CN1320957C - Method of screening chromatographic stuffing from pearlite - Google Patents
Method of screening chromatographic stuffing from pearlite Download PDFInfo
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- CN1320957C CN1320957C CNB021170088A CN02117008A CN1320957C CN 1320957 C CN1320957 C CN 1320957C CN B021170088 A CNB021170088 A CN B021170088A CN 02117008 A CN02117008 A CN 02117008A CN 1320957 C CN1320957 C CN 1320957C
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- perlite
- distilled water
- protein
- chromatographic
- stuffing
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Abstract
The present invention relates to a method of screening chromatographic stuffing for separating and purifying cell-free whooping cough protein from home-made perlite, which comprises: (1), edible perlite is selected and sieved for obtaining prescreening material with the granularity of 125 to 250 mu m; (2), the prescreening material and added distilled water of the step (1) are weighed; the addition quantity of the distilled water has the standard that the perlite is sufficiently suspended; then, the distilled water is rested for 2 to 5 minutes for discarding upper floaters; (3), the perlite obtained in the step (2) is used as stuffing, is further packed in a column, and screened out; a cylinder of a disposable syringe is used as a chromatographic column; the bottom is uniformly padded by a synthetic fiber; 1 ml of perlite is placed as a column bed; after the perlite is placed in the column, the distilled water is added; when flow speed is larger than 5 to 10 second/drip, the perlite is discarded again; the rest is chromatographic stuffing for separating and purifying cell-free whooping cough protein. Home-made perlite of raw material used in the method has the advantages of sufficient material sources and low price. The method has the advantages of simplicity, convenience, rapidness, time saving, labor saving and energy saving.
Description
Technical field
The present invention relates to screen perlitic method, particularly relate to the method for from pearlite, screening the chromatographic stuffing that is applicable to separation and purification acellular pertussis protein.
Technical background
Perlite is the natural peral rock that volcanic action, volcanism forms, and rapid expansion forms expanded perlite more than 20 times under 1100 ℃ of high temperature, and through unique processing, the technology calcining is pulverized manufacturings such as grinding and selection by winnowing and formed.It is rich in and remains silent and open pore (as Fig. 3), has that unit weight is light, thermal conductivity factor is little, an expanded perlite of nontoxic, tasteless, aseptic, odorless, is a kind of new type filter aid.It is widely used in the liquid filtering in the industrial production such as beer, beverage, monosodium glutamate, medicine, edible oil, oil, chemical industry, sewage disposal.As: Paracell is maximum in the world filter aid manufacturer, and company of Greif section (Grefco Inc) and No. three metal mining industry Co., Ltd. of Japan have developed perlitic product cooperatively.The many producers of China, for example: the packet header of Inner Mongol, Chifeng, Xinyang, Henan Province etc., some manufacturer has made perlite become first-class in the world product through studying for many years and reforming, is the desirable substitute of diatomite.Perlite is as filter aid, in application such as Inner Mongol brewery and packet header brewery and North China medicine company groups.
Summary of the invention
The object of the invention is in order to save foreign exchange, is raw material with the pearlite, therefrom filters out the method for the chromatographic stuffing that is used for separation and purification acellular pertussis protein; Provide a kind of and save time, laborsaving, the energy-conservation method of from perlite, screening chromatographic stuffing.
The object of the present invention is achieved like this: method of screening the chromatographic stuffing that is used for separation and purification acellular pertussis protein from perlite provided by the invention may further comprise the steps and screens:
(1) at first select to meet the edible perlite that detects index and sieve, obtaining granularity is the primary dcreening operation material at 125-250 μ m;
(2) take by weighing the primary dcreening operation material that step (1) obtains, add distilled water, the distilled water addition left standstill 2-5 minute afterwards so that perlite fully suspends is as the criterion, and selected the perlite material of sedimentation, discarded the top floating thing;
(3) perlite that step (2) is obtained is further adorned the post screening, syringe with disposable syringe is chromatographic column (1*5cm), evenly fill up with synthetic fibers the bottom, the perlite that distilled water suspends is adorned post, bed volume is 1ml, distilled water flows through perlitic flow velocity and is discarded again greater than the 5 seconds/perlite that drips then, and remaining perlite is can be as the chromatographic stuffing of separation and purification acellular pertussis protein.
