43 results about "Trypsinogen" patented technology

The invention discloses a method for extracting trypsin from animal pancreas. According to the technical scheme of the invention, the zymogen in pancreas cells is extracted by using a sulfuric acid solution; subsequently according to the principle of isoelectric precipitation, the pH is adjusted, and the trypsinogen is deposited and precipitated in a classified salting-in and salting out mode by using ammonium sulfate. By utilizing the method, 6g trypsin can be extracted from 1kg of pancreas.

The present invention relates to novel Prolipase-Bovine trypsinogen (PLBTR) fusion proteins, the genes encoding them, and the production and uses thereof. More specifically, the present invention relates to methods of producing in optimal quantities PLBTR fusion proteins which comprise a heterologous polypeptide which is normally susceptible to autocatalytic activity. More particularly, the present invention relates to fusion proteins which comprise an heterologous polypeptide, such as a serine protease, fused to a lipase signal sequence, which can be expressed by recombinant host cells in desired amounts. The present invention further relates to polynucleotides encoding such fusion proteins, to expression vectors for expression of such fusion proteins, to host cells transformed with such polynucleotides / vectors, and to methods of generating such fusion proteins.

The invention provides a trypsinogen-2 chemiluminescent immunoassay kit which comprises: 1) a trypsinogen-2 calibrator; 2) a trypsinogen-2 antibody or streptavidin-coated microplate; 3) an enzyme-labelled trypsinogen-2 antibody when a trypsinogen-2 antibody-coated microplate is included in 2), or an enzyme-labelled trypsinogen-2 antibody and a biotin-labelled trypsinogen-2 antibody when a streptavidin-coated microplate is included in 2); 4) a chemiluminescent substrate liquid; and 5) a concentrated washing liquid. The invention also provides a method for preparing the kit. The kit of the invention has the advantages of simple operation, high sensitivity, rapid reaction, etc.

The present invention relates to a method for the manufacture and purification of recombinant trypsinogen and trypsin in E. coli and yeast, using high yield expression vectors with and without secretion leader sequences. The invention further relates to an improved method and apparatus for carrying out protein refolding specifically useful for processing trypsinogen that has accumulated intracellularly in the form of inclusion bodies.

The invention relates to production and application of high stability recombinant trypsin, and discloses a method for producing the recombinant trypsin, which comprises the following steps of: (1) performing recombinant expression on a fusion protein, wherein the fusion protein sequentially comprises 40-200 amino acid sequences and trypsinogen sequences from an N terminal to a C terminal, and is solubly expressed; and (2) cutting off the 40-200 amino acid sequences and a leader sequence at the N terminal of the fusion protein to obtain the recombinant trypsin. The method realizes the soluble expression of the trypsin, and the high activity and high stability trypsin can be obtained.

The invention discloses a trypsinogen-2 content detection kit and a preparation method thereof. The detection kit consists of reagent I and reagent II which are independent of each other, wherein the reagent I comprises the following components: a biological buffer, a preservative, inorganic salt and water; the reagent II comprises the following components: latex granules coated by trypsinogen-2 antibody, the biological buffer, the preservative and water. The trypsinogen-2 content detection kit adopts the latex granules with the particle size of 100-300nm to prepare the latex granules coated by the trypsinogen-2 antibody, thus effectively guaranteeing the sensitivity of the kit, the detection linearity range, the measurement value accuracy and the bottle opening stability; furthermore, the latex granules coated by the antibody are prepared by a chemical cross-linking method, so that the combination stability between the antibody and the latex granules can be guaranteed, and the stability of the detection kit is improved. The detection kit has the advantages of being good in stability, high in sensitivity, high in specificity, simple and convenient to operate, etc.

The invention relates to a preparation method of recombinant trypsin, and relates to the field of genetic engineering. The invention provides an expression vector and an engineering bacterium comprising trypsinogen. According to the method provided by the invention, plasmids with recombinant trypsinogen gene sequence are introduced into Escherichia coli cells, such that recombinant engineering bacteria are constructed; through fermentation, the expression of recombinant trypsinogen is induced; and through renaturation, enterokinase digestion transformation, and separation purification, trypsin with activity is obtained. The method provided by the invention is characterized in that the recombinant trypsin preparation process is simple, and the cost is low.

The invention relates to a method for expressing a bovine-derived trypsinogen gene in Pichia pastoris by gene recombination. The recombinant bovine trypsinogen is expressed in Pichia pastoris by the novel promoter and secreted into the culture medium; the zymogen is self-activated, or treated by enterokinase or trypsinase or the like to prepare the active bovine trypsinase; and the prepared active bovine trypsinase can be used for producing the recombinant human insulin and human insulin analogs. The method can avoid the defects in the constitutive promoter glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the induction-type promoter alcohol oxidase (AOX1) promoter.

