222 results about "Trypsin Digestion" patented technology

The invention relates to a human amnion mesenchymal stem cell serum-free culture medium and a culture method thereof. The culture medium is formed by adding human serum albumin, human transferrin, human insulin and sodium selenite into a DMEM / F12 basic culture medium. The culture method for the culture medium comprises the following steps of: digesting human amnion by using trypsin, then digesting the human amnion by using collagenase IV and deoxyribonuclease I, and filtering the mixture to obtain single cell suspension; and adding the human serum albumin, the transferrin, the insulin and the sodium selenite into the DMEM / F12 basic culture medium in a ratio of VDMEM to VF12 of 1:1, and putting human amnion mesenchymal stem cells in a 37 DEG C CO2 incubator with saturated humidity and volume fraction of 5 percent under the serum-free condition, wherein culture in vitro and amplification are realized by solution change and transfer of culture, potentiality of multi-direction differentiation is maintained, and the amplified cells can be induced in vitro to form cartilage cells, osteoblasts and adipocytes. The culture medium and the culture method have the characteristics of no other animal sources, wide source and no limitation of ethics.

The present invention is directed to a simple method for absolute quantification of plasma vitellogenin from two or more different fish species such as Rainbow trout and Atlantic salmon, or Atlantic cod and haddock. In the case of Rainbow trout and Atlantic salmon, plasma samples obtained from control and β-estradiol induced fish were digested with trypsin. A characteristic ‘signature peptide’ was selected and analyzed by high performance liquid chromatography coupled to an electrospray quadrupole-time-of-flight tandem mass spectrometer, using a deuterated homologue peptide as an internal standard. The hybrid tandem mass spectrometer was operated in a ‘pseudo’ selected reaction monitoring mode by which three diagnostic product ions were monitored for identification and quantification purposes. The reproducibility (coefficient of variation ˜5%) and sensitivity (limit of quantification of 0.009 mg / mL) achieved by this simple assay allow it to be considered as an alternative to immunological assays. In the case of Atlantic cod and haddock, the amino acid sequence of the vitellogenin protein has not yet been determined, but, the Atlantic cod vitellogenin has been characterized using a ‘bottom-up’ mass spectrometric approach. Vitellogenin synthesis was induced ‘in vivo’ with β-Estradiol, and subjected to trypsin digestion for characterization by matrix-assisted laser desorption / ionization-Quadrupole-Time-of-flight tandem mass spectrometry. A peptide mass fingerprint was obtained and ‘de novo’ sequencing of the most abundant tryptic peptides was performed by low energy collision induced dissociation-tandem mass spectrometry. Thus, the sequences of various tryptic peptides have been elucidated. It has also been determined that Atlantic cod vitellogenin shares a series of common peptides with the two different known vitellogenin sequences of Haddock, a closely related species. There are also disclosed novel isolated signature peptides, namely Thr-Tyr-Phe-Ala-Gly-Ala-Ala-Ala-Asp-Val-Leu-Glu-Val-Gly-Val-Arg, Asp Leu Gly Leu Ala Tyr Thr Glu Lys, Phe Phe Gly Gln Glu Ile Ala Asn Ile Asp Lys, Glu Ile Val Leu Leu Gly Tyr Gly Thr Met Ile Ser Lys and Tyr Glu Ser Phe Ala Val Ala Arg.

The invention discloses a method for constructing a kidney cell line of scophthalmus maximus, which comprises the following steps of: preparing a cell culture fluid first, taking a kidney tissue of the scophthalmus maximus as a material, using 0.5 percent trypsinase to digest the kidney tissue for 30 minutes at room temperature after the rinsing and shearing, adopting an 200-mesh nylon gauze to filter and collect cells, split charging the cells with the quantity of 5-10*10 counts / bottle into culture bottles with the growth area of 25 cm, and placing the culture bottles at a temperature of 24 DEG C for culturing; replacing the cell culture fluid with half quantity each four days, and using 0.25 percent trypsinase digest the cells for passage when primary cells grow to be monolayered; and performing passage once each 8 to 10 days, wherein the content of serum in the cell culture fluid is reduced to 10 percent from 20 percent when the 8th generation is reached, and the cell line is successfully established at the moment. The kidney cell line of the scophthalmus maximus obtained by utilizing the method is fibroid and can be continuously transferred for more than 40 generations; the cell line can be directly applied to pathogeny characteristics research, vaccine development and functional gene research; and the construction method is suitable to construct kidney cell lines of other fishes.

