273 results about "Wild type protein" patented technology

Provided is a compound useful as an active ingredient in a pharmaceutical composition for treating cancer.　The present inventors have examined a compound useful as an active ingredient in a pharmaceutical composition for cancer treatment, and have completed the present invention by finding that: a heterocyclic compound represented by formula (I) and formula (I-I) has excellent an action of inducing degradation of a mutant KRAS protein and a wild-type KRAS protein, and in particular, an action of inducing degradation of a KRAS protein having a G12C mutation, G12D mutation, G12V mutation, or G13D mutation; and said heterocyclic compound has mutant KRAS inhibition and the inhibitory activity of KRAS amplified by a wild-type KRAS gene, and in particular, inhibitory activity of KRAS having a G12C mutation, G12D mutation, G12V mutation, or G13D mutation. The heterocyclic compound according to the present invention or a salt thereof can be used as a therapeutic agent for cancer.

The invention provides a porcine epidemic diarrhea virus S protein mutant, a nucleic acid molecule, a subunit vaccine and application. Compared with wild type porcine epidemic diarrhea virus S protein, the porcine epidemic diarrhea virus S protein mutant has the advantage that amino acid at the 1076th site, amino acid at the 1077th site, amino acid at the 1016th site and amino acid at the 1017th site are mutated into proline. The natural trimer which is stable and maintains the conformation of the S protein before fusion is constructed, the safe and efficient PEDV subunit vaccine is obtained, and the use effect and immunogenicity of the porcine epidemic diarrhea vaccine can be improved; moreover, compared with a wild type S protein, the porcine epidemic diarrhea virus S protein mutant disclosed by the invention has a higher expression quantity.

The present invention belongs to the technical field of bioengineering. Provided are an IscB mutein and the use thereof. Compared to the wild-type IscB protein, the IscB mutein comprises arginine at at least one of the following amino acid positions: position 84, position 96, position 102, position 111, position 159, position 368 and position 386; preferably, the IscB mutein further comprises arginine at position 401 and position 456. The relatively strong interaction between the IscB mutein and a double-stranded DNA target improves the editing activity for mammalian cell lines, which can greatly enhance the application potential of the IscB mutein as a new-generation underlying tool for gene editing, and promote the use of the IscB mutein in the aspects of precision medicine, animal model construction and crop breeding.

The invention discloses an E3 ubiquitin enzyme mutant as well as modification and a protein targeted degradation system and application thereof, and particularly relates to a series of targeted protein degradation enhanced E3 ubiquitin enzymes (Trim21-N) and a modification method thereof, the E3 ubiquitin enzymes (Trim21-N) are obtained by modifying a wild type Trim21 (wtTrim21) protein structural domain, and the E3 ubiquitin enzymes comprise Trim21-a, Trim21-b, Trim21-c, Trim21-d, Trim21-e and Trim21-f. The efficiency of degrading the target protein through the ubiquitin proteasome is remarkably improved, and the method has the characteristics of strong endogenous and exogenous universality, high specificity, ubiquitin proteasome dependence, low cytotoxicity, strong cell compatibility and the like on the degradation of the target protein. The invention provides E3 ubiquitin enzyme (Trim21-N) based on a targeted protein degradation enhancement version and an exogenous fluorescence report degradation system of the E3 ubiquitin enzyme (Trim21-N), preferably Trim21-e with the optimal degradation effect, and the E3 ubiquitin enzyme (Trim21-N) is named as delta Trim21. The invention further provides an endogenous protein degradation system based on the delta Trim21. The method has the characteristics of high specificity, rapidness and small module, so that the method can be used for researching various biological and disease mechanisms, including the fields of cell targeted therapy, cell mechanism exploration, genetic engineering and the like.

