CN1088584A - From body fluid, extract the production technique of Fiberonectin - Google Patents
From body fluid, extract the production technique of Fiberonectin Download PDFInfo
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- CN1088584A CN1088584A CN92113640A CN92113640A CN1088584A CN 1088584 A CN1088584 A CN 1088584A CN 92113640 A CN92113640 A CN 92113640A CN 92113640 A CN92113640 A CN 92113640A CN 1088584 A CN1088584 A CN 1088584A
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- fiberonectin
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Abstract
The invention belongs to the production technique of extracting Fiberonectin in the biology medical science.The present invention uses independent phenylmethylsulfonyl fluoride arrestin enzymic activity, and with sepharose gelatinum affinity chromatography technology one step chromatography, from people and animal body fluid (blood, amniotic fluid, tissue juice), extract the macromole glycoprotein that can be applicable to biology medical research, experiment, clinical medicine and make-up and beauty---and Fiberonectin (Fibronectin, Fn).Production technique of the present invention is extracted Fiberonectin, and is with low cost, product purity height, production technique simple and stable, extraction yield height.
Description
(Fibronectin Fn), has another name called fibronectin to Fiberonectin, fine sticking element etc.Also use following title: CIG in the document, anti-gelatin factor, microfibrillar protein, opsonic protein, fibroblast surface antigen(FSA), soluble fibroblast antigen(SF-antigen), galactoprotein a, cell surface protein(CSP), cell attachment factor (CAF), cell spreading factor, large external transformation-sensisive(LEFS) protein, Zeta(Z) etc.
Fiberonectin is a kind of high molecular cool insoluble protein, the about 45K of molecular weight, be present in the body fluid (blood, amniotic fluid, tissue juice) of people and animal and cell surface and between in the matter, having inducing cell differentiation, propagation, connect a cell and a matter, participate in multiple functions such as blood coagulation, wound healing and body defence, is the important component of body.
Nearly 20 years (1972～1992) international and domestic patent documentations and looking into newly of scientific literature are shown, more existing in the past extract methods (Methods in Enzymol 1982 such as Ruoslahti E of Fiberonectins; J Biochem 1981 such as 82:803 and Isumera M; 90:1), existing abroad two patents (Ep38462-A, 1986; WO63483,1988).Domestic still do not have and the similar patent application of the present invention.
The method of existing extraction Fiberonectin is used multiple a large amount of proteinase inhibitor, simultaneously in extraction, adopt three steps or above chromatographic step of three steps, the agents useful for same medicine is increased, cost rises, and the extraction formality is numerous and diverse, and the production rate of Fiberonectin is not high.Existing extracting method is confined to blood, and raw material sources are restricted.
The objective of the invention is at the deficiencies in the prior art part, a kind of production technique of extracting Fiberonectin from body fluid is provided.The present invention has not only reduced cost, has simplified extraction step, has increased the output capacity of product, and has improved the purity of product.The present invention can be widely used in the multiple body fluid (comprising blood, amniotic fluid and tissue juice) of people and multiple animal, has opened up more extensive more cheap raw material sources.
In order to realize the foregoing invention purpose, the production technique that the present inventor takes is: (1) obtains body fluid; (2) add phenylmethylsulfonyl fluoride, low temperature ultracentrifugation; (3) application of sample is in prefabricated cyanogen bromide sepharose gelatinum chromatography column, buffer solution elution Fiberonectin; (4) the Fiberonectin filtration of Ti Quing, measurement, aseptic freeze-dried; (5) the Fiberonectin biological function that extracts detects.The present inventor notices that Fiberonectin shows very strong stability during evolution, the structure proximate of Fiberonectin in people and the various animal body fluid, and main biological function is identical.This provides inexpensive widely raw material for obtaining of Fiberonectin of the present invention.The present invention is according to molecular weight, the condensation character of Fiberonectin.And there is the physicochemical property of strong affinity in Fiberonectin and scleroproein, gelatinum, virgin rubber unit, designed production technique of the present invention.