The invention relates to a production and freeze-drying method and application for supernate of a culture solution of a mesenchymal stem cell. a culture solution supernate freeze drying agent can be used with skin care products simultaneously after being dissolved again, the function of improving a skin is achieved, the production method adopts umbilical cord tissues of a healthy person as raw materials, the umbilical cord tissues are digested through collagenase and cultured outside the body, amplification and trypsin digestive passage are conducted, an NuncTM cell factory system with ten layers or four layers is utilized to replace a traditional cell culture bottle finally, the cost is greatly reduced, needed space is reduced, a large number of mesenchymal stem cells are obtained in shorter culture time, low concentration fetal bovine serum (3%FBS) is reserved in cell culture media to make the condition of the cells healthier compared with that of the cells without serum, the quality of culture supernatant fluid is guaranteed, the culture supernatant fluid can be stored under room temperature after vacuum freeze drying is conducted on the culture supernatant fluid directly, and activity of various growth factors of the culture supernatant fluid is maintained.

The invention relates to a construction method of a human amniotic mesenchymal stem cell bank. The construction method comprises the following steps: taking a human amnion for detection, flushing and washing the human amnion with a phosphate buffered solution, then smashing the human amnion, diluting with the phosphate buffered solution, digesting with trypsin, digesting with collagenase IV and deoxyribonuclease I, and filtering to obtain a single-cell suspension; by adding human serum albumin, transferring, insulin and sodium selenite into a DMEM / F12 basal culture medium with the ratio of VDMEM to VF12 being 1 to 1, placing human amnion mesenchymal stem cells in an incubator under the serum-free condition, and then performing liquid exchange and culture transfer; subjecting the mesenchymal stem cells obtained through in vitro culture and proliferation to liquid nitrogen refrigeration, and preserving the cells according to the gender of newborn infants, the ABO / Rh type and the HLA type; establishing cell information files, so that the human amniotic mesenchymal stem cell bank is constructed. The method has the characteristics of no other animal derivation, wide source range and no ethic limitation. With adoption of the method, the human amniotic mesenchymal stem cells can be provided for cell therapy and other application.

The invention discloses a method for isolation and serum gradient switching culture of human umbilical cord mesenchymal stem cells. The method comprises the following steps: digesting human umbilical cord with collagenase I until Wharton's jelly is fully digested, collecting digested single cells, inoculating the single cells into alpha-MEM medium, carrying out suspension culture until 80-90% of cells are fused, and digesting with trypsin; inoculating the suspension of trypsin-digested cells into alpha-MEM medium, carrying out suspension culture until 80-90% of cells are fused, digesting with trypsin, subcuturing and inoculating the subcultured cells into alpha-MEM medium, and carrying out subculture repeatedly. The concentration of fetal bovine serum in alpha-MEM medium in the steps of subculture decreases with the increase of the passage number until fetal bovine serum disappears. According to the method, the material human umbilical cords are easily accessible; the preparation of human umbilical cord mesenchymal stem cells is rapid and safe, and is not subject to ethical restrictions; and the subsequent culture is independent on the characteristics of fetal calf serum, resulting in greatly reduced culture cost and risk in clinical use. The method of the invention has a good application prospect.

The invention relates to a construction method of a fin cell line of anabarilius grahami and belongs to the technical field of freshwater aquatic organism cell culture. The method comprises that the anabarilius grahami tail fin tissue cell serves as a material, a tissue block method is adopted, and the primary culture is strated, culturing is achieved in a dulbecco modified eagle medium (DMEM) / F12 containing fetal calf serum and human basic fibroblast growth factors and having a PH value ranging between 7.0 and 7.4; and subculture is performed by using a trypsin digestion method. The method has the advantages that 1, the minimally invasive processing is adopted, on the premise that the fish is alive, and the operation is easy and feasible; 2, the anabarilius grahami fin muscle cell line (AGF) is in a fibroblast cell patternin a fiber structure, continuous passage can be achieved, the line can be applied to biological characteristic research directly, and requirement for anabarilius grahami molecular cell level theoretical research is met; and 3, the construction method is also applicable to cell lines of constructed fin rays of other fishes.