The present invention provides a phosphorylated Tau protein mutant and application thereof. The provided phosphorylated Tau protein mutant comprises at least one mutation substitution or deletion at E117, A145, G204, S305, A384, and L408 relative to the amino acid sequence SEQ ID NO.1 of the phosphorylated wild-type Tau protein, has improved thermal stability, and the thermal stability is significantly higher than that of the wild-type Tau protein during long-term storage. The phosphorylated Tau protein mutant can be used as a standard product and has greater utilization value.

The invention relates to a mutant of an RSV pre-fusion conformation F protein, the mutant comprises amino acid mutation relative to an amino acid sequence of a wild type RSV F protein, the amino acid mutation is that amino acids at positions 392 and 493 or near the 493 are substituted by C, and a disulfide bond is formed between mutated positions 392C and 493C or near the 493C. The invention also provides a related DNA (deoxyribonucleic acid) coding sequence, a recombinant plasmid, an mRNA (messenger ribonucleic acid) sequence and a corresponding RSV vaccine. By introducing disulfide bonds at proper positions and combining with cavity filling, electrostatic mutation, proline mutation and the like, the expression and stability of the F protein with conformations before and after fusion and trimerization conformations are remarkably improved, and the F protein is remarkably inhibited from being converted into conformations after fusion. The mutant is also applied to preparation of RSV vaccines and detection of respiratory syncytial virus infection.

The invention provides a system and a method for screening Cas protein mutants capable of identifying different PAM sequences, and belongs to the technical field of bioengineering. The system comprises a Cas protein mutant screening vector and an sgRNA expression vector, the Cas protein mutant screening vector comprises the following expression elements: a Cas protein coding gene, a plasmid replicon and a first resistance screening tag expression cassette; the sgRNA expression vector comprises the following expression elements: a plasmid replicon, a second resistance screening tag expression cassette, a third resistance screening tag expression cassette and an sgRNA expression cassette. According to the system and the method, aiming at the problem that the efficiency of identifying PAM sequences except NGG by wild type spCas9 protein is low, an active Cas9 mutant capable of identifying PAM sites except NGG is screened out, and the application field and the editing efficiency of a CRISPR gene editing system can be greatly expanded.

The invention provides an Akkermann Amuc1100 protein mutant as well as a preparation method and application thereof, wild Akkermann Amuc1100 protein is subjected to mutation modification through a molecular biological technical means, and the bioactivity of the Amuc1100 protein mutant subjected to molecular modification is obviously improved compared with that of the wild protein; compared with a wild type Amuc1100 protein, the wild type Amuc1100 protein has a better effect when being applied to the aspects of regulating intestinal immunity, relieving acute pancreatitis, promoting browning of white adipose cells, inhibiting obesity, relieving ulcerative colitis and the like, and a very good application prospect is provided for developing bioactive medicines with higher efficiency.

The invention relates to the field of plant molecular biology and forest genetic engineering, and particularly provides a PagRAP2.3 protein point mutant and application thereof in drought resistance of poplar. The mutant PagRAP2.3 MA is obtained by mutating methionine at the first site and cysteine at the second site of a wild type PagRAP2.3 protein into methionine and alanine. The invention further discloses a preparation method of the mutant PagRAP2.3. An expression vector containing the PagRAP2.3 MA gene is constructed, poplar 84K is transformed through an agrobacterium-mediated method, and a transgenic line with stable expression is obtained. Functional verification results show that overexpression of PagRAP2.3 MA can significantly increase the plant height of the poplar and promote plant growth, but the sensitivity to moisture is enhanced under drought stress. The mutant can be used for regulating and controlling the growth and development of forest trees and evaluating the drought resistance, and has a forestry breeding application prospect.

Provided is a respiratory syncytial virus (RSV) vaccine. The nucleic acid comprises a polynucleotide for encoding a mutant of an RSV F protein. Compared with a wild-type RSV F protein, the mutant at least comprises one or more of the following mutations: a disulfide bond mutation, a cavity-filling mutation and an electrostatic mutation.

