CN102977203A - Epithelial cell division chalone, and preparation method and application thereof - Google Patents
Epithelial cell division chalone, and preparation method and application thereof Download PDFInfo
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- CN102977203A CN102977203A CN201210485772XA CN201210485772A CN102977203A CN 102977203 A CN102977203 A CN 102977203A CN 201210485772X A CN201210485772X A CN 201210485772XA CN 201210485772 A CN201210485772 A CN 201210485772A CN 102977203 A CN102977203 A CN 102977203A
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Abstract
The invention belongs to the technical field of biochemical separation and purification, and in particular relates to an epithelial cell division chalone, and a preparation method and an application thereof. A high-speed tissue pounding machine is replaced by a colloid grinder, a method for producing pigskin into uniform slurry satisfies the requirements of industrial production, and the protein yield is improved by above 70%. Water extraction liquid is subjected to ultrafiltration concentration and is added with ethanol to be subjected to fractional precipitation, the ethanol use amount is reduced by 97% compared with the ethanol use amount of an original technology which adopts ethanol to precipitate directly, so that the production cost is lowered; and cation exchange chromatography is adopted during the purification process, so that the specific activity of the epithelial cell division chalone is greatly improved. The epithelial cell division chalone can effectively inhibit epithelial cell division, has obvious functions in relieving and stopping itching of patients suffering from psoriasis and also has a certain treatment value to skin cancer.
Description
Technical field
The invention belongs to field of biochemical separation and purifying technology, split element and its preparation method and application but be specifically related to a kind of epidermic cell.
Background technology
But epidermic cell splits a kind of endogenic protein cell factor that element (being called for short the skin chalone) is the epidermic cell generation of animal, and it can act on the epidermic cell of animal, and the mitotic division that suppresses epidermic cell makes it to rest on the G1 phase of cell growth cycle.20 beginnings of the century just the someone find to exist in the organism and suppress fissional material, the someone finds that a kind of extract of tissue is added in the homologue and can suppress this histiocytic division later on.Point out in the control of nineteen sixty Bulough and Laurence epithelial cell division in the research body that a single-minded inhibitor of endogenic cell can block the growth cycle of cell, this inhibitor is a kind of endogenous material that is produced by cell, play in vivo the negative regulation effect, therefore propose " Chalone " this noun and name this class material, refer to that they play a part to slow down or interrupt in vivo.Chalone is because the source is different, and target cell is different and many kinds are arranged, but but but but splitting element, epidermic cell such as lymphocyte splits that element, granulocyte split element, liver cell is split element etc.They have the general character of 4 aspects: (1) in a organized way specificity (2) nothing plants group specificity (3) no cytotoxicity (4) reversibility.In view of above-mentioned specificity, chalone should play restraining effect for some unusual divisions, propagation.Obviously have potential application prospect, but but but for example lymphocyte splits to have and may develop into anti-rejection, liver cell and split and have the function, the epidermic cell that suppress the liver cancer cell Fast Growth and split element and have a large amount of effects that generate of some type psoriasis patients scurf that suppress.
Summary of the invention:
The problem that the present invention need to solve is the extraction purifying to the skin chalone, does larger improvement and makes it to be more suitable for suitability for industrialized production, makes that technique is more reasonable, yield is higher, better quality, more stable.Active HaCat people's immortalization epidermic cell strain meta assay method of using makes more convenient operation, science, reliability, comparability stronger
Technical scheme of the present invention
But epidermic cell of the present invention splits element by the animal skin separation and purification, and molecular weight is 30000-50000.