The invention discloses a preparation method of deciduous tooth mesenchymal stem cells, comprising the steps of: cleaning and sterilizing the tissue surfaces of deciduous teeth with normal saline and 75% alcohol, preserving the deciduous teeth in deciduous teeth preserving fluid; acquiring dental pulp from the deciduous teeth, adding dental pulp digestive fluid to the dental pulp for digestion; obtaining a unicell suspension; adding cell culture fluid, carrying out centrifuging and cleaning, adding precipitated cells into cell culture fluid, putting the cell culture fluid in a carbon dioxide incubator for culturing; when the primary cell fusion reaches 70%, adding cell washing fluid, shaking the culture bottle for washing cells, sucking and abandoning the cell washing fluid, adding cell digestive fluid for digestion, adding cell culture fluid to terminate digestion, repeatedly beating upon the bottle bottom until the cells completely fall off, adding cell culture fluid, inoculating into a culture bottle, putting the culture bottle in the carbon dioxide incubator for culturing, regarding the cells as P1 generation mesenchymal stem cells; when the P1 generation cell fusion reaches 70-80%, carrying out trypsin digestion, collecting digestive cells, carrying out centrifuging and inoculation; after 3d, carrying out trypsin digestion again, collecting digestive cells, carrying out centrifuging, counting and inoculation, and when the P3 generation cell growth reaches 80%, gathering and cryopreserving the cells.

The invention relates to an erythroculter ilishaeformis spermatogonia stem cell separation and culture method. The method comprises the following steps: (1) collecting spermatic tissues: collecting spermary of erythroculter ilishaeformis with an age of seven months in a sterile environment, then rinsing spermary in PBS containing double-antibody, and finally grinding the spermary to disperse the spermary tissues; (2) digesting and separating spermatogonia stem cell: adding IV type collagenase (0.1%) into the dispersed spermary tissues, subjecting the digestive fluid to centrifugal separation, collecting the precipitate, digesting the precipitate by trypsin (0.25%), stopping the digestion by a culture medium containing FBS (10%), and collecting the cells; (3) carrying out primary culture of spermatogonia stem cell: using a DMEM / F12 complete medium containing cell factors to re-suspend the cells obtained in the step (2), adjusting the cell concentration, then paving the suspension liquid on a 24-hole cell culture plate coated by gelatin, and culturing the cells at a temperature of 26 DEG C. Trough the provided method, the in-vitro growth of primary cells of erythroculter ilishaeformis spermatogonia stem cell becomes easier, and an effective cell platform is provided for the research on reproduction and growth of erythroculter ilishaeformis.

The invention relates to a method for establishing and identifying a cynoglossus semilaevis testis cell line, which comprises the following steps: adopting a MEM (Minimum Essential Medium), adding 20 percent of fetal calf serum, 0.1 percent of B-mercaptoethanol, 2 ng / ml of human alkaline fibroblastic growth factors, 1 ng / ml of leukemia inhibiting factors, 1 nmol of sodium pyrurate and 1 nmol of glutamine and preparing into a complete medium; inoculating a tissue block into a culture bottle, overturning and inversely placing the bottle to culture for 3 h and positively placing the culture bottle after the tissue block is attached onto a wall, so that the tissue block is soaked into the culture medium for culture. A trypsin digestion method is adopted for subculturing cells. One cynoglossus semilaevis testis cell line is established by adopting the above method and is subcultured to fiftieth generations. Meanwhile, the invention also provides a method for identifying a chromosome of the established cell line and the level of a molecular marker and a method for identifying the sensitivity of the cell line to related viruses. The method for establishing the cynoglossus semilaevis testis cell line can be used for culturing other fish gland cells and establishing the cell line, thereby having wider promotion and application prospect.

The invention relates to a turbot fin cell line forming method. It includes the following steps: using pectoral fin and pelvic fin tissue as raw material; shearing the fin tissue adopting trypsin, hyaluronidase, II type collagenase multiple step digestion process to gain free fin cell and loosen tissue block; and culturing in L-15 culture solution containing 20% fetal calf serum, carboxymethyl chitin, N- acetyl glucose hydrochloride, glucosamine hydrochloride, human epidermic cell growth factor, human alkali fibroblast growth factor, and tooth bothid gill cell culture solution; adopting trypsin digestion process to do sub-culturing. The technology is scientific. The formed sub-culturing cell reaches the 62th generations. The formed turbot fin cell is not transfected by any virus or oncogene, and can hopefully be directly applied to correlation theory study and viral vaccine development.

