32 results about "Sortase A" patented technology

The present invention discloses a combined vaccine for inhibiting and / or preventing type A streptococcal infection. The present invention provides a composition for inhibiting streptococcus and / or preventing streptococcal infection, and the composition comprise an immunogen. The immunogen is one of the following 1)-4): 1) the immunogen comprises a sortase A and a C5a protease; 2) the immunogen comprises a sortase A solution and a C5a protease solution; 3) the immunogen comprise a fusion protein containing sortase A and a fusion protein containing C5a protease; and 4) the immunogen comprises a junctional complex of sortase A and polysaccharide and a junctional complex of C5a protease and polysaccharide. The specific amino acid sequence of the sortase A is the N-terminal amino acid residues from site No.22 to No.189 of a sequence 2; and the amino acid sequence of the C5a protease is a sequence 4 in the sequence table. The combined vaccine can be applied to prevention and treatment of mucosal system infections caused a variety of Gram-positive bacteria.

The present invention relates to a self-cleaving fusion protein including a target protein, a peptide consisting of amino acid sequence represented by LPXTG, a domain of Sortase A having cleaving function, and a tag, which are sequentially positioned from the amino terminal; a nucleic acid encoding the same; an expression vector including the nucleic acid of the present invention; and a cell transformed with the expression vector of the present invention. In addition, the present invention relates to a method for refining a target protein including culturing, dissolving, and purifying the transformed cell, and a method for preparing a therapeutic antibody-drug conjugate by using the purifying method.

The invention disclsoes a method for synthesizing cyclic peptides through an enzyme method and belongs to the technical field of cyclic peptide synthesis. The method comprises: mixing linear peptides and sortase A at the mol ratio of 25 to 1, and reacting at 37 DEG C to prepare the cyclic peptides. The yield of the cyclic peptides is 70 percent to 93 percent; the method has the advantages of high yield, simple steps and the like and is green and environmentally friendly. The prepared RGD (arginine-glycine-aspartic acid) linear peptides and the cyclic peptides have a property of inhibiting the mutual effect of integrin alphavbeta3 and extracellular matrixes; the prepared AKRHHGYKRKFH linear peptides and cyclic peptides have a property of inhibiting candida albicans, pseudomonas aeruginosa and staphylococcus aureus and have wide application value.

The invention discloses a method for constructing Escherichia coli capable of efficiently secreting and expressing transpeptidase Sortase A, and belongs to the field of gene engineering. According tothe present invention, the efficient secretion and expression of SortaseA in Escherichia coli is successfully achieved, and the problems that the transpeptidase Sortase A cannot be secreted and expressed and the expression level of the transpeptidase Sortase A in cells is low in the existing report are solved; at the shake flask level, the yield of the obtained sortedaseA can achieve 90.2 mg / L, and the extracellular enzyme activity can achieve 34.2 U / mL; and the foundation is established for the subsequent industrial production of SortaseA.

The invention discloses the application of salvianolic acid A in the preparation of medicines for treating Staphylococcus aureus infectious diseases. Specifically, the present invention discloses the application of salvianolic acid A in the treatment of Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus, alone or in combination with antibiotics. The purpose of the present invention is to introduce salvianolic acid into the treatment of Staphylococcus aureus infection, by using salvianolic acid A to inhibit the important virulence factor Sortase A, significantly reduce the pathogenicity of Staphylococcus aureus, and contribute to the body's immune system The removal of germs. The present invention also clarifies the molecular mechanism of salvianolic acid A inhibiting Staphylococcus aureus Sortase A, provides a reliable basis for salvianolic acid A to treat Staphylococcus aureus infection, and provides an important precursor compound for the development of new therapeutic drugs. It lays a certain foundation for the development of similar traditional Chinese medicines. Compared with the treatment with antibiotics, the treatment with salvianolic acid A has the characteristics of no drug resistance and high cure rate.

