37 results about "Ribosome display" patented technology

The polynucleotide construct of (1) or (2) below is used to perform ribosome display, CIS display and / or mRNA display in order to screen a Fab against an antigen of interest: (1) a polynucleotide construct which monocistronically comprises a ribosome-binding sequence, Fab first chain-coding sequence, linker peptide-coding sequence, Fab second chain-coding sequence and scaffold-coding sequence in this order, and further comprises at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself, and (2) a polynucleotide construct which comprises a Fab first chain-expressing cistron and a Fab second chain-expressing cistron each containing a ribosome-binding sequence, a Fab first chain-coding sequence or Fab second chain-coding sequence, and a scaffold-coding sequence in this order, the first Fab-expressing cistron further comprising at its 3′-end a ribosome stall sequence, said Fab second chain-expressing cistron further comprising at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself.

The present invention relates to a method of identifying a protein that binds to a target molecule and has intracellular functionality. This method includes providing a construct comprising a deoxyribonucleic acid molecule encoding the protein which binds to the target molecule, with the deoxyribonucleic acid molecule being coupled to a stall sequence. A host cell is transformed with the construct and then cultured under conditions effective to form, within the host cell, a complex of the protein whose translation has been stalled, the mRNA encoding the protein, and ribosomes. The protein in the complex is in a properly folded, active form and the complex is recovered from the cell. This method can be carried out with a cell-free extract preparation containing ribosomes instead of a host cell. The present invention also relates to a construct which includes a deoxyribonucleic acid molecule encoding a protein that binds to a target molecule and an SecM stalling sequence coupled to the deoxyribonucleic acid molecule. The deoxyribonucleic acid molecule and the SecM stalling sequence are coupled with sufficient distance between them to permit expression of their encoded protein, within the cell, in a properly folded, active form.

The invention provides a method for recovery of cDNA from mRNA, comprising reverse transcription (RT) of mRNA using a RT primer which includes a sequence based on the 5′ consensus region of the mRNA which is identical or similar to 5′ consensus region of the mRNA and which includes a sequence capable of specifically hybridising to the 3′ region of the mRNA, followed by polymerase chain reaction (PCR) using a single primer to generate ss cDNA, ds cDNA and amplify the cDNA. Primers for use in methods of the invention and kits for performing methods of the invention are also provided. The methods of the invention can partially or fully automated.

Compositions, methods, and kits are provided for efficiently generating and screening humanized antibody with high affinity against a specific antigen. The library of humanized antibody is generated by mutagenizing a chimeric antibody template that combines human antibody framework and antigen binding sites of a non-human antibody. Alternatively, the library of humanized antibody is generated by grafting essential antigen-recognition segment(s) such as CDRs of the non-human antibody into the corresponding position(s) of each member of a human antibody library. This library of humanized antibody is then screened for high affinity binding toward a specific antigen in vivo in organism such as yeast or in vitro using techniques such as ribosome display or mRNA display. The overall process can be efficiently performed in a high throughput and automated manner, thus mimicking the natural process of antibody affinity maturation.

The invention relates to a method for generating and selecting Fab fragments using ribosomal display and cell-free protein synthesis. The invention also provides a ribosomal display reaction system for generating Fab fragment complexes. The compositions of a Fab fragment complex and a library thereof are also provided.

The polynucleotide construct of (1) or (2) below is used to perform ribosome display, CIS display and / or mRNA display in order to screen a Fab against an antigen of interest: (1) a polynucleotide construct which monocistronically comprises a ribosome-binding sequence, Fab first chain-coding sequence, linker peptide-coding sequence, Fab second chain-coding sequence and scaffold-coding sequence in this order, and further comprises at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself; and (2) a polynucleotide construct which comprises a Fab first chain-expressing cistron and a Fab second chain-expressing cistron each containing a ribosome-binding sequence, a Fab first chain-coding sequence or Fab second chain-coding sequence, and a scaffold-coding sequence in this order, the first Fab-expressing cistron further comprising at its 3′-end a ribosome stall sequence, said Fab second chain-expressing cistron further comprising at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself.

The invention relates to an in vitro molecular directed evolution method for reshaping antibody, which integrates ribosome displaying technology with DNA reorganization technology by designing reshaped antibody molecules, thus directly obtaining the extracorporal molecules of the single-chain antibody reshaped orientation.

A combined ribosome-display and phage-display method and kit for carrying out the method are provided. The method includes screening a ribosome-display library of binding agents to identify binding agents that interact with one or more target molecules of interest, converting the RNA encoding the binding agents to a phage-display format by amplification and primer extension, and the screening the phage-display library to enrich for binding agents that interact with one or more target molecules of interest.

