptosis through the double-target-spot function in apoptotic pathway and ensure stronger antitumor effect.

The invention provides a preparation method for a soluble truncated human tumor necrosis factor-related apoptosis inducing ligand (TRAIL) active protein, comprising the following steps of: (A) designing and synthetizing an oligomerization deoxyribonucleic acid (DNA) single-stranded segment according to the amino acid sequence of the human TRAIL protein issued by a Genebank by the preference of a bacterium genetic code; (B) synthetizing a complete double stranded DNA by three-time polymerase chain reaction (PCR); (C) carrying out amplification by utilizing a T-carrier, and inserting the amplified double stranded DNA segment into an expression carrier and screening a positive transformant; (D) expressing the truncated human TRAIL protein in escherichia coli at low temperature; (E) purifying the protein by using a three-step method; and (F) determining the truncated TRAIL protein. The invention realizes that the truncated human TRAIL protein is efficiently expressed in an escherichia coli cell and a purification preparation technique thereof and overcomes the problem that an infusible inclusion body is easy to form in the prior art. The method has the advantages of simple operation, short fermentation time and easy purification, and moreover, the protein is ensured not to have any modification, and the protein reaches electrophoretically pure. The invention can be directly used for the research work of biochemistry and molecular biology and the oncology and the preclinical stage of tumor treatment or the development of clinical drugs.

The invention, relating to the field of biotechnology, provides TRAIL fusion protein, a DNA sequence encoding the fusion protein, a vector containing the DNA sequence, a host cell or a transgenic animal containing the vector, a preparation method of the fusion protein and applications of the fusion protein. The TRAIL fusion protein from N-terminal to C-terminal comprises crosslinking region, humanized leucine zipper sequence and humanized TRAIL protein, humanized TRAIL extracellular region or fragments of humanized TRAIL extracellular region, can observably increase stability, prolong the half life in animal body and improve treatment effect, and has extensive application prospect.

The invention provides a TRAIL secretion mesenchyma stem cell and a purpose thereof for encephaloma treatment, and relates to the field of gene recombination and stem cell application. Particularly, the invention provides a building body for expressing a secretion type TRAIL protein soluble fragment and a slow virus expression vector containing the building body. The invention also provides the mesenchyma stem cell integrating the building body on the genome; the TRAIL protein fragment can be expressed and secreted. The invention also provides application of the building body or the vector or the mesenchyma stem cell to encephaloma treatment.

A method and compound for enhancing the lethality of an anti-cancer therapy, such as radiation, chemotherapy, or TRAIL protein, are disclosed. The compound is composed of morpholino subunits joined by phosphorodiamidate linkages, and has a targeting sequence that is complementary to an AUG start, IRES, or splice-donor region of the transcript for human X-linked inhibitor of apoptosis protein (XIAP). The method includes exposing cancer cells to the compound.

An application of the colibacillus expressing TRAIL protein in preparing the medicines for treating tumor is disclosed. Said colibacillus with tumor target and carrying said TRAIL protein can be located in the tumor tissue to make the TRAIL protein to play its stronger suppression role to tumor.

The invention provides a molecular design and preparation technique of a novel multifunctional targeted nanocapsule anticancer drug. The technique comprises the following steps: wrapping paclitaxel with a carboxylated block copolymer F127-COOH and P123 to form a nano micelle, and forming a silicon-dioxide-shell stable micelle structure in the micelle by using tetramethoxysilane to form drug-carrying nanocapsules; and coupling carboxyl groups on the nanocapsule surface with amino groups on the TRAIL protein surface by using a coupling agent to form the surface-TRAIL-protein-coupled drug-carrying nanocapsules. The technique has the advantages of favorable repetitiveness and mild conditions. The prepared multifunctional targeted nanocapsule anticancer drug has the advantages of high stability, wide anticancer spectrum and obvious curative effects.