The invention discloses a preparation method for an antibody-monomethyl auristatin E (MMAE) conjugate. The method comprises the following steps: (1) a monoclonal antibody with an LPXTG sequence at each C terminus of an anti-MART-1 protein presentation peptide EAAGIGILTV / HLA-A2 composite, and monomethyl auristatin E or a monomethyl auristatin E derivative with an oligo-glycine joint are provided; (2) under the catalysis of a Sortase A enzyme, the LPXTG sequences and the oligo-glycine joint are subjected to a transpeptidation reaction, and the monoclonal antibody is coupled with and the monomethyl auristatin E or the monomethyl auristatin E derivative with the oligo-glycine joint; and (3) after the reaction is completed, separation purification is performed, and therefore the antibody-monomethyl auristatin E conjugate is obtained. Through site-direct coupling, the method provided by the invention makes per molecule of the antibody have four molecules of the MMAE, has good uniformity, andexhibits good tumor killing capacity in vivo experiments; and compared with current target spots of ADCs (antibody-drug conjugates), CLA12-vcMMAE ADCs expand the selection areas of the current targetspots of the ADCs, demonstrate the view that an intracellular protein can also be taken as the target spot of the ADCs through presentation of MHC (major histocompatibility complex) I molecules.

The invention discloses a fusion protein vaccine capable of inhibiting Streptococcus and / or preventing Streptococcus infection. With fusion protein obtained through linkage of sortase A and a cholera toxin B subunit through connecting peptide, the Th17 cell activation level is remarkably increased, the effects of preventing pathogenic bacteria from settling and quickly removing the pathogenic bacteria can be realized, and the fusion protein has protecting effects on Group A Streptococcus of different serotypes and has the superiority of high efficiency, broad spectrum and low cost. With the fusion protein, the use amount of immunogen is reduced, the production technology is simplified, and the production cost is reduced. Meanwhile, the vaccine adopts the way of mucosal immunity, has the characteristics of being free of tissue damage, free of local side effects and convenient to use, and is easy to popularize and use.

Herein is reported a method for the enzymatic production of a polypeptide comprising the step of incubating i) a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue) or LPXTA (SEQ ID NO: 41, wherein X can be any amino acid residue), ii) a second polypeptide that has i) a glycinyl, an alaninyl, or a cysteinyl compound at its N-terminus, or ii) an oligoglycine, or oligoalanine, or a cysteine amino acid residue followed by one to three glycine or alanine amino acid residues at its N-terminus, or iii) a lysine amino acid residue within its 5 N-terminal amino acid residues, and iii) a third polypeptide with sortase A activity, in a deep eutectic solvent and thereby producing a polypeptide.

The invention relates to application of tectorigenin in preparation of a Listeria monocytogenes sortase A inhibitor. SrtA enzyme activity experiments, biofilm experiments and cell invasion experiments prove that tectorigenin can effectively inhibit SrtA catalytic activity and has a certain inhibition effect on the pathogenicity of Listeria monocytogenes; and a mouse listeria monocytogenes systemic infection model treatment experiment proves that tectorigenin has a good treatment effect on infection caused by listeria monocytogenes.

Herein is reported a method for producing an enzymatic conjugation product of two polypeptides comprising incubating of a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 20, wherein X can be any amino acid residue), a second polypeptide has an oligo-alanine Am (m=2 (SEQ ID NO: 26), or 3 (SEQ ID NO: 27), or 4 (SEQ ID NO: 28), or 5 (SEQ ID NO: 29)) amino acid sequence at its N-terminus, a third polypeptide with sortase activity which is derived from Staphylococcus aureus Sortase A, and recovering the conjugate from the reaction mixture and thereby producing the enzymatic conjugation product of two polypeptides.

The invention discloses an enzyme and a method for marking the surface of cell membrane and studying cell-cell interaction, and relates to a mutant of Staphylococcus aureus sortase A evolved through genetic engineering, which can realize labeling mediated by proximity effect Based on this reaction, the labeling of non-genetically modified cell membrane surfaces can be achieved, and cell-cell interactions can be accurately captured and labeled in vivo and in vitro.

Herein is reported a polypeptide comprising the amino acid sequence of SEQ ID NO: 38 as sole Listeria monocytogenes derived polypeptide and its use in conjugating polypeptides.