The subject invention concerns novel and translatable materials and methods for expansion of stem cells, such as mesenchymal stem cells (MSC), that significantly improve translational success of the cells in the treatment of various conditions, such as stroke. The subject invention utilizes cell self-aggregation as a non-genetic means to enhance their therapeutic potency in a microcarrier bioreactor. The subject invention integrates a cell aggregation process in a scalable bioreactor system. In one embodiment of the method, thermally responsive microcarriers (TRMs) are utilized in conjunction with a bioreactor system. Cells are cultured in a container or vessel in the presence of the TRMs wherein cells adhere to the surface of the TRMs. Once cells are adhered to the TRMs they can be cultured at a suitable temperature for cell growth and expansion, e.g., at about 37° C. After a period of time sufficient for cell growth and expansion on the TRMs, the cell culture temperature is reduced so that the cells detach from the TRMs. The detached cells are allowed to form cell clusters that are then cultured under conditions such that the clusters aggregate to form 3D aggregates. The 3D aggregates can be collected and treated to dissociate the cells (e.g., using enzymatic treatment, such as trypsinization). Dissociated cells can then be used for transplantation in methods of treatment or for in vitro characterization and study.

The invention discloses a constructing method and application of a koi brain cell line. The constructing method includes: placing koi brain tissue in digestive liquid A containing collagenase and trypsin for digestion; collecting cells; adding into digestive liquid b containing EDTA-4Na for digestion; performing primary culture and secondary culture after the cells which are digested are collected to obtain the koi brain cell line. Aiming at features of koi brain tissue cells, a digestion method combining collagenase with trypsin is adopted to separate tissue cells for primary culture, the koi brain cell line is constructed successfully and continuously transferred to more than 100 generations already, and a lot of koi brain source cells can be provided; the cells can maintain good growing state, so that the cells can be freeze-stored. The koi brain cell line is sensitive to koi herpes viruses with cytopathy occurring after being exposed to the viruses, and proliferated virus particles can be found through electron microscope observation. And the koi brain cell line can be directly applied to pathogenic characteristic study and vaccine development.

The invention relates to a construction method and ultralow temperature freezing and storing method of a sinocyclocheilus grahami saccus olfactorius cell line, and belongs to the technical field of cell culture and ultralow temperature freezing and storing method of the biological cell of freshwater aquatic life. A sinocyclocheilus grahami saccus olfactorius tissue is used as a raw material to be cultured in an L-15 culture solution at the pH (Potential of Hydrogen) value of 7.0-7.2 and comprising fetal calf serum by means of a tissue explant and subculture is carried out by means of trypsin digestion. The method specifically comprises the preparation of a cell culture solution, primary culture and subculture. The construction method disclosed by the invention has the following beneficial effects: (1) primary culture takes short time, and cell mass is large and can be applied to chromosome analysis; (2) the constructed sinocyclocheilus grahami saccus olfactorius cell line is in fiber like morphology, can be subcultured continuously and directly applied to a research on biological characteristics, and satisfies the demands of the storage, the theoretical research and the application of sinocyclocheilus grahami germplasm resources; (3) the construction method is also applicable to the construction of saccus olfactorius cell lines of other fishes.

The object of the present invention aims at the disadvantages of the prior art enzymatic hydrolyzate bitterness, salty taste, large molecular weight, non-concentrated molecular weight distribution, and high cost, and provides a method for extracting donkey-hide gelatin peptides. Or neutral protease or trypsin for enzymatic hydrolysis, the enzymatic solution is concentrated and refined technology and spray-dried to obtain the donkey-hide gelatin peptide product. The method has the advantages of simple operation, good taste, low cost, environmental protection and high efficiency, and is very suitable for large-scale industrial production.

Four novel Bacillus thuringiensis strains, which are deposited at the BCCM-LMG under accession nos. LMG P-12592, LMG P-12593, LMG P-12594, and LMG P-13493, produce new crystal proteins during sporulation that are toxic to Lepidoptera, more particularly against Noctuidae such as Spodoptera spp. and Agrotis ipsilon, against Pyralidae such as Ostrinta nubilalis, and against Yponomeutidae such as Plutella xylostella, and that are encoded by a novel gene. The crystal proteins contain protoxins, which can yield a toxin as trypsin-digestion product. A plant, the genome of which is transformed with a DNA sequence that comes from either one of the strains and that encodes its respective toxin, is resistant to Lepidoptera. Each strain, itself, or its crystals, crystal proteins, protoxin or toxin can be used as the active ingredient in an insecticidal composition for combatting Lepidoptera.