The invention discloses a lytic polysaccharide monooxygenase mutant as well as a preparation method and application thereof. In order to solve the problems of low expression level, poor stability and the like of the existing lysing polysaccharide monooxygenase in a microbial expression system, the lysing polysaccharide monooxygenase RsLPMO9 is mutated to obtain a multi-site mutant of which the amino acid sequence is shown as SEQ ID NO.2, and the expression quantity of the multi-site mutant is obviously higher than that of a wild type protein RsLPMO9. The analysis of the thermal stability shows that the thermal stability of the multi-site mutant is obviously improved compared with that of the wild protein RsLPMO9, and the lytic polysaccharide monooxygenase mutant provided by the invention has an application prospect in the aspects of degrading biomass or plant polysaccharide and the like.

The invention discloses a PET plastic degrading enzyme LCC mutant as well as a protein crystal and application thereof, and relates to the technical field of biology, the LCC mutant is obtained by modification on the basis of a wild PET plastic degrading enzyme LCC, and the amino acid sequence of the LCC mutant is shown as SEQ ID NO.1. Compared with a wild type LCC, the LCC mutant has higher thermal stability, the Tm value of the LCC mutant is 91.30 DEG C, the protein Tm value of the wild type LCC is 86.76 DEG C and is increased by 4.54 DEG C compared with that of the wild type LCC, good activity is still kept, and the yield of the mutant LCC-M4 is 2 times higher than that of the wild type LCC. In addition, the invention further provides a protein crystal and a structure of the LCC mutant, and a research basis is provided for further modifying the PET plastic degrading enzyme LCC with high activity and high stability.

Provided is a mutant of a respiratory syncytial virus (RSV) pre-fusion conformational F protein. The mutant comprises an amino acid mutation relative to the amino acid sequence of a wild-type RSV F protein, the amino acid mutation being amino acids at a position 392 and a position 493 or a position near the position 493 being substituted by C, and a disulfide bond being formed between a position 392C and a position 493C or a position near the position 493C after the mutation. Also provided are a related DNA coding sequence, recombinant plasmid and mRNA sequence and a corresponding RSV vaccine. By introducing a disulfide bond at a suitable position, and combining same with cavity filling, electrostatic mutation, proline mutation and the like, the expression and stability of an F protein having pre- and post-fusion conformations and a trimerized conformation are significantly improved, and the conformation of the F protein is significantly inhibited from being converted into the post-fusion conformation. The mutant is also applied to preparation of an RSV vaccine and RSV infection detection.

The invention discloses a recombinant protein for detecting an anti-myelin sheath related glycoprotein MAG antibody and a detection reagent of the recombinant protein, and belongs to the technical field of neural immunodiagnostics, five amino acid sites in MAG protein are subjected to specific amino acid point mutation replacement to obtain an MAG protein mutation sequence, the MAG protein mutation sequence is fused with GlcATP enzyme and HNK-1ST enzyme full-length sequences to construct a lentiviral expression vector, and the recombinant protein and the detection reagent are used for detecting the anti-myelin sheath related glycoprotein MAG antibody. According to the present invention, three genes are constructed, and are stably transfected in a host cell so as to stably express the three genes in one cell at the same time, such that the constructed recombinant protein and the wild type MAG protein have similar anti-MAG IgM antibody binding domain, and on the premise of not using GlcATP enzyme and HNK-1ST enzyme small molecule agonists, the sensitivity and the specificity of the detection of the MAG autoimmune antibody by using the CBA method are improved; the method is suitable for preparing related detection reagents for in-vitro detection of the IgM type anti-MAG antibody.

The invention relates to the field of nucleic acid editing, in particular to the technical field of regularly clustered interval short palindromic repeat (CRISPR). In particular, the invention relates to a mutant of a Cas effector protein, a fusion protein comprising the mutant, and nucleic acid molecules encoding the same. The invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing). The invention also relates to methods for nucleic acid editing (e.g., gene or genome editing) using the proteins or fusion proteins comprising the invention. Compared with a wild type Cas protein, the Cas protein mutant disclosed by the invention has better activity, such as higher cleavage activity, stronger target site recognition capability and higher target sequence editing activity.