At first gather people and multiple animal, as the body fluid of ox or horse or pig or other animals, as blood or amniotic fluid or tissue juice; add 1/10 3～10% citron sodium anti-freezing (blood) immediately; use phenylmethylsulfonyl fluoride (commercially available) the arrestin enzymic activity of 0.1～0.3mol/L simultaneously, remain under 0～4 ℃ of low temperature, and the low temperature ultracentrifugation; 0 ± 1 ℃; 7,500～25,000 RPM; 10～40 minutes, get supernatant liquor.
The supernatant liquor application of sample is on buffer A (mainly containing 0.5～1mol/L Ttris-HCL, 0.1～0.2mol/L PMSF, 0.5～5%NaCl(solution) equilibrated gelatinum sepharose chromatography column, and sepharose and gelatinum are the commercially available prod.Chromatography column mainly contains sodium tetraborate 0.1～0.2mol/L in advance with cyanogen bromide-activated in the activation solution, coupling buffer mainly contains thanomin 0.5～2mol/L, sodium azide 0.1～1mol/L, and above reagent is the commercially available prod.With ZY-1 type ultraviolet spectrophotometer (280nm) monitoring, wash to contain the urea buffer B, contain the urea buffer B and mainly contain Tris-HCl 0.5～1mol/L, PMSF 0.1～0.2mol/L, urea 3-8mol/L.In two hours, 50ml volumetrical sepharose gelatinum affinity column can add 350～500ml sample.Again with containing high density Na
+Buffer B wash Fiberonectin.Whole affinity chromatography process should be carried out at 0～5 ℃.It is pure that the Fiberonectin that extracts can reach electrophoresis, is stored in the urea buffer B of 3～8mol/L.Fiberonectin is used spectrophotometric determination through 0.22 μ m filtering with microporous membrane degerming, and the 280nm place absorbs, and calculates Fiberonectin output, packing, aseptic lyophilize.
The Fiberonectin that extracts is with 1～1.5% agarose constant voltage, 5～8V/cm electrophoresis, and the result is single electrophoresis band, and it is pure that the Fiberonectin purity that proves this extraction reaches electrophoresis, proves that through SDS continuous gradient electrophoresis its molecular weight is 45K.Fiberonectin serum with anti-people and anti-horse, anti-ox carries out immunodiffusion respectively, presents precipitation line clearly as a result between antigen-antibody, proves that extract is a Fiberonectin.Use the various kinds of cell activity experiment, comprise that methods such as various kinds of cell is cultivated, the active detection of macrophage phagocytic, the metering of cell dynamic form prove its biological function.
Production technique of the present invention is simple, with low cost than art methods, good reproducibility, extraction yield height.The conjugated protein extraction yield of plasma fibronectin is 50mg/100ml.
The Fiberonectin level is not normal in many discovering, body, can cause numerous disease to take place, as tumour, hepatopathy, joint disease, diabetes, nervous system disorders and septicemia, and diffuse intravascular coagulation, shock etc.Fiberonectin also plays key effect in the healing of wound.Experimental animal model proves that Fiberonectin has positive effect in trauma care.With the conjugated protein rat experiment skin wound surface that is applied to of small amount of fibers, speed of wound healing obviously speeds as Nishida.With Fiberonectin collyrium eye drip treatment cornea stain ulcer, second day epidermis raised growth of medication, ulcer healing after three weeks, no scar is left over, and does not also have other side effects.In addition, the effect of the Blood clotting of Fiberonectin, treatment diffuse intravascular coagulation (DIC), septicemia is also very obvious.Therefore, Fiberonectin has huge value of exploiting and utilizing and application prospect.
The Fiberonectin that the present invention extracts can be used as research, the teaching of biology medical science, the diagnostic reagent of Clinical Laboratory, also can be used as the important medium of cell cultures.The Fiberonectin that the present invention extracts can be used for treating diseases such as wound, burn, septicemia and tumour, also can be used for beauty and skin care and oral cavity health toothpaste is medium.Present domestic still do not have scleroproein batch process and sale.
Claims (2)
1, a kind of production technique of from body fluid, extracting Fiberonectin, comprise and obtain body fluid, proteolytic enzyme suppresses and the hypervelocity low-temperature centrifugation, sepharose gelatinum affinity chromatography, Fiberonectin filtration, the metering, aseptic freeze-dried of extracting, and the detection of the biological function of Fiberonectin, it is characterized in that:
(1) obtain body fluid after, add 1/10 3-10% liquor sodii citratis anti-freezing immediately, use the phenylmethylsulfonyl fluoride arrestin enzymic activity of 0.1～0.3mol/L simultaneously, and remain on 0.4 ℃, then at 0 ± 1 ℃, with 7,500～25,000 rev/min rotating speed, ultracentrifugation 10～40 minutes is got supernatant liquor.