(1) but, epidermic cell split the element the preparation method: get fresh porcine skin and eliminate appended fatty tissue, clean, be twisted into the pork skin gruel with mincer, weigh, add three times of water and with colloidal mill homogenate is worn in the pork skin gruel, stir 3 hours, filtration, filter residue added diploid ponding restir 2 hours again, merged secondary filtrate.Filtrate degreasing fat by the ultrafiltration membrance filter of cut-off molecular weight 10000, is concentrated into the 3%-5% of initial volume, will concentrate rear ultrafiltration reservation liquid and precipitate with ethanol, gets 30%-80% the partly phosphoric acid buffer dissolving of usefulness PH7.5 of precipitation, the centrifuging and taking supernatant.It is positively charged that supernatant liquor is transferred to PH acid this moment of skin chalone, can exchange on SP-Sepharose ion exchange column, and a large amount of foreign proteins can pass and by flush away, the phosphoric acid buffer wash-out of using PH8.0 instead can get elution peak 1, using the PH8.0 phosphoric acid buffer wash-out that contains 0.5 M NaCl instead can get elution peak 2(tomographic results and see Fig. 1 again), survey active proof activity mainly in peak 1, the part activity is also arranged in the peak 2, and SP-Sepharose F.F is a kind of cationite, the skin chalone is positively charged under acidic conditions, can exchange on chromatography column, all electronegative protein can not exchange on post, all in passing the peak, by the phosphoric acid buffer wash-out of PH6.5; Then use the phosphoric acid buffer of PH8.0 instead, because PH8.0 is higher than the iso-electric point of skin chalone, the skin chalone is electronegative, therefore most of skin chalone is washed, again with containing the phosphoric acid buffer of 0.5M NaCl, because ionic strength increases, the high albumen of other iso-electric points is also by wash-out.The determination of activity result: the specific activity of sample is 2975u/mg before the upper prop, and it is very large but specific activity is very low to pass peak area, only has 989u/mg, and elution peak 1 area is very little but specific activity is very high, reaches 244285u/mg and improves 82 times before than upper prop.The less small part skin chalone that still has of elution peak 2 areas, specific activity is 62160u/mg.
(2), the determination of activity of skin chalone: adopt HaCat people's immortalization epidermic cell strain (available from the multiple auspicious Bioisystech Co., Ltd in Shanghai) cell cultures in RPMI1640 nutrient solution (containing calf serum 10%), in 37 ℃, CO2 incubator, cultivate, when treating that cell grows to 80% left and right sides of confluent culture bottle wall with 0.25% trypsinase with cell dissociation, make 3 * 10
4/ ml.Cell suspension every hole in 96 well culture plates adds 160 μ l cell suspensions, cultivates to add skin chalone 40 μ l after 20 hours.Dilution is decided according to the content of skin chalone in the solution, can be from the horizontal doubling dilution of every hole microgram to nanogram or a gram level, and control group adds 40 μ l nutrient solutions.Various concentration are done three holes and are repeated, continue to cultivate and add 5mg/ml MTT 20 μ l after 72 hours, cultivated again 4 hours, go supernatant to add methyl-sulphoxide 100 μ l colour developing, at microplate reader OD492nm place reading, the reading of three repeating holes is averaged, and with the mean value calculation inhibition percentage of control group, it is an active micro-unit (mu) that regulation suppresses 1%.Inhibition percentage is figure to the protein concn in every hole, can gets a S type curve, get lower platform, the intermediate value of getting lower platform is active site.The activity unit that the protein content/hole of active site is contained is called specific activity (u/mg), and specific activity represents the purity of skin chalone, and the activity of the high expression of specific activity skin chalone is also high.
Specific activity (u/mg)=
(3), the skin chalone is used: skin chalone (specific activity is greater than 2000u/mg) after the degerming is added in the ointment, for the psoriasis patients external application, patient Huang (psoriasis vulgaris) is the patient in more than 30 year, the general skin decreases, once used multiple therapy methods, can only of short durationly alleviate, skin chalone on probation is treated left three weeks of leg, sooner or later twice outer wiping every day, right leg in contrast, three all rear left-leg scurfs disappear entirely, skin recovers elasticity, smooth, only stay the calm trace of red pigments, also no longer scratch where it itches, and untreated right leg still is covered with very thick scurf, hard, coarsely (the results are shown in accompanying drawing 3) as before.Under the doctor cooperates, tried out the skin chalone also once for the multidigit patient.Curative effect is fine after having three inpatients to treat in four patients of Nanjing hospital, and non-inpatient's curative effect is not obvious.10 patients of Tianjin hospitalize, evident in efficacy, it is better that the import of doctor's feedback ratio contains the drug effect of hormone.Curative effect is also very remarkable after two the psoriasis in plaques patients medication of Nanjing.The skin chalone not only has the effect that suppresses division to people's immortalization epidermic cell, the A431 skin cancer cell also there is obvious restraining effect, with the skin chalone test of a partial purification goods specific activity 500u/mg, the level of every hole 450ng, the growth of A431 cell suppressed 40%.A431 skin cancer cell 1 * 10
4/ ml, every hole adds 160 μ l cell suspensions in 96 orifice plates, cultivates 20 hours 37 ℃ of CO2 incubators, adds skin chalone 40 μ l, skin chalone concentration from 625ng/ml doubling dilution to 19.8ng.Cultivate after 72 hours and develop the color with mtt assay, the OD492nm reading calculates inhibition percentage with the control group reading, and to protein mass mapping in every hole, visible skin chalone has very strong restraining effect to the A431 skin cancer cell, illustrates that the skin chalone can be used for treating skin carcinoma.The results are shown in accompanying drawing 4.The skin chalone that the present invention produces can be prepared into the medicine for the treatment of psoriatic or skin carcinoma.