In order better to be used for the chromatographic stuffing of separation and purification acellular pertussis protein, can be to chromatographic stuffing, further screening also comprises:
Step (4) in the post of step (3) with 4 kinds of PH8.0 Tris-Hcl buffer solutions (containing NaCl), stepwise elution in order: will get acellular pertussis protein solution 1ml, being added to the pearlite that step (3) handles well is in the chromatographic column of filler, the nature flow velocity, collect outflow liquid with the 10ml test tube, after sample flows to end, the outflow liquid of collecting with the Tris-Hcl buffer solution (containing NaCl) of following 4 kinds of PH8.0 stepwise elution in order, test tube with the number of finishing is collected eluent, and every pipe is collected 1ml;
The Tris-Hcl buffer solution that described 4 kinds of PH8.0 contain NaCl is as follows:
1.25mM Tris-Hcl buffer solution 5ml
2.50mM the Tris-Hcl buffer solution contains 0.2M NaCl 5ml
3.50mM the Tris-Hcl buffer solution contains 0.6M NaCl 5ml
4.50mM the Tris-Hcl buffer solution contains 1.0M NaCl 5ml.
Step (5) is surveyed the UV absorption A280 of eluent protein: each pipe that obtains from step (4) is collected 1ml eluent protein, carries out ultraviolet absorptivity and measure on ultraviolet specrophotometer, and this protein has absorbance A at the 280nm place
280, the record reading.With A
280Numerical value be ordinate, the pipe number of collecting eluent is abscissa mapping, the effect of separating as can be seen in the drawings.Those are not opened Separation of Proteins with wash-out the time flow velocity slow perlite be eliminated again.Flow velocity perlite fast, good separating effect is a chromatographic stuffing when selecting wash-out for use, separating resulting as shown in Figure 1, as can be seen from Figure 1, the acellular pertussis protein that pearlite separates has 4 absworption peaks.Peak 1, peak 2, peak 3, peak 4.
Also comprise and carry out the purity that electrophoresis is identified isolated protein: the eluent of each albumen absworption peak is collected in one respectively in the step 4, with the pure pertussis protein of standard is contrast, and the sample at the peak that obtains 1 to peak 4 carries out the purity that the SDS-polyacrylamide gel is identified isolated protein.The results are shown in Figure 2, as can be seen from Figure 2, peak 1 contains 69k and some heteroproteins, and peak 2, peak 3 are respectively PT and FHA.Wherein the lipidated protein at peak 2, peak 3 reaches more than 80%, does not contain foreign protein substantially.Peak 4 is the heteroproteins of separating, and discards not, according to effect of separating purification and elution speed, determines which kind of perlite is as chromatographic stuffing.The purity of identifying isolated protein with electrophoresis proves protein 69k, and PT and FHA are the Main Ingredients and Appearances of pertussis protein, have also proved the feasibility and the reliability of this method.
And can do following separation and purification acellular pertussis experimental protein and test:
1. survey the effect of hemagglutination activity check pearlite separation and purification pertussis protein.
2. identify the purity of isolated protein with the SDS-polyacrylamide gel.
3. according to effect of separating purification and elution speed, determine which kind of perlite is as chromatographic stuffing.
Advantage of the present invention:
Provided by the invention filtering out from pearlite is used for the method that separation and purification acellular pertussis protein is done chromatographic stuffing, raw materials used pearlite ample supply and prompt delivery, 1.5-3.0 yuan of low price per kilogram, the per kilogram perlite is 6 dollars abroad.This method is easy to save time fast, laborsaving, energy-conservation.The pearlite stable performance, being used for separation and purification acellular pertussis protein, to do chromatographic stuffing easy to operate.