A method is disclosed wherein an antigenic B-cell epitope is discovered by in vitro transcription and translation of pre-determined sequence on thermostable protein scaffolds detected by antibodies to a native protein. Immune reactivities of IgE B-cell epitopes from pre-determined IgE sequences from the constant region, engaged in binding to high affinity IgE Fc receptors, placed in a loop of green fluorescent protein (GFP) were shown immuno-reactive with anti-IgE. Moreover, the antigenic B-cell epitope can be further optimized by molecular evolution, selected by conformer antibody and receptor on solid phase via ribosome display. Alternatively, a random aptameric antigenic sequence inserted into a loop of the protein scaffold, mimicking a pre-determined sequence of a native protein, can be selected by solid phase conformer antibody and receptor via ribosome display. High affinity binding of B-cell epitopes selected from the random aptameric library with antibodies to native pre-determined B-cell epitope of a native protein can be optimized by molecular evolution with error-prone RT-PCR by ribosome display.

The present invention relates to a method of identifying a protein that binds to a target molecule and has intracellular functionality. This method includes providing a construct comprising a deoxyribonucleic acid molecule encoding the protein which binds to the target molecule, with the deoxyribonucleic acid molecule being coupled to a stall sequence. A host cell is transformed with the construct and then cultured under conditions effective to form, within the host cell, a complex of the protein whose translation has been stalled, the mRNA encoding the protein, and ribosomes. The protein in the complex is in a properly folded, active form and the complex is recovered from the cell.

The invention discloses an anti-human PD-1 protein antibody as well as an encoding gene and an application thereof. The antibody comprises a heavy chain variable region and a light chain variable region, wherein amino acid sequences of three hypervariable regions CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are shown as GFTFSSYAMS, AISGSGGTTYYADSVKG and AWAPFVNSIVVVTANDL respectively; amino acid sequences of three hypervariable regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are shown as RASQGISTWLA, AASSLQS and QQANSFPI respectively. A human single-chain antibody library is established, single-chain antibodies are screened with a ribosome display technology, and the obtained anti-human PD-1 protein antibody can be specially bonded with human PD-1 protein, has high affinity, can be used for detecting cells expressing PD-1, can be used as a fully-human anti-human PD-1 antibody for treating human diseases and effectively prevents and treats tumor diseases.

According to the present invention, a composition possessing cell-free protein synthesis activity with reduced contaminating lipopolysaccharide, and a method for producing a protein using the same are provided. When ribosome display is performed using the composition and method for protein production of the present invention, the background that is caused by non-specific binding is reduced, so that a nucleic acid that encodes the desired polypeptide can be selected with high accuracy and high efficiency.

The invention relates to a high-efficiency pH-selective anti-tumor polypeptide and its application, and belongs to the technical field of research and development and application of anti-tumor drugs. The present invention uses ribosome display technology to construct a series of histidine-rich polypeptides with membrane cleavage and anti-tumor activity, and uses the special acidity coefficient of histidine imidazole side chain to construct a series of polypeptides that are effective for normal physiological conditions and tumors. Selective anti-tumor polypeptides in characteristic acidic conditions. They have killing effects on both lung cancer cells and breast cancer cells, and can selectively kill tumor cells under acidic conditions; inhibit the growth of tumors in mouse xenograft models, especially highly selectively inhibit tumor cells under acidic conditions Tumor growth. Through microscope observation, lactate dehydrogenase experiment, fluorescence quenching and polypeptide circular dichroism, it is proved that these polypeptides increase their permeability by inserting and cleaving the cell membrane, resulting in decreased cell activity and death.

The invention provides a human genetic engineering antibody TRD 109 which is screened by utilizing a molecular biological technology-ribosome display technology, aims to TNF-alpha and realizes high-efficiency expression of the antibody to a prokaryotic system. A biological characteristic research demonstrates that the TRD 109 is a human genetic engineering antibody which has high affinity, better stability, simple structure and easy construction and can specifically neutralize the TNF-alpha, inhibit the generation of multiple kinds of cell factors, block the generation and development of AR in the source and is a human anti TNF-alpha antibody which can reduce the infiltration of eosinophils in a mouse nasal mucous membrane and relieve mouse allergic rhinitis symptom; and a gene and a geneproduct of the human anti TNF-alpha antibody can be used for preparing a medicaments for diagnosing and treating diseases caused by overexpression of the TNF-alpha, such as preparing a medicament for treating diseases including allergic rhinitis and rheumatoid arthritis.

The invention discloses a dodecapeptide ZA capable of inhibiting the hepatitis C virus from infecting human body cells and a preparation method and application thereof. The preparation method comprises the following steps: firstly preparing an E2-GST fusion protein; then preparing an E2-GST fusion protein antibody; thirdly establishing a random DNA library; fourthly preparing a ribosome library; and fifthly screening the polypeptide bonded with the hepatitis C virus E2 glycoprotein through the ribosome display library so as to obtain the dodecapeptide (ZA) specially bonded with the E2 protein. Due to the high affinity of the polypeptide ZA with the hepatitis C virus E2 glycoprotein, the polypeptide ZA can remarkably alleviate the intrusion of HCV into the human body cells and DC cells, inhibit the integration of the E2 protein with human body cells, and have a high inhibition capacity. The dodecapeptide ZA is used for preparing the medicines capable of treating and preventing hepatitis C virus infection. The polypeptide is applied to a kit for detecting the hepatitis C virus envelope antigen.