The construction of human corneal endothelium cell line with corneal endothelium as material includes the steps of: the first adherent culture of human corneal endothelium cell inside DMEM / F12 culture liquid with ox embryo serum in 20 wt%, chondroitin sulfate oxidizing and degrading matter, epidermal cell growth factor, basic fibroblast growth factor and ox eye auxin to obtain pure corneal endothelium cell; and the subsequent secondary culture in trypsinization process. The technological process is scientific and reasonable, and up to now, passage cell of 106-th generation has been obtained. The human corneal endothelium cell line of the present invention has no any virus or cancer genetic transfection, has no any tumorigenicity, and is expected to be used in direct artificial cornea production and clinical application.

The invention relates to a method for constructing a brown spotted rockfish fin cell line. The method is as follows: brown spotted rockfish fin tissue are taken as a material, the hyaluronidase with the concentration of 0.5% and collagenase II with the concentration of 0.2% combining digestion method is used for initiating primary culture, and the material is cultured in a DMEM / F12 culture medium which has the PH value of 7.0-7.4 and contains fetal calf serum, alkaline fibroblast growth factor, insulin-like growth factor I and carboxymethyl chito-oligosacaride, and the trypsin digestion method is adopted for subculturing. The brown spotted rockfish fin cell line constructed by the method is passed to the sixty-first generation at present. The method has scientific and reasonable process, and is expected to be applied to separating, identifying and reproducing fish viruses, preparing viral vaccines, studying the interaction between viruses and host cells, etc.

It is intended to provide an osteogenesis promoter capable of promoting osteogenesis by promoting the differentiation of osteoblasts, and foods, drinks, drugs or feeds for promoting osteogenesis. Namely, an osteogenesis promoter capable of promoting osteogenesis by promoting the differentiation of osteoblasts which comprises, as the active ingredient, lactoperoxidase and / or a digestion product obtained by digesting lactoperoxidase with a protease such as trypsin; and foods, drinks, drugs or feeds for promoting osteogenesis containing lactoperoxidase and / or its digestion product.

The invention relates to a method for establishing a schizothorax wangchiachii heart cell line and performing ultralow temperature cryoprservation on the schizothorax wangchiachii heart cell line, belonging to the technical field of freshwater aquatic organism cell culture and ultralow temperature cryopreservation. The method comprises the following steps of taking heart tissues of schizothorax wangchiachii as materials, and culturing the materials in an M199 culture solution by adopting a tissue block method and a two-enzyme digestion method, wherein the M199 culture solution contains fetal calf serum and cell growth factors and the pH value of the M199 culture solution is 7.0-7.2; and performing secondary culture by adopting a trypsase digestion method. The method specifically comprises the steps of preparing a cell culture solution, performing primary culture and performing secondary culture. The method has the beneficial effects as follows: 1, the operation is simple and convenient; 2, damages caused by a pure tissue block method to heart cells are reduced; 3, the form of the established schizothorax wangchiachii heart cell line is in the shape of fibroblasts, and the established schizothorax wangchiachii heart cell line grows well, can realize continuous passage culture, can be directly applied to biological characteristic researches, and meets requirements for storage, theoretical study and application of genetic resources of the schizothorax wangchiachii; and 4, the establishing method is also suitable for establishing heart cell lines of other types of fishes.

A proto-microcarrier for arthrodial cartilage is prepared from fresh joint cartilage through freeze drying, pulverizing, sieving adding trypsin to the particles, oscillating at 37 deg.C for 24-48 hr, washing with distilled water three times, adding TritonX-100, oscillating at 4 deg.C for 48-72 hr, flushing with distilled water for 48 hr, centrifugal separation of deposit, mixing with distilled water, freeze drying, UV irradiating, Co60 irradiation, digestion by trypsin, stirring, pulverizing and washing with distilled water three times.