Description of drawings
Fig. 1 is the collection of illustrative plates of pearlite separation and purification pertussis protein
Peak 1. contains the heteroproteins chromatographic column outflow liquid of 69k among the figure;
Peak 2.PT; Peak 3.FHA; The heteroproteins that peak 4. separates;
The SDS-PAGE figure of Fig. 2 perlite separation and purification pertussis protein
Among the figure: 1. peak 1 sample among sample protein 2. Fig. 1 on the chromatographic column
3. peak 3 samples, 5. standard samples among peak 2 samples 4. Fig. 1 among Fig. 1
6. peak 4 samples among Fig. 1
The electron microscope photo scanning of Fig. 3 chromatographic stuffing pearlite (amplifying 500 times photo)
The specific embodiment
Embodiment 1:
Get and contain SiO
270-80%, Al
2O
3The homemade edible class perlite of 10-20% with the different sieve in aperture, is got granularity and is respectively the primary dcreening operation material at the perlite material of 125 μ m;
Pearlite 1g with obtaining in the step 1 puts into the 25ml beaker respectively, adds 20ml distilled water, after stirring, leaves standstill 2-5 minute, discards the top floating thing, and selecting the fast perlite of sinking speed is material, further adorns the post screening; Syringe with disposable syringe is a chromatographic column, evenly fill up with synthetic fibers the bottom, the perlite 1ml that packs into, flow velocity is discarded again greater than the perlite of 5-7 second/drip during the dress post, and remaining is can be as the chromatographic stuffing of separation and purification acellular pertussis protein.
Embodiment 2:
The chromatographic stuffing that is used for separation and purification acellular pertussis protein that filters out from pearlite that obtains with embodiment 1 carries out the experiment of separation and purification acellular pertussis protein:
With ready acellular pertussis protein solution 1ml, being added to the pearlite of handling well among the embodiment 1 is in the filler chromatographic column, the nature flow velocity, collect outflow liquid with the 10ml test tube, after sample flows to end, flow out liquid and be with the described 4 kinds of buffer solutions of PH8.0 Tris-Hcl buffer solution (containing NaCl): stepwise elution in order, with the test tube collection eluent of the number of finishing, every pipe is collected 1ml, carries out separation and purification acellular pertussis protein.Described buffer solution is:
1.25mM Tris-Hcl buffer solution 5ml;
2.50mM the Tris-Hcl buffer solution contains 0.2M NaCl 5ml;
3.50mM the Tris-Hcl buffer solution contains 0.6M NaCl 5ml;
4.50mM the Tris-Hcl buffer solution contains 1.0M NaCl 5ml;
Embodiment 3:
Survey the UV absorption A280 of eluent protein: what obtain from embodiment 2 respectively manages eluent, in the absorbance A of ultraviolet specrophotometer 280nm place mensuration protein
280, the record reading.With A
280Numerical value be ordinate, the pipe number of collecting eluent is abscissa mapping, the effect of separating as can be seen in the drawings.Those are not opened Separation of Proteins with wash-out the time flow velocity slow perlite be eliminated again.Flow velocity perlite fast, good separating effect is a chromatographic stuffing when selecting wash-out for use, separating resulting as shown in Figure 1, and as can be seen from Figure 1, the acellular pertussis protein that pearlite separates has 4 absworption peaks.Peak 1, peak 2, peak 3, peak 4.
Embodiment 4:
Get and contain SiO
270-80%, Al
2O
3The homemade edible class perlite of 10-20% with the different sieve in aperture, is got granularity and is done the primary dcreening operation material at the perlite material of 250 μ m;
Pearlite 1g with obtaining in the step 1 puts into the 25ml beaker respectively, adds 20ml distilled water, after stirring, leaves standstill 2-5 minute, discards the top floating thing, and selecting the fast perlite of sinking speed is material, further adorns the post screening; Syringe with disposable syringe is a chromatographic column, evenly fill up with synthetic fibers the bottom, the perlite 1ml that packs into, flow velocity is discarded again greater than the perlite of 5-7 second/drip during the dress post, and remaining is further to screen as the chromatographic stuffing of separation and purification acellular pertussis protein.