(2) gelatinum is coupled on the sepharose of cyanogen bromide-activated, activation solution mainly contains 0.1～0.2mol/L sodium tetraborate, then with mainly containing 0.5～1mol/L Tris-HCl, the buffer A balance sepharose gelatinum affinity column of 0.1～0.2mol/L PMSF and 0.5～5%NaCl solution.Add sample in affinity column.The ultraviolet spectrophotometer monitoring is with the buffer B wash-out Fiberonectin that contains 3～8mol/L urea.Whole affinity chromatography process should be carried out at 0～5 ℃.
2, a kind of production technique of extracting Fiberonectin from body fluid according to claim 1 is characterized in that: gather people and multiple animal, as the body fluid of horse or sheep or pig or ox, as blood or amniotic fluid or tissue juice as raw material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92113640A CN1088584A (en) | 1992-12-22 | 1992-12-22 | From body fluid, extract the production technique of Fiberonectin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92113640A CN1088584A (en) | 1992-12-22 | 1992-12-22 | From body fluid, extract the production technique of Fiberonectin |
Publications (1)
| Publication Number | Publication Date |
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| CN1088584A true CN1088584A (en) | 1994-06-29 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN92113640A Pending CN1088584A (en) | 1992-12-22 | 1992-12-22 | From body fluid, extract the production technique of Fiberonectin |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7323332B2 (en) | 2002-12-27 | 2008-01-29 | Shimadzu Corporation | Preparation method of insect cell extract solution for cell-free protein synthesis, the insect cell extract solution and cell-free synthesis method of protein using the insect cell extract solution |
| CN1697972B (en) * | 2002-08-13 | 2010-09-29 | N-Dia公司 | Device and method for detecting amniotic fluid in vaginal secretion |
| CN104984739A (en) * | 2015-06-17 | 2015-10-21 | 武汉汇研生物科技股份有限公司 | Preparing method and application of gelatin affinity chromatography media |
| US11353464B2 (en) | 2013-01-02 | 2022-06-07 | Qiagen Sciences, Llc | Methods for predicting time-to-delivery in pregnant women |
| CN115887275A (en) * | 2022-11-25 | 2023-04-04 | 广州远想生物科技股份有限公司 | Freeze-dried essence and preparation method thereof |
-
1992
- 1992-12-22 CN CN92113640A patent/CN1088584A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1697972B (en) * | 2002-08-13 | 2010-09-29 | N-Dia公司 | Device and method for detecting amniotic fluid in vaginal secretion |
| US7323332B2 (en) | 2002-12-27 | 2008-01-29 | Shimadzu Corporation | Preparation method of insect cell extract solution for cell-free protein synthesis, the insect cell extract solution and cell-free synthesis method of protein using the insect cell extract solution |
| CN100462443C (en) * | 2002-12-27 | 2009-02-18 | 株式会社岛津制作所 | Method for preparing insect cell extract for synthesizing cell-free protein, the insect cell extract and method for synthesizing the cell-free protein by using the insect cell extract |
| US7745171B2 (en) | 2002-12-27 | 2010-06-29 | Shimadzu Corporation | Cell-free protein synthesis method using insect cell extract solution |
| US11353464B2 (en) | 2013-01-02 | 2022-06-07 | Qiagen Sciences, Llc | Methods for predicting time-to-delivery in pregnant women |
| CN104984739A (en) * | 2015-06-17 | 2015-10-21 | 武汉汇研生物科技股份有限公司 | Preparing method and application of gelatin affinity chromatography media |
| CN104984739B (en) * | 2015-06-17 | 2018-01-16 | 武汉汇研生物科技股份有限公司 | A kind of preparation method and applications of gelatin affinity chromatography medium |
| CN115887275A (en) * | 2022-11-25 | 2023-04-04 | 广州远想生物科技股份有限公司 | Freeze-dried essence and preparation method thereof |
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