The present invention provides a benzofuran compound, a preparation method and applications thereof, wherein the structure is represented by a general formula (I-1) or (I-2), R1, R3 and R4 are any one selected from hydrogen, C1-C5 straight or branched chain alkyl, hydroxyl, an aldehyde group, an acetyl group, a carboxyl group, a cyano group, an amino group, a nitro group, fluorine, chlorine, bromine, an amide group, an ester group, an alkoxy group, an aromatic group, and a heteroaromatic group, R2 is any one selected from hydrogen, C1-C5 straight or branched chain alkyl group, a hydroxyl group, an aromatic group, and a heteroaromatic group, and m and n are integers of 0-5. According to the present invention, the Staphylococcus aureus protease Sortase A substrate polypeptide fragment -LPXTG- is adopted as the structural simulation object, the benzofuran structure is adopted as the simulation substrate proline, and the linked amide hydrophobic fragment segment is used to stimulate the substrate leucine residue to design the novel protease Sortase A inhibitor.

The invention discloses a preparation method of an antibody-conjugated medicine, the antibody-conjugated medicine and applications. The preparation method comprises the following steps: firstly, underthe catalysis of Sortase A enzyme, leading LPXTG sequence and oligoglycine linker to generate transpeptidation reaction, so that the anti-CD20 monoclonal antibody is connected with a first linker arm; and then leading an azide group linker and alkynyl linker to generate cycloaddition reaction, so that the first linker arm is connected with a second linker arm. The antibody-conjugated medicine isprepared by utilizing an enzyme-chemical method, so that the usage amount of expensive medicines can be reduced, and the cost can be saved. By adopting difunctional small molecules, the steric hindrance of Sortase A enzyme in transpeptidation reaction can be reduced, and the flexibility is good. The conjugation sites and quantity on the antibody medicines can be efficiently controlled, the uniformity is enhanced, and the in-vivo and in-vitro anti-tumor activity can be improved on the basis of keeping the original antibody affinity.

Herein is reported a method for the enzymatic production of a thioester comprising incubating i) a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue), ii) a second polypeptide that has at its N-terminus a cysteine amino acid residue or is a cysteinyl compound, and iii) a third polypeptide with sortase A activity.

The present invention discloses a combined vaccine for inhibiting and / or preventing type A streptococcal infection. The present invention provides a composition for inhibiting streptococcus and / or preventing streptococcal infection, and the composition comprise an immunogen. The immunogen is one of the following 1)-4): 1) the immunogen comprises a sortase A and a C5a protease; 2) the immunogen comprises a sortase A solution and a C5a protease solution; 3) the immunogen comprise a fusion protein containing sortase A and a fusion protein containing C5a protease; and 4) the immunogen comprises a junctional complex of sortase A and polysaccharide and a junctional complex of C5a protease and polysaccharide. The specific amino acid sequence of the sortase A is the N-terminal amino acid residues from site No.22 to No.189 of a sequence 2; and the amino acid sequence of the C5a protease is a sequence 4 in the sequence table. The combined vaccine can be applied to prevention and treatment of mucosal system infections caused a variety of Gram-positive bacteria.

The invention discloses a preparation method and an application method of a double-network hydrogel for three-dimensional cell culture, which belongs to the field of biomedical materials. Firstly, a methacrylated hyaluronic acid grafted with a Sortase A enzyme-specific substrate short peptide is synthesized. (HAMA) conjugate (HAMA‑P), the substrate is enzymatically cross-linked with a certain concentration of Sortase A to obtain an injectable hyaluronic acid single network hydrogel. The method has the advantages of readily available raw materials, mild reaction conditions, short reaction time, and the like. Then, enzymatically and optically double-crosslinked hyaluronic acid-gelatin double network hydrogels were prepared. (GelMA) hydrogel as the second reinforcement network. The double-network hydrogel prepared by the invention provides a suitable scaffold and three-dimensional microenvironment for cell adhesion and growth, and has good application prospects in injectable tissue engineering, 3D printing, and three-dimensional cell culture.

The invention discloses a highly efficient mutant of staphylococcus aureus sortase A. Starting from the Staphylococcus aureus sortase A gene and a known mutant gene, a mutant library was constructed by error-prone PCR and site saturation mutation. Using a screening platform based on fluorescence resonance energy transfer, after multiple rounds of screening and mutation A series of sortase A mutants with improved catalytic activity comprising D124G, Y187L, E189R and / or F200L mutations were obtained by integration. These sortase A mutants can not only improve the efficiency of catalyzing the reaction between LPXTG and the atypical substrate α-Gly, but also improve the efficiency of catalyzing the reaction between LPXTG and the typical substrate α-Gly n The efficiency of the reaction between.