The invention relates to the technical field of biology and aims to provide an establishment and application method of an immortalized Hu sheep rumen epithelial cell line. The method comprises the following steps: taking rumen epithelial tissues of newborn Hu sheep, cutting and cleaning the tissues, and digesting them with trypsin digestion liquid; purifying cells by a differential adherence method to obtain primary Hu sheep rumen epithelial cells; transfecting virus liquid carrying SV40T antigen gene into primary Hu sheep rumen epithelial cells to obtain infected Hu sheep rumen epithelial cells; and further subculturing the infected Hu sheep rumen epithelial cells to obtain an immortalized Hu sheep rumen epithelial cell line. A test cell model can be provided for rumen physiological regulation and nutrient absorption mechanism research of Hu sheep. The culture method is simple, the growth speed is high, and the Hu sheep rumen epithelial cell line with physiological functions and capable of continuous passage can be obtained. By utilizing the cell line, sampling of living Hu sheep can be reduced, and the problems of long primary cell culture time, high cost and complicated processat present are solved.

The invention discloses a method for obtaining human endometrial mesenchymal stem cells from curettage samples, which comprises the following steps: 1) mixing artificial abortion products and collection liquid according to the volume ratio of 1:0.9-1.1; obtaining curettage samples; 2 ) Separation and culture of endometrial stem cells: after the curettage sample is incubated on a shaker, it is filtered; the filtrate is obtained; then the fragmented tissue and monocytes in the blood are collected by density gradient centrifugation; then the cells are cultured, expanded and Purification; 3) After the P1 generation cells obtained in the above steps covered the bottom of the bottle, digest the cells with trypsin to collect the cells, resuspend the cells with pre-cooled freezing solution to obtain a cell suspension, and freeze the above cell suspension, The resulting frozen samples were stored in liquid nitrogen storage tanks. The method of the invention can obtain target cells with high purity and large quantity, that is, endometrial stem cells.

The invention relates to a constructing method of a human amniotic membrane epithelial stem cell library. The method comprises the following steps: digesting a human amniotic membrane through trypsin, and carrying out filtering to prepare single-cell suspension; adding human epidermal growth factors, human transferrins, human insulin, sodium selenite, alanyl-L-glutamine dipeptide, alanine, asparaginate, aspartic acid, glutamic acid, glycine, proline and serine into a DMEM / F12 culture medium, putting cells into a CO2 incubator at temperature of 37 DEG C, saturated humidity and volume percentage of 5 percent for culturing, liquid change and passage under a serum-free condition; and putting cells obtained through in-vitro culture and amplification into liquid nitrogen for cryopreservation, and constructing a cell information file for retrieval, namely building the human amniotic membrane epithelial stem cell library. The human amniotic membrane epithelial stem cell library is used for storing human amniotic membrane epithelial stem cells and has the characteristics of wide source and no ethical restriction, and a cell culture and storage medium is animal origin-free. The human amniotic membrane epithelial stem cell library can supply the epithelial stem cells for cell treatment and other applications.

The invention discloses a separation culture method of the primary hepatocyte of a jian carp. The separation culture method comprises the following steps: selecting a healthy jian carp without injury, collecting blood from the caudal vein of the jian carp, then sterilizing the surface of a fish body, placing into a super clean bench, and aseptically collecting the liver of the jian carp; rinsing by using a PBS solution which contains double antibodies, adding a trypsin digestion solution, wherein the temperature of digestion treatment is 26-28 DEG C, and the time of the digestion treatment is 15-20 minutes; after digestion is finished, adding to a culture medium A to finish the digestion, filtering the trypsin digestion solution by using a filter screen of 200 meshes, and collecting filter liquor; and respectively centrifugalizing 50 grams of the filter liquor and 30 grams of the filter liquor for 5 minutes, then washing twice by using a culture medium B, removing a supernatant to obtain a precipitation, namely an extracted hepatocyte, preparing a cell suspension, and planking for culture. The experimental method disclosed by the invention is simple and fast and can save the separation time to a great extent. The separation time adopted by an experiment is about 40 minutes, and the obtained hepatocyte has the advantages of good growth condition, small erythrocyte quantity and cell survival rate of about 95%.