The present invention compared with prior art its beneficial effect is
(1), now technique is cut into 1cm with the pigskin degrease
2Fritter adds water high-speed tissue mashing machine's homogenate, filter, filter residue again homogenate once, merging filtrate.The present invention uses colloidal mill instead, and pigskin is worn into homogenate fully, and extraction rate of protein improves more than 70%, is more suitable for suitability for industrialized production.
(2), existing process water extract directly adds ethanol, 30%-85% precipitation, the solubilising protein yield is the 1.09g/kg pigskin, the per kilogram pigskin needs 43L ethanol.The present invention is with the ultrafiltration of water extract elder generation; molecular weight is removed less than 10,000 inactive protein matter; concentrated simultaneously extract volume to 3%-5%; use again 30%-70% ethanol precipitation; saved ethanol more than 97%; the protein yield is the 3.28g/kg pigskin; yield improves 3 times; specific activity improves 6 times; gross activity has improved 300 times; owing in purge process, removed the material of unborn epithelical cell growth factor class in the pigskin; so gross activity far surpasses the gross activity in the initial water extract, has reduced production cost; saved manpower; also saved in a large number because using the necessary protection facility that increases of inflammable articles such as the transportation of ethanol; reclaim; the equipment of the various necessity such as dangerous goods store and supervisor's expense.The comparative result lattice that see the following form:
| ? | Former technique | Improve technique |
| Starting raw material (pigskin) consumption | 0.4kg | 5.56kg |
| High-speed tissue mashing machine's water extracting albumen yield | 7.285g/kg pigskin | ? |
| Colloidal mill homogenate water extracting albumen yield | ? | The 13g/kg pigskin |
| Protein yield behind the ultrafiltration and concentration | ? | 7.83g/kg pigskin |
| 30%-85% ethanol precipitating proteins yield | 1.09g/kg pigskin | 3.28g/kg pigskin |
| Specific activity u/mg | 495 | 2975 |
| Gross activity u/kg pigskin | 52300 | 16130000 |
| Consume ethanol L/kg pigskin | 37.7 | 0.93 |
(.3), existing technique purifying aspect is by DEAE Mierocrystalline cellulose chromatography, Sephacry s-200 gel-filtration is again by DEAE-Sephadex column chromatography.The present invention has improved 82 times before by SP-Sepharose chromatography elution peak 1 a specific activity upper prop, and is simple and effectively.The chromatography collection of illustrative plates is seen accompanying drawing 1
(.4), measure active with the strain of HaCat people's immortalization epidermic cell, at first cell quantity can provide in a large number, cellularity is also more stable controlled, because the cell of sufficient amount is arranged, the skin chalone of test can be done a series of dilutions, be diluted to nanogram or a gram level from the microgram level always, make intermediate value that a complete S type curve gets lower platform and be active site and be science very.Existing technique is only surveyed 4 concentration, is difficult to capture real active site, and especially high specific activity sample microgram level does not measure.Activity determination method, HaCat cell 3 * 10 now are described as an example of SP-Sepharose chromatography elution peak 1 example
4/ ml cell suspension, the every hole of 96 well culture plates adds 160 μ l, in 37 ℃ of incubators of CO2, cultivate and add 40 μ l skin chalones after 20 hours, skin chalone concentration is decided according to active height, can be from the horizontal doubling dilution of every hole microgram to a gram level, cell continues to cultivate adding 20 μ l MTT(5mg/ml after 72 hours), cultivate again after 4 hours and remove supernatant, add 100 μ l methyl-sulphoxides, at microplate reader OD492nm place reading, the reading of the getting repeating hole mean value calculation inhibition percentage with control group of averaging, mapping obtains a curve to protein concn with inhibition percentage, makes lower platform, get intermediate value, be 8.55mu such as intermediate value among Fig. 2, the albumen at intermediate value place is 35pg, and calculating specific activity is 244285u/mg.