This invention relates to the biotechnology area, and provides a kind of TRAIL chimeric protein, DNA sequence encoding this chimeric protein, vectors comprising this DNA, host cells or transgenic animals that contain one of the vectors, and preparation methods for the said chimeric protein and its applications. The said TRAIL fusion protein from the N to C terminal comprises human leucine zipper sequence, human TRAIL protein, human TRAIL extracellular domain or a fragment thereof. The chimeric protein has significantly enhanced stability, prolonged half-life in animals, increased efficacy and thus has broad application future.

The industrial extracting, renaturation and purifying process of recombined TRAIL inclusion body protein includes crushing, washing and other pre-treatment of the recombinant colibacillus thallus expressing TRAIL to obtain TRAIL inclusion body of 80 % over purity; dissolving the TRAIL inclusion body with denaturant to obtain denaturated solution; renaturating the solution of the denaturated inclusion body solution via diluting course, two-step dialysis to eliminate residual denaturant to obtain renaturating solution containing TRAIL active protein, superfiltering and concentrating the renaturating solution, column chromatography to separate and to collect qualified eluted liquid as the destination product, which is one kind of renaturated recombinant TRAIL protein with similar activity with soluble expression protein, in the yield over 40 %.

The invention belongs to the field of genetically engineered drugs and provides a mutant protein MuR5S4TR of TRAIL, and a preparation method and use thereof. The 2-10th bits of an N-terminal amino acid sequence of the mutant protein are composed of a transmembrane peptide sequence RRRRR (R5) and a binding sequence AVPI of an apoptosis inhibitor XIAP, and the 11-169th bits are TRAIL protein peptide segments (123-281aa). The specific sequence is shown in SEQ ID NO:2.

The invention discloses an exosome, a method for preparing the same and application of the exosome to preparing medicines for skin superficial tumor. The method particularly includes carrying out Trail gene transformation on Escherichia coli so as to express Trail proteins; extracting the exosome of the Trail proteins; carrying out lysozyme treatment, then modifying nano-particles by the aid of target peptides and loading a photosensitizer indocyanine green (ICG). The ICG can be triggered by near-infrared light to emit heat and release singlet oxygen after the nano-particles are delivered by means of skin penetration, and tumor cells can be synergistically killed by the nano-particles and the Trail proteins. The exosome, the method and the application have the advantages that the shortcomings of the traditional methods can be overcome by the aid of the method for preparing the exosome, the method is high in yield and medicine-loading rate, short in cycle and low in cost, and the medicines are easy and convenient to load; reports of application of exosomes to delivery by means of skin penetration are unavailable at present, the exosome can efficiently penetrate skin, and accordinglynovel strategies can be provided to treating skin diseases; in addition, therapy for curing melanoma is clinically available, the exosome can be combined with photo-thermal and biological therapy after penetrating the skin, accordingly, development and metastasis of the melanoma can be synergistically suppressed, and excellent in-vivo treatment effects can be realized.

Provided is a nucleic acid fragment, the nucleotide sequence thereof being shown in SEQ ID NO: 1. Provided is a recombinant protein encoded by said nucleic acid fragment, and a preparation method and use of said recombinant protein. Also provided are a conjugate of a recombinant protein modified by PEG, and a preparation method and use of said recombinant protein. The recombinant protein has anti-tumor activity, and acts in synergy with a proteasome inhibitor; the PEG modification of the recombinant protein, while retaining anti-tumor drug effects, also has improved stability and a longer half-life.

The invention discloses a recombinant protein with improved stability and activity, obtained by using adenovirus fibrin fragments as a trimer motif and adopting TRAIL fusion expression and used as an antitumor therapy drug. The invention also provides a method for expression, purification and activity detection of the fusion protein. The method concretely comprises the following steps: 1, designing and constructing fusion gene, and carrying out low-temperature inducible expression by using an Escherichia coli system; 2, purifying the target protein by using a nickel column affinity chromatography technology, and removing an His label by using a thrombin digestion site to obtain the final target product; 3, detecting the killing activity of the fusion protein to tumor cells and the toxicity of the fusion protein to normal cells in vitro; 4, detecting the stability of the fusion protein under different conditions; and 5, detecting the in vivo antitumor activity of the fusion protein in a nude mouse model. Two adenovirus fibrin fragments are used as the TRAIL fusion protein of the trimer motif. The invention provides a method for simply and rapidly obtaining the high-stability TRAIL protein. The method can be applied to antitumor researches of the TRAIL protein. The abuses of poor stability and low activity of the TRAIL protein are solved.