The invention provides a production technology of bovine trypsin. By means of a gene recombination technology, bovine trypsinogen or inclusion body protein of bovine trypsinogen fusion protein are expressed out in escherichia coli, and then acidizing is carried out to denature and renature the inclusion body protein into folded bovine trypsinogen or bovine trypsinogen fusion protein. By the adoption of a prokaryotic expression system, the expression quantity is high, denaturation, renaturation and follow-up steps are simple and easy to implement, and the technological cost is low. The prepared bovine trypsin with activity can be used for production of recombinant human insulin and insulin analogues.

The invention provides a Vero cell line for stably secreting and expressing bovine trypsinogen (S.pro-try) and application thereof to influenza virus culture and influenza virus vaccine production. The Vero cell line is named as VTY55, has the preservation number of CGMCC No.12680, and belongs to the technical field of biology. The invention also provides a method for building the Vero-S.pro-try cell line. The Vero-S.pro-try monoclonal cell line VTY55 provided by the invention has the advantages that on one hand, the cell foundation is laid for the large-scale influenza vaccine production in future, and the crucial problems of vaccine productivity expansion and quality improvement can be solved; on the other hand, the cell line can also be used as a study tool in other aspects such as laboratory effective ingredient separation culture, influenza virus monitoring, anti-influenza-virus medicine screening, neutralizing antibody determination and the like; wide application prospects are realized.

The present invention relates to transgenic animal models of chronic pancreatitis. The present invention also provides methods for generating animal models by introducing mutated trypsinogen genes into the germline of animals and screening methods for identifying biologically active compound.

The invention discloses a method for extracting chymotrypsinogen and trypsinogen through fractional precipitation. The principle is that high-concentration saline ions can compete with protein for water molecules in a protein solution so as to damage a hydrated sheath on the surface of the protein and reduce the solubility, and accordingly, the protein can be precipitated from the solution. Ammonium sulfate is most widely used due to large solubility, small temperature coefficient and low probability of protein denaturation. Therefore, the invention adopts an ammonium sulfate fractional precipitation method to research and improve the technology for extracting the chymotrypsinogen and the trypsinogen, the yield of the prepared chymotrypsinogen is improved to 0.5%-0.6% especially by adopting affinity chromatography, and the trypsinogen as a by-product is obtained simultaneously.

The present invention relates to novel Prolipase-Bovine trypsinogen (PLBTR) fusion proteins, the genes encoding them, and the production and uses thereof. More specifically, the present invention relates to methods of producing in optimal quantities PLBTR fusion proteins which comprise a heterologous polypeptide which is normally susceptible to autocatalytic activity. More particularly, the present invention relates to fusion proteins which comprise an heterologous polypeptide, such as a serine protease, fused to a lipase signal sequence, which can be expressed by recombinant host cells in desired amounts. The present invention further relates to polynucleotides encoding such fusion proteins, to expression vectors for expression of such fusion proteins, to host cells transformed with such polynucleotides / vectors, and to methods of generating such fusion proteins.

The invention discloses the application of mogroside IIE in preparing trypsin inhibitors, and belongs to the technical field of medicines. After a long period of extensive trials, the inventor of thepresent application finds that the mogroside IIE can inhibit the activity of trypsin at the acinar cell background, and the inhibition has dependence on time and concentration. In addition, the mogroside IE can inhibit the activation of trypsinogen by an acute pancreatitis inducer-cerulein. At the same time, that the mogroside IE inhibits the activation of the trypsinogen is related to cathepsin B, so that the mogroside IE can be used to prepare trypsin inhibitors.

The present invention relates to novel Prolipase-Bovine trypsinogen (PLBTR) fusion proteins, the genes encoding them, and the production and uses thereof. More specifically, the present invention relates to methods of producing in optimal quantities PLBTR fusion proteins which comprise a heterologous polypeptide which is normally susceptible to autocatalytic activity. More particularly, the present invention relates to fusion proteins which comprise an heterologous polypeptide, such as a serine protease, fused to a lipase signal sequence, which can be expressed by recombinant host cells in desired amounts. The present invention further relates to polynucleotides encoding such fusion proteins, to expression vectors for expression of such fusion proteins, to host cells transformed with such polynucleotides / vectors, and to methods of generating such fusion proteins.