(5) prior art method therefor yield is not high, need to use a large amount of ethanol, not only cost high and also when scale operation security measures, ethanol reclaim very large paying of the need such as storage.Chicken embryonic epidermis primary cell is used in determination of activity, and not only technical requirements is high, and material is under one's control, and the gained cell is limited, and the comparability of between-lot is relatively poor.The present invention has done significant improvement to above-mentioned each side, makes it to be more suitable for large-scale mass production.The skin chalone adopts and is diluted to nanogram level even a gram level from the microgram horizontal series, calculate its inhibition percentage, concentration is charted, get the specific activity of skin chalone concentration calculating skin chalone when suppressing intermediate value, it is more reasonable that this method is surveyed inhibition percentage than fixed concentration, because the activity of skin chalone and concentration relationship are not linear relation but parabolic relation
Description of drawings
Fig. 1, SP-Sepharose F.F tomographic map
Fig. 2, HaCat people's immortalization epidermic cell, it is active that upper lower platform graphing method is measured the skin chalone
Fig. 3, skin chalone treatment psoriatic effect
Fig. 4, skin chalone are to the effect of skin cancer cell A431 growth-inhibiting
Embodiment
1, but epidermic cell splits the preparation method of element
(1) gets fresh clean pigskin 7Kg removal fatty tissue and to get altogether 5.96Kg, be twisted into meat gruel with electrical meat mincer, get the 0.4Kg meat gruel and undertaken by former technique, as a comparison.Extract the skin chalone 5.56Kg adopt new technology.
(.2), the gruel of 0.4Kg pork skin is added water with the high-speed homogenization of high-speed tissue mashing machine 30 seconds totally three times, fine cloth filters, and filter residue adds homogenate waterborne again, filters, merges altogether 1.88L of secondary filtrate, and Tot Prot is 2.914g, and the per kilogram pigskin gets 7.285g protein.1.88L filtrate is added 95% ethanol precipitation by 1:0.46 makes alcohol concn reach 30%, refrigerator overnight, next day filter at low temperature filtrate, press 5.5 times of filtrate volumes and add ethanol, make to reach 85% and place and to spend the night, low-temperature centrifugation must precipitate, and is dissolved in the 0.02M phosphoric acid buffer of PH7.5, it is 427.93mg that the centrifuging and taking supernatant gets the 32.5ml total protein concentration, is equivalent to the per kilogram pigskin and gets 1.07g protein.
(3), the gruel of 5.56Kg pork skin is added water 24Kg, wear into homogenate by colloidal mill, slowly stirred 3 hours, fine cloth filters, and filter residue adds water again and stirs 2 hours merging filtrates, to get altogether 41.2L, and total protein is 72.306g, and the per kilogram pigskin gets 13g.41.2L filtrate is concentrated by the membrane ultrafiltration of cut-off molecular weight 10,000, exceed after measured non-activity of liquid, it is that 1.2L protein concentration 36.3mg/ml total protein concentration 43.56g per kilogram pigskin gets 7.83g that ultrafiltration keeps liquid.Ultrafiltration keeps liquid and adds ethanol to 30%-70%, and resolution of precipitate liquid total protein concentration 18.24g is equivalent to the per kilogram pigskin and gets 3.28g.30%-70% ethanol precipitation is dissolved in upper SP-Sepharose cation-exchange chromatography post (Φ 2cm, high 8.5cm) applied sample amount 55ml in the 0.02M PH6.5 phosphoric acid buffer, contains 222.31mg protein, be equivalent to the 0.0641Kg pigskin.Must pass the peak by above-mentioned buffer solution elution, use PH8.0 phosphoric acid buffer wash-out instead and get peak 1, use the above-mentioned buffer solution elution that the contains 0.5M NaCl peak 2 of getting back instead, activity mainly in peak 1, also has activity in the peak 2 after measured.Peak 1 specific activity than chromatography before sample improve 82 times, peak 2 and improve 21 times, the gross activity loss is more, but still is higher than former technique more than 3 times.
2, but epidermic cell splits the determination of activity of element
HaCat people's immortalization epidermic cell is cultivated in containing the RPMI RPMI-1640 of 10% calf serum, cultivates 6 days in 37 ℃ of CO2gas incubator, and cell proliferation is about about 80% to the culturing bottle wall, removes nutrient solution, makes 3 * 10 with 0.25% tryptic digestion
4The suspension of cell/ml.Add 160 μ l cell suspensions in the every hole of 96 orifice plates, cultivate adding skin chalone 40 μ l after 20 hours.Skin chalone concentration is decided on the active height of skin chalone generally can be from every hole horizontal doubling dilution of microgram to the nanogram level.As through SP-Sepharose chromatography elution peak 1, activity is very high then to need to be diluted to a gram level.For example former technique specific activity is 495u/mg, and the novel process specific activity is 2975u/mg; Specific activity is 2975u/mg before SP-Sepharose chromatography upper prop, and passing the peak specific activity is 989u/mg, and elution peak 1 specific activity is 244285u/mg, and elution peak 2 specific activities are 62160u/mg.