The invention opened a method to express the soluble TRAIL protein which includes cloning the extracellular gene of the TRAIL into the expression carrier with the PL promotor of the ª™bacteriophage; Transforming the cI+ host bacteria; expressing the recombinative TRAIL protein by the low-temperature induction. Also it can add the Zn2+ or the synthetic inhibiter of the prokaryotic protein into the medium in the inducing stage. The recombinative TRAIL protein is soluble, so it can simplify the isolation process.

The invention relates to a method for gene modification of mesenchymal stem cells through a tripolymer TRAIL fusion protein, and application. A nucleic acid sequence of the tripolymer TRAIL fusion protein is shown as SEQ ID NO:1. The tripolymer TRAIL fusion protein can form a triple-helix parallel coiled coil structure domain in a molecular structure through disulfide bond covalent binding, and thus the protein is assisted in being folded into a tripolymer. The tripolymer structure domain is subjected to covalent bond binding, therefore, the N tail end of the TRAIL protein is additionally provided with the structure domain, the stability and the antineoplastic activity of the TRAIL tripolymer can be enhanced significantly.

The invention mainly relates to the field of genetic engineering drugs, in particular to a mutant cDNA sequence obtained by mutating valine at position 114, glutamate at position 116, glycine at position 118, proline at position 119 and glutamine at position 120 in an amino acid sequence at positions 114-281 of an extracellular fragment of a wild-type TRAIL protein respectively into arginines, so as to allow amino acids at positions 114-121 of the TRAIL protein to form a 8-consecutive arginine sequence, and then by gene synthesis and PCR mutation and splicing; and the TRAIL mutant has excellent therapeutic effect for a variety of tumors of different types, and is a new generation of promising drug for highly efficiently inducing tumor cell apoptosis.

The invention discloses recombination human HM-TRALL protein with an amino acid sequence shown in the SEQ ID NO.1. The method comprises the steps that a human recombination HM-TRALL protein expression carrier containing a His6-Myc label is built at first, recombination bacteria for expressing human recombination HM-TRALL protein are built through the carrier, recombinant expression is carried out on the recombination human HM-TRALL protein through IPTG induction, extraction and purification are carried out, and high-purity HM-TRAIL protein is obtained. The recombination human HM-TRALL protein can selectively induce apoptosis of breast cancer cells in vitro, and will not cause normal breast cell apoptosis. The prepared recombination human HM-TRALL protein has the advantages of being good in stability and high in dissolving degree, can obviously induce apoptosis of tumor cells and is good in application prospect.

The invention belongs to the biotechnological field, and particularly relates to tripolymer TRAIL protein and application thereof.The tripolymer TRAIL recombination protein is prepared through the steps that a 114-281 amino acid sequence of TRAIL and a vector PET are subjected to gene cloning to prepare TRAIL recombination protein (HZ-TRAIL for short) with an N-terminal histidine label and an isoleucine zipper motif recombination gene pET23dw-His-ILZ-hTRAIL114-281; the HZ-TRAIL is modified by polyethylene glycol to enable PEG to be coupled to the N-terminal amino of the HZ-TRAIL protein through a covalent bond, and then the polyethylene glycol-modified TRAIL recombination protein, that is, the PEG-HZ-TRAIL protein is obtained.The TRAIL recombination gene is improved into the novel PEG-HZ-TRAIL through a PEGylation technique to produce the high-activity TRAIL protein of a tripolymer, and the tripolymer TRAIL protein has the advantage of being high in pharmacological efficacy on resisting tumors and rheumatoid arthritis.

A method for production of anti-tumor TRAIL includes inserting a TRAIL molecule, encoded by a viral vector irreversibly derived from a cell line, into a carrier cell, thereby obtaining a stably TRAIL-producing carrier cell, and wherein the TRAIL molecule includes a soluble molecule.