The invention relates to a construction method of a muscle cell line of anabarilius grahami and belongs to the technical field of freshwater organism cell culture. The method comprises the steps of firstly preparing cell culture fluid, ensuring that anabarilius grahami muscle tissue serves as a material, inoculating in a culture bottle of 25 square centimeters, and placing a culture box at a temperature of 28 DEG C for culture; replacing the culture liquid in half volume every 3 days, and performing passage by using a trypsin digestion method after a cell grows into a monolayer; and performing passage every 5 days, when the passage of a tenth generation is achieved, the serum concentration is the culture fluid is reduced to 10%, and the cell line is constructed successfully. The method has the advantages that 1, the operation is easy and feasible; 2, in the constructed anabarilius grahami muscle cell line (AGM), the cell growth is good, the cells are in a fibroblast cell pattern, above 50 generations can be passed to continuously, and the method can be applied to studies of anabarilius grahami cell biological characteristics and immune resistance gene functions directly; and 3, the method is also applicable to muscle cell lines of other fishes.

A method for utilizing an affinity column to analyze holoprotein in the technical field of protein group comprises the following steps: compounding affinity separating materials, obtaining an affinity separating material base, extracting biology sample holoprotein, screening affinity material combinations whose absorbing protein category accounts for 20 to 80% of holoprotein from the affinity separating material base obtained from the first step, selecting one or a plurality of affinity material combinations from the affinity material combinations obtained from the second step for respectively filling affinity columns, then, utilizing the obtained affinity columns, adopting a cascade combination analysis method, a series combination analysis method, or a cascade and series mixed combination method to analyze biology sample holoprotein, obtaining a plurality of groups of samples whose protein complexity is simplified, respectively carrying out parenzyme digestion and LC-MS / MS analysis to the samples obtained from the third step, and thereby finishing the holoprotein analysis of biology sample holoprotein. The method of the invention can overcome the technical defect that high abundance protein submerges low abundance protein signals, and improves the extremity property protein detectable rate.

The invention discloses trypsin digestive juice, a preparation method and a ready-to-use phlegm digesting device. The trypsin digestive juice comprises the main components: trypsin and a solution stabilizer, wherein the solution stabilizer is selected from at least one of the following substances: dextrin, mycose and bovine serum albumin. The trypsin digestive juice disclosed by the invention not only can be stably stored under a working pH condition, but also can be stably stored at room temperature and working concentration, so that the storage cost is greatly lowered, and the trypsin digestive juice is ready-to-use and convenient and efficient.

The invention relates to a construction method of a sinocyclocheilus grahami grahami fin cell line and belongs to the technical field of freshwater aquatic organism cell culture. The method comprises that a sinocyclocheilus grahami grahami fin ray tissue serves as a material, a tissue block method is adopted, and culturing is achieved in a L-15 culture solution containing fetal calf serum and human basic fibroblast growth factors and having a PH value ranging between 7.2 and 7.4; subculture is performed by using a trypsin digestion method; and the subculture particularly comprises preparation of cell culture fluid, fin tissue minimally invasive sampling, primary culture and continuous culture. The method has the advantages that 1, the minimally invasive processing is adopted, on the premise that the fish is alive, and the operation is easy; 2, the sinocyclocheilus grahami grahami fin cell line is in a fibroblast shape, continuous passage can be achieved, the line can be applied to biological characteristic research directly, and requirements for sinocyclocheilus grahami grahami germplasm conservation and theoretical research and application are met; and 3, the construction method is also applicable to cell lines of constructed fin rays of other fishes.

The invention aims at providing an establishment method of an ovary cell line of cynoglossus semilaevis, which comprises the following steps: 1) removing the outermost membrane of the ovary tissue of cynoglossus semilaevis under a sterile condition, cutting the tissue into small blocks in a culture solution, flushing the tissue blocks with a PBS solution and digesting with a trypsin solution, wherein the solution after trypsin digestion contains cells, cell clusters and tissue blocks not completely digested; and 2) centrifuging the solution to remove the supernatant trypsin, and suspending the cells, cell clusters and small tissue block precipitates with a culture solution; after a suspension is obtained, inoculating into a culture plate treated by the type-I collagen; adding a culture solution, and culturing in a biochemical incubator at 24 DEG C; and performing subculture to finish establishment of the ovary cell line. In the establishment method provided by the invention, an ovary cell line of cynoglossus semilaevis is successfully established by use of the ovary tissue of cynoglossus semilaevis, the established cell line can be continuously passed, the passage cells obtained by the method can be passed for over 60 generations, and a great quantity of ovary cells can be provided.