The present invention relates to RSV F protein mutants and their applications, belonging to the field of vaccines. Based on the amino acid sequence of wild-type RSV F protein, the present invention has carried out modifications in multiple aspects and in various ways, including disulfide bond mutations, proline mutations, cavity filling mutations, electrostatic mutations, p27 sequence modifications, and / or trimerization domain modifications, and designed a variety of RSV F protein mutants. Then, through gene synthesis, expression, and ELISA detection, the present invention tested the pre-fusion conformation (pre-F) ratio and stability under high and low temperature conditions of the RSV F protein mutants, and screened out several RSV F protein mutants with a relatively high pre-fusion conformation ratio and stable structure. Finally, candidate antigen representatives were selected for immunogenicity detection, which proved that they have good immunogenicity and can be used for the development of RSV vaccines, the preparation of RSV-specific antibodies, or RSV detection reagents.

High affinity SIRP-α reagent are provided, which (i) comprise at least one amino acid change relative to the wild-type protein; and (ii) have an increased affinity for CD47 relative to the wild-type protein. Compositions and methods are provided for modulating phagocytosis in a mammal by administering a therapeutic dose of a pharmaceutical composition comprising a high affinity SIRPα reagent, which blocks the physiological binding interaction between SIRPα and its ligand CD47.

The invention discloses Vip3 protein with improved activity and application thereof. Vip3Aa mutant protein is obtained by single-point mutation of a residue Y19 in a structural domain I or a residue V383 in a structural domain III; the Vip3Ca mutant protein is obtained by carrying out single-point mutation on a residue K383 in the structural domain III. The insecticidal activity of the Vip3 mutant protein disclosed by the invention on various lepidoptera pests is obviously improved compared with that of a wild protein.

The present invention relates to a MeCP2 gene-specific trans-splicing ribozyme, and a use thereof. The trans-splicing ribozyme according to the present invention replaces the RNA of a mutant MeCP2 gene, which causes Rett syndrome, with a wild type MeCP2 RNA, so as to reduce the expression of the mutant MeCP2 protein and increase the expression of the wild type MeCP2 protein, and enables regulated expression so that wild type MeCP2 expression can be prevented from exceeding physiological levels, and thus can be effectively used for preventing or treating Rett syndrome.

The invention aims to provide a method and equipment for predicting protein complex affinity change caused by mutation, and the method comprises the following steps: utilizing a first sub-network in a twin network to predict protein complex affinity change caused by mutation on the basis of wild type protein complex information and mutation information; determining wild type multi-modal coding information comprising wild type sequence coding information and discretely coded wild type geometric feature coding information; determining affinity information of the wild type through a decoder; determining mutant affinity information through the corresponding second sub-network; and determining affinity change information. By using discrete coding and multi-modal information, the model is helped to cope with the condition that the local structure is not well optimized, so that the model can obtain a more accurate affinity change prediction result by using complex information with low precision. And protein surface information is introduced, so that the model is helped to better learn the influence of a cavity formed by a protein interaction interface on the affinity change, and the affinity change prediction accuracy is further improved.

The invention relates to the technical field of biology and biology, in particular to a screening method of thermostable mutation sites of a G protein coupled receptor and application of the thermostable mutation sites of the G protein coupled receptor, and the screening method comprises the following steps: S1, replacing a third intracellular ring segment in a wild type G protein coupled receptor with a third intracellular ring sequence of a kappa opioid receptor; marking a SmBiT peptide fragment label on the wild type G protein coupled receptor; s2, mutating a wild type G protein coupled receptor according to a mutation site in the G protein coupled receptor mutant to be detected; s3, incubating the G protein coupled receptor modified in the step S2 at different temperatures to obtain modified G protein coupled receptor samples treated at different temperatures; s4, carrying out mixed incubation on the modified G protein coupled receptor samples treated at different temperatures and a nano antibody-LgBiT compound, then adding luciferase to act as a substrate, and detecting; the method is high in screening efficiency, low in cost and short in time.