The invention discloses a gene drug for combined treatment on tumors, and a use thereof. The gene drug is a composition for preventing and / or treating tumors. The composition is composed of 1), Endostatin protein coding gene-containing recombinant viruses and TRAIL protein coding gene-containing recombinant viruses, or 2), an Endostatin protein and a TRAIL protein, or 3), Endostatin protein coding gene-containing recombinant vectors and TRAIL protein coding gene-containing recombinant vectors. An experiment proves that in the gene drug provided by the invention, an Endostatin gene for inhibiting tumor neovascularization and a TRAIL gene for promoting tumor cell apoptosis respectively carry signal peptides and are used for construction of adenovirus high-expression vectors. The gene drug can be used for treating a cervical cancer, a liver cancer, a breast cancer and a lung cancer.

The present invention relates to: a recombinant lentiviral vector comprising a gene encoding a TRAIL protein and a CD protein; and a cell that is transfected with the lentivirus produced by using the vector. A host cell transfected with the recombinant lentivirus of the present invention maintains a high cell proliferation rate and overexpresses a TRAIL protein and a CD protein. Thus, a mesenchymal stem cell transfected with the lentivirus may be usefully employed as a cell therapeutic agent.

The invention mainly relates to the field of genetic engineering drugs, in particular to a mutant cDNA sequence obtained by mutating valine at position 114, glutamate at position 116, glycine at position 118, proline at position 119 and glutamine at position 120 in an amino acid sequence at positions 114-281 of an extracellular fragment of a wild-type TRAIL protein respectively into arginines, so as to allow amino acids at positions 114-121 of the TRAIL protein to form a 8-consecutive arginine sequence, and then by gene synthesis and PCR mutation and splicing; and the TRAIL mutant has excellent therapeutic effect for a variety of tumors of different types, and is a new generation of promising drug for highly efficiently inducing tumor cell apoptosis.

The present invention belongs to the field of genetic engineering drugs, and provides a mutein MuR5S4TR of TRAIL, and a preparation method and use thereof. The N-terminal positions 2-10 of the amino acid sequence of the mutein consist of the transmembrane peptide sequence RRRRR (R5) and the binding sequence AVPI of the apoptosis inhibitor XIAP, the positions 11-169 are the TRAIL protein peptide segment (123-281 aa), and the specific sequence is as shown in SEQ ID NO: 2.

The invention discloses a recombinant protein with improved stability and activity, obtained by using adenovirus fibrin fragments as a trimer motif and adopting TRAIL fusion expression and used as an antitumor therapy drug. The invention also provides a method for expression, purification and activity detection of the fusion protein. The method concretely comprises the following steps: 1, designing and constructing fusion gene, and carrying out low-temperature inducible expression by using an Escherichia coli system; 2, purifying the target protein by using a nickel column affinity chromatography technology, and removing an His label by using a thrombin digestion site to obtain the final target product; 3, detecting the killing activity of the fusion protein to tumor cells and the toxicity of the fusion protein to normal cells in vitro; 4, detecting the stability of the fusion protein under different conditions; and 5, detecting the in vivo antitumor activity of the fusion protein in a nude mouse model. Two adenovirus fibrin fragments are used as the TRAIL fusion protein of the trimer motif. The invention provides a method for simply and rapidly obtaining the high-stability TRAIL protein. The method can be applied to antitumor researches of the TRAIL protein. The abuses of poor stability and low activity of the TRAIL protein are solved.

Provided is a nucleic acid fragment, the nucleotide sequence thereof being shown in SEQ ID NO: 1. Provided is a recombinant protein encoded by said nucleic acid fragment, and a preparation method and use of said recombinant protein. Also provided are a conjugate of a recombinant protein modified by PEG, and a preparation method and use of said recombinant protein. The recombinant protein has anti-tumor activity, and acts in synergy with a proteasome inhibitor; the PEG modification of the recombinant protein, while retaining anti-tumor drug effects, also has improved stability and a longer half-life.