.3, application example
Nearest two examples are diagnosed as the psoriasis in plaques women, and course of disease more than ten year is though through treating still frequent recurrence, show as that plaquelike scurf layer thickens, scurf comes off in a large number, scratch where it itches in the affected part.Through (every gram ointment contains 2mg skin chalone, and sooner or later respectively wipe the affected part once every day, and scurf removes to the greatest extent after one month, and it is soft that skin recovers, and no longer scratches where it itches with skin chalone ointment.
Claims (6)
1. but an epidermic cell splits element, it is characterized in that separation and purification from animal skin, and molecular weight is 30000-50000.
2. but the described a kind of epidermic cell of claim 1 splits the preparation method of element, it is characterized in that being made of following steps:
(1), with fresh clean degrease pigskin as raw material, by mincer pigskin is made the pork skin gruel;
(.2), with the water of three times of pigskin weight as extracting solution, add in the pork skin gruel, grind to form homogenate by colloidal mill, stirring 2-3 hour, filtration below 10 ℃, filter residue added diploid ponding restir 2 hours again, merged secondary filtrate, and 4 ℃ of hold over night are removed the upper strata grease;
(3), degrease filtrate by the cut-off molecular weight 10000 ultrafiltration membrance filter, be concentrated into 3% of initial volume;
(4), molecular weight is kept liquid through 30--80% ethanol precipitation, again dissolving greater than 10000 ultrafiltration;
(5), with supernatant liquor by SP-Sepharose F.F cation-exchange chromatography post, the skin chalone exchange on chromatography column, changes PH increase ionic strength, the skin chalone is by wash-out.
3. but split the preparation method of element according to. the described a kind of epidermic cell of claim 2, it is characterized in that using colloidal mill to grind pigskin, make the pigskin of difficult homogenate obtain abundant fragmentation.
4. but the described a kind of epidermic cell of claim 1 splits the activity determination method of element, it is active to it is characterized in that measuring the skin chalone with Hacat plancenta hominis cultural method, in the epidermic cell nutrient solution, add the skin chalone, suppress the growth of epidermic cell, detect the inhibition percentage of skin chalone with mtt assay, get the skin chalone from the microgram level, with doubling dilution to nanogram level even a gram level, mapping draws upper lower platform to inhibiting rate with protein concn in every hole, and the intermediate value of getting lower platform is active site.
5. will suppress 1% and be defined as a little mu of activity unit, the activity unit of every milligram of protein is specific activity (u/mg protein)
But the described a kind of epidermic cell of claim 1 splits the application of element in preparation treatment psoriasis.
6. but the described a kind of epidermic cell of claim 1 splits the application of element in preparation treatment skin carcinoma medicine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106279406A (en) * | 2016-08-05 | 2017-01-04 | 南京必优康生物技术有限公司 | The active component tetranectin of skin chalone G1 isolated and purified in Corii Sus domestica and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1362416A (en) * | 2001-12-29 | 2002-08-07 | 南京大学 | Production process of epidermal cell inhibitor with animal skin and its use |
| CN1554665A (en) * | 2003-12-29 | 2004-12-15 | �Ϻ���ͨ��ѧ | Production process and use of epidermal cell colyone |
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2012
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1362416A (en) * | 2001-12-29 | 2002-08-07 | 南京大学 | Production process of epidermal cell inhibitor with animal skin and its use |
| CN1554665A (en) * | 2003-12-29 | 2004-12-15 | �Ϻ���ͨ��ѧ | Production process and use of epidermal cell colyone |
Non-Patent Citations (2)
| Title |
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| 朱颉 等: "表皮抑素特征及五肽分子与增生性瘢痕", 《中国临床康复》, vol. 7, no. 14, 25 June 2003 (2003-06-25), pages 2076 - 2077 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106279406A (en) * | 2016-08-05 | 2017-01-04 | 南京必优康生物技术有限公司 | The active component tetranectin of skin chalone G1 isolated and purified in Corii Sus domestica and application thereof |
| CN106279406B (en) * | 2016-08-05 | 2019-08-20 | 南京必优康生物技术有限公司 | The active constituent tetranectin of the skin chalone G1 isolated and purified in pigskin and its application |
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