This invention discloses a recombinant vector, recombinant bacteria, and fermentation method for high-yield GHK production, belonging to the field of biomedical technology. The recombinant expression vector of this application comprises a bicistronic expression cassette controlled by a T7 / lac heterozygous promoter, sequentially containing the encoding (GHK). n The vector encodes the first cistron of the polypeptide and the second cistron, where n is an integer from 2 to 200. When this vector is transformed into *E. coli* BL21, the resulting recombinant bacteria can simultaneously express (GHK) after IPTG induction. n Polypeptides and trypsinogen. During fermentation, trypsinogen is activated and enables the processing of (GHK). n In situ enzymatic cleavage of peptides efficiently releases GHK. This invention integrates the traditional multi-step process into a "one-step fermentation," simplifying the production process and reducing purification difficulty. Examples show that the GHK yield can reach up to 3.9 g / L, providing an efficient and convenient new strategy for the large-scale biomanufacturing of GHK.

Pharmaceutical composition containing a mixture of proenzymes and enzymes, containing proenzymes trypsinogen and chymotrypsinogen and enzymes ct-amylase and lipase as active substances, and one or more pharmaceutically acceptable excipients, for simultaneous, separate and subsequent administration of the composition in parenteral or transmucosal way, the composition has anti-proliferative and anti-metastatic effects to cancer tumours and is intended for therapeutic, prophylactic and anti-metastatic use in mammals.

The invention discloses a porcine trypsinogen mutant and an expression of the porcine trypsinogen mutant in pichia pastoris. The porcine trypsinogen mutant is cut by enterokinase to obtain a novel porcine trypsinogen mutant. According to the invention, PGAP is taken as the promoter, methanol induction is avoided, harm of methanol and pollution to the environment are reduced, and the method is safe and convenient. In addition, compared with wild type porcine trypsin, the porcine trypsin mutant has higher stability and activity.

The utility model relates to the field of biological detection, in particular to a urinary trypsinogen-2 determination kit, which is characterized in that a fluorescence labeled urinary trypsinogen-2 antibody is contained in a fluorescence labeled pad and can be quickly combined with urinary trypsinogen-2 in a sample; due to the design of the fluorescent labeled pad and the nitrocellulose membrane, the detection reaction is rapidly carried out, and the detection time is shortened; the sample pad, the fluorescent label pad, the compact glass fiber pad, the nitrocellulose membrane and the absorbent paper are reasonably arranged on the test strip, so that uniform flow of liquid on the test strip is ensured, and the detection efficiency is improved; a user only needs to dropwise add a sample into the sample adding opening, operation is easy and fast, complex pretreatment steps are not needed, even non-professional personnel can easily operate, and the use threshold is lowered. The problems that a traditional urinary trypsinogen-2 detection method is complex in operation and long in consumed time, needs professional technicians to carry out and limits wide clinical application of the traditional urinary trypsinogen-2 detection method are solved.

The invention discloses the application of mogroside IIE in preparing trypsin inhibitors, and belongs to the technical field of medicines. After a long period of extensive trials, the inventor of thepresent application finds that the mogroside IIE can inhibit the activity of trypsin at the acinar cell background, and the inhibition has dependence on time and concentration. In addition, the mogroside IE can inhibit the activation of trypsinogen by an acute pancreatitis inducer-cerulein. At the same time, that the mogroside IE inhibits the activation of the trypsinogen is related to cathepsin B, so that the mogroside IE can be used to prepare trypsin inhibitors.

Pharmaceutical composition containing a mixture of proenzymes and enzymes, containing proenzymes trypsinogen and chymotrypsinogen and enzymes α-amylase and lipase as active substances, and one or more pharmaceutically acceptable excipients, for simultaneous, separate and subsequent administration of the composition in parenteral or transmucosal way, the composition has anti-proliferative and anti-metastatic effects to cancer tumours and is intended for therapeutic, prophylactic and anti-metastatic use in mammals.

The present invention relates to methods for producing a mammalian trypsin in a Fusarium venenatum host strain, comprising (a) cultivating the Fusarium venenatum host strain in a culture medium underconditions suitable for expression of the mammalian trypsin and secretion thereof into the medium, wherein the Fusarium venenatum host strain comprises a nucleic acid construct comprising a nucleic acid sequence encoding the mature coding sequence of a mammalian trypsin operably linked to nucleotides 58 to 129 of SEQ ID NO: 1 encoding the signal peptide and propeptide of Fusarium oxysporum trypsinogen; and (b) recovering the mammalian trypsin from the medium. The present invention also relates to constructs comprising a nucleic acid sequence encoding the mature coding sequence of a mammalian trypsin operably linked to nucleotides 58 to 129 of SEQ ID NO: 1 encoding the signal peptide and propeptide of Fusarium oxysporum trypsinogen, and to vectors and Fusarium venenatum host strains comprising such constructs.