The invention relates to an RSV F protein soluble fragment, an autotrimer structural domain and application thereof, and belongs to the technical field of recombinant protein. Based on an amino acid sequence of a wild RSV F protein, a soluble and rigid sequence is screened out, a soluble fragment is constructed, disulfide bond mutation pair modification is performed on the basis of the soluble fragment to obtain a self-derived trimer structural domain sequence, and the self-derived trimer structural domain sequence is used for replacing a transmembrane region and an intracellular region of the RSV F protein. Therefore, trimerization can be realized, stable pre-fusion conformation can be maintained, and soluble expression in cells can be realized at the same time. The invention also provides an RSV F protein mutant containing an autotrimer structural domain sequence, the generation and the stability of the pre-F conformation trimer are proved, a representative is selected from the pre-F conformation trimer for animal experiments, and it is proved that the pre-F conformation trimer has good immunogenicity and can be used for developing RSV vaccines and preparing RSV specific antibodies or RSV detection reagents.

The invention relates to the field of nucleic acid editing, in particular to the technical field of regularly clustered interval short palindromic repeat (CRISPR). In particular, the invention relates to a mutant of a Cas effector protein, a fusion protein comprising the mutant, and nucleic acid molecules encoding the same. The invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing). The invention also relates to methods for nucleic acid editing (e.g., gene or genome editing) using the proteins or fusion proteins comprising the invention. Compared with a wild type Cas protein, the Cas protein mutant disclosed by the invention has better activity, such as higher cleavage activity, stronger target site recognition capability and higher target sequence editing activity.

The invention discloses an LbCas12a protein mutant as well as a preparation method and application thereof, the LbCas12a protein mutant is obtained by performing K390A or K945A mutation on a wild type LbCas12a protein, and the amino acid sequence of the wild type LbCas12a protein is as shown in SEQ ID NO. 1. According to the present invention, the LbCas12a protein is subjected to directional modification, the alanine mutation occurs at the K390 / K945 site, the LbCas12a-K390A protein mutant and the LbCas12a-K945A protein mutant are prepared, the protein mutants obtained based on the method provide a series of significant advantages in function, and the solid foundation is laid for the application of the protein mutants in multiple fields. Through Michaelis-Menten kinetic analysis, it is observed that when the LbCas12a-K390A / K945A protein mutant prepared through the method and the wild type LbCas12a protein target the same dsDNA target, the catalytic efficiency of the LbCas12a-K390A / K945A protein mutant is 42.1 times that of the wild type LbCas12a, and the catalytic efficiency of the LbCas12a-K390A / K945A protein mutant is 707.9 times that of the wild type LbCas12a, and the catalytic efficiency of the LbCas12a-K390A / K945A protein mutant is 707.9 times that of the wild type LbCas12a. The results show that K390 and K945 are mutated into alanine, so that the affinity of the LbCas12a protein to a substrate can be increased to a certain extent, and the LbCas12a protein has higher trans-cleavage activity.

The invention relates to the technical field of biological pharmacy, in particular to a high-yield mutant streptococcus protein G and application thereof. Compared with a wild type protein G, the mutant recombinant protein G provided by the invention has the advantages that the yield in an expression system in escherichia coli is multiplied, the cost for preparing a G Column filler is greatly reduced, and the problem of high cost of a monoclonal antibody is solved. The mutant recombinant protein G retains the binding function of wild protein G to IgG protein, can greatly reduce the manufacturing cost of a G Column affinity chromatography column, is more stable and not easy to degrade, has more recycling times while reducing the production cost of the protein G affinity chromatography column, and has wide application prospects in an antibody purification process.