With ready acellular pertussis protein solution 1ml, being added to the pearlite of handling well among the embodiment 1 is in the filler chromatographic column, the nature flow velocity, collect outflow liquid with the 10ml test tube, after sample flows to end, flow out liquid and be with the described 4 kinds of buffer solutions of PH8.0 Tris-Hcl buffer solution (containing NaCl): stepwise elution in order, with the test tube collection eluent of the number of finishing, every pipe is collected 1ml, carries out separation and purification acellular pertussis protein.Described buffer solution is:
1.25mM Tris-Hcl buffer solution 10ml;
2.50mM the Tris-Hcl buffer solution contains 0.2M NaCl 5ml;
3.50mM the Tris-Hcl buffer solution contains 0.6M NaCl 5ml;
4.50mM the Tris-Hcl buffer solution contains 1.0M NaCl 5ml;
Embodiment 5:
Electrophoresis is identified the purity of isolated protein: the eluent of each albumen absworption peak is collected in one respectively among the embodiment 3 and 4, is contrast with the pure pertussis protein of standard, and the sample at peak 1 to peak 4 carries out the SDS-polyacrylamide gel electrophoresis to be identified, the results are shown in Figure 4.As can be seen from Figure 2, peak 1 contains 69k and some heteroproteins, and peak 2, peak 3 are respectively PT and FHA.Wherein the lipidated protein at peak 2, peak 3 reaches more than 80%, does not contain foreign protein substantially.Peak 4 is the heteroproteins of separating, and discards not. protein 69k, PT and FHA are the Main Ingredients and Appearances of pertussis protein.Identify that with electrophoresis the purity of isolated protein has proved the feasibility and the reliability of this method, illustrates that also the granularity of perlite chromatographic stuffing is applicable to separation and purification acellular pertussis protein at 125-250 μ m.
Embodiment 6: electron-microscope scanning
Separation and purification acellular pertussis protein, the perlite chromatographic stuffing of fast, the good separating effect of flow velocity is fixed on the electron microscopic sample rake ion sputtering gold, the S-570 of Hitachi during wash-out
Scanning electron microscopic observation, and take a picture, the results are shown in Figure 3.As can be seen from Figure 3, the pearlite surface imperfection of screening, porous.
Claims (1)
- One kind from perlite the screening chromatographic stuffing method, it is characterized in that: carry out according to the following steps:(1) at first select to meet the edible perlite that detects index and sieve, obtaining granularity is the primary dcreening operation material at 125-250 μ m;(2) take by weighing the perlite primary dcreening operation material that step (1) obtains, add distilled water, the distilled water addition left standstill 2-5 minute afterwards so that perlite fully suspends is as the criterion, and selected the perlite material of sedimentation;(3) perlite that step (2) is obtained is further adorned the post screening, syringe with disposable syringe is a chromatographic column, evenly fill up with synthetic fibers the bottom, the 1ml perlite of packing into is the post bed, add distilled water behind the dress post, flow velocity by perlite water is discarded again greater than the 5 seconds/perlite that drips, the chromatographic stuffing that remaining is as separation and purification acellular pertussis protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021170088A CN1320957C (en) | 2002-04-27 | 2002-04-27 | Method of screening chromatographic stuffing from pearlite |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021170088A CN1320957C (en) | 2002-04-27 | 2002-04-27 | Method of screening chromatographic stuffing from pearlite |
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| Publication Number | Publication Date |
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| CN1453064A CN1453064A (en) | 2003-11-05 |
| CN1320957C true CN1320957C (en) | 2007-06-13 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021170088A Expired - Fee Related CN1320957C (en) | 2002-04-27 | 2002-04-27 | Method of screening chromatographic stuffing from pearlite |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103592198A (en) * | 2013-11-28 | 2014-02-19 | 厦门大学 | Determination method for polar components in edible oil |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1097350A (en) * | 1993-07-13 | 1995-01-18 | 河北省崇礼县助滤剂厂 | Pearlite filter aid |
| CN1137563A (en) * | 1995-06-06 | 1996-12-11 | 屠勇 | Composite pearlstone filter aid for beer |
| KR20020026332A (en) * | 2002-03-12 | 2002-04-09 | 장현태 | Adsorbent for the removal of odor using silerlite and perlite |
-
2002
- 2002-04-27 CN CNB021170088A patent/CN1320957C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1097350A (en) * | 1993-07-13 | 1995-01-18 | 河北省崇礼县助滤剂厂 | Pearlite filter aid |
| CN1137563A (en) * | 1995-06-06 | 1996-12-11 | 屠勇 | Composite pearlstone filter aid for beer |
| KR20020026332A (en) * | 2002-03-12 | 2002-04-09 | 장현태 | Adsorbent for the removal of odor using silerlite and perlite |
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| Publication number | Publication date |
|---|---|
| CN1453064A (en) | 2003-11-05 |
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