This invention discloses a method for catalyzing acetal reactions using bovine trypsinogen, belonging to the field of organic chemical engineering. Experiments revealed that bovine trypsinogen possesses two catalytic activities: one is a natural-like activity catalyzing transesterification reactions, and the other is a hybrid activity catalyzing acetal reactions. The results demonstrate that bovine trypsinogen has better catalytic ability than bovine trypsin, amino acids, or other proteins, and can be used to catalyze acetal reactions, including tandem reactions of transesterification and acetalization, thus solving the problems of low reaction efficiency, harsh reaction conditions, difficult operation, and severe pollution associated with existing acetal reactions.

The present invention relates to compositions, methods, uses and kits for treating cancer. The present invention relates to methods for minimising the progression of cancer in a subject, the method comprising administering to the subject therapeutically effective amounts of chymotrypsinogen and trypsinogen, thereby minimising the progression of cancer in the subject. In particular, the methods provide a means for treating cancer by reducing the number of cancer stem cells in the subject.

The invention discloses an immunochromatographic test strip for rapidly detecting acute pancreatitis and a preparation method thereof. The test strip is formed by sticking a sample pad, a glass fiber membrane labeling pad, a nitrocellulose coating membrane and absorbent paper to a substrate in sequence through lap joint, wherein a highly specific monoclonal antibody of trypsinogen-2 labeled by a biotin-avidin-colored latex complex is coated on the glass fiber membrane labeling pad; the nitrocellulose coating membrane comprises a detection region and a control region; the detection region is coated by another specific monoclonal antibody of trypsinogen-2 with epitope different from that of the labeled monoclonal antibody on the glass fiber membrane labeling pad; and the control region is coated by an anti-mouse antibody. Compared with radioimmunoassay and enzyme linked immunosorbent assay (ELISA), the test strip has the advantages of safety, simpleness and convenience in operation, suitability for single human / sample detection, rapidness and the like, dispenses with special skills, is free of trauma, is easy in result reading and can realize timely in-situ and family self-detection of acute pancreatitis.

The invention relates to the field of biotechnology, and provides a recombinant trypsin purifying method. The method comprises the steps of firstly activating the recombinant trypsinogen generated by yeast fermentation, and then purifying with macroporous adsorption resin and cation exchange chromatography to obtain pure trypsin. A process of dilution or ultrafiltration of the fermentation liquid for directly purifying trypsinogen in the yeast fermentation liquid is avoided, and separate enzyme digestion pretreatment on the zymogen is not required, thus the purifying steps are saved, the operation is simple, and the yield is relatively high; moreover, the chromatography filler is cheap, and the method is suitable for large-scale production. Experiments indicate that the purity and activity of the recombinant trypsin are both relatively high, and the recombinant trypsin can be used as a raw material for industrial production.

The invention discloses a method for extracting chymotrypsinogen and trypsinogen through fractional precipitation. The principle is that high-concentration saline ions can compete with protein for water molecules in a protein solution so as to damage a hydrated sheath on the surface of the protein and reduce the solubility, and accordingly, the protein can be precipitated from the solution. Ammonium sulfate is most widely used due to large solubility, small temperature coefficient and low probability of protein denaturation. Therefore, the invention adopts an ammonium sulfate fractional precipitation method to research and improve the technology for extracting the chymotrypsinogen and the trypsinogen, the yield of the prepared chymotrypsinogen is improved to 0.5%-0.6% especially by adopting affinity chromatography, and the trypsinogen as a by-product is obtained simultaneously.

The invention provides a recombinant yeast expression system of trypsin free from enterokinase activation, a construction method and application of the recombinant yeast expression system, modified trypsinogen coding nucleic acid, a recombinant yeast host cell, active trypsin and a production system. The propeptide sequence is connected with a mature enzyme sequence; and a mature enzyme sequence, wherein a basic amino acid residue is contained between the propeptide sequence and the mature enzyme sequence. According to the technical scheme, the problem that in the prior art, recombinant trypsin production must depend on exogenous enterokinase for activation can be effectively solved.

The invention provides a production technology of bovine trypsin. By means of a gene recombination technology, bovine trypsinogen or inclusion body protein of bovine trypsinogen fusion protein are expressed out in escherichia coli, and then acidizing is carried out to denature and renature the inclusion body protein into folded bovine trypsinogen or bovine trypsinogen fusion protein. By the adoption of a prokaryotic expression system, the expression quantity is high, denaturation, renaturation and follow-up steps are simple and easy to implement, and the technological cost is low. The prepared bovine trypsin with activity can be used for production of recombinant human insulin and insulin analogues.