The invention discloses preparation and application of pseudorabies virus circular RNA (Ribonucleic Acid). The circular RNA comprises a coding element for coding any one of gD protein, gB protein, gC protein and gK protein of the pseudorabies virus; preferably, the coding element encodes a pseudorabies virus gD protein. Further, the encoding element encodes gD wild-type protein, gD truncated protein, gD mutant protein and gD truncated mutant protein; preferred coding elements encode gD truncated plus muteins. Further, the encoding element encodes an optimized nucleotide sequence of pseudorabies virus gD truncated and mutant protein. Further, the circular RNA comprises an optimized translation initiation element. Researches show that the circular RNA provided by the invention can induce protein expression in an animal body, generate a high-level neutralizing antibody and successfully activate T cell immunity, especially CD8 + T cell immune response, and a basis is provided for preparing a circular mRNA vaccine for preventing and / or treating pseudorabies.

The invention belongs to the technical field of characterization of target analytes, and provides application of PANX2 protein and a mutant thereof as biopore protein. The bioporous protein is used for detecting one or more of metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic agents, adenosine triphosphate and monosaccharides. After one or more amino acids of the wild type PANX2 protein are mutated into common amino acids except original amino acids, compared with the wild type PANX2 protein, the PANX2 protein mutant can be normally expressed and is stable in property, a current signal is obviously improved during detection, the up-down amplitude of the current signal is longer, and burrs are fewer.

The present invention discloses mutants of wild-type RSV F protein. The binding experiment of pre-fusion conformation trimer-specific monoclonal antibody AM14 shows that the mutant provided in the embodiment of the present disclosure can have strong binding activity with AM14 monoclonal antibody, while the wild-type RSV F protein before artificial mutation has no binding activity with AM14 monoclonal antibody. The results of accelerated stability test show that the mutant provided in the embodiment of the present disclosure is placed in a 37°C environment for 4 weeks, and the binding activity of AM14 monoclonal antibody has no obvious change. The results of animal immunization experiments show that compared with the wild-type RSV F protein before artificial mutation, the mutant provided in the embodiment of the present disclosure can induce the production of higher titers of neutralizing antibodies.

The present invention belongs to the field of antibody technology, and specifically relates to anti-herpes zoster virus glycoprotein E monoclonal antibodies and binding epitope analysis methods. VZV gE protein is used as an immunogen to immunize mice, determine the protein titer, perform fusion experiments, and subclone experiments to obtain monoclonal antibodies. The analysis method is as follows: determine that the antibody recognition epitopes are all conformational epitopes; analyze the complete structure of the VZV gE protein, predict the disulfide bond position and antigenic epitope; mutate and identify the cysteine ​​residues in the VZV gE sequence predicted to form disulfide bonds or affect the formation of disulfide bonds one by one; transfect and express the recombinant mutant plasmid, detect the expression of mutant and wild-type proteins; detect the activity of monoclonal antibodies; detect the activity of monoclonal antibodies corresponding to the mutant proteins with the same expression amount; and detect the affinity of monoclonal antibodies. Application: pharmaceutical composition, reagent, test strip or kit, biological product. The present invention fills the gap in the current analysis of conformational epitopes of VZV gE protein antigens.

The invention relates to an RSV F protein mutant and application thereof, and belongs to the field of vaccines. On the basis of the amino acid sequence of wild RSV F protein, multiple aspects and multiple modes of modification are carried out, including disulfide bond mutation, proline mutation, cavity filling mutation, electrostatic mutation, p27 sequence modification and / or trimerization structural domain modification, and multiple RSV F protein mutants are designed. And then, through gene synthesis, expression and ELISA detection, the pre-conformation (pre-F) proportion of the RSV F protein mutants and the stability under high-temperature and low-temperature conditions are tested, and a plurality of RSV F protein mutants with relatively high pre-conformation proportion and stable structures are screened out. Finally, candidate antigen representatives are selected for immunogenicity detection, and it is proved that the candidate antigen representatives have good immunogenicity and can be used for developing RSV vaccines and preparing RSV specific antibodies or RSV detection reagents.