CN1552726A - Purification of tetramucoprotein as protein label for early dianosis of oophoroma - Google Patents
Purification of tetramucoprotein as protein label for early dianosis of oophoroma Download PDFInfo
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- CN1552726A CN1552726A CNA031364896A CN03136489A CN1552726A CN 1552726 A CN1552726 A CN 1552726A CN A031364896 A CNA031364896 A CN A031364896A CN 03136489 A CN03136489 A CN 03136489A CN 1552726 A CN1552726 A CN 1552726A
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Abstract
A process for purifying the tetramucoprotein as the protein marker for the early diagnosis of ovary cancer includes such steps as primary separating by saturated ammonium sulfate deposition method, medium purifying by ion exchange chromatography and affinity chromatography, and fine purifying by polyacrylamide gel electrophoresis method. Its advantages are high purity, high activity and high specificity.
Description
The invention belongs to medical science and field of biology, particularly, the present invention relates to a kind of purification process of oophoroma early diagnosis protein marker TN.
At present the sickness rate of ovarian cancer in China's gynecological cancer accounts for the 3rd and rise to first by the eighties.Ovarian cancer is the malignant tumour that curative ratio is minimum in the gynecological tumor, mortality ratio is the highest in worldwide always.Ovarian cancer is difficult for being found in early days, has the ovarian cancer first visit patient more than 80% to reach an advanced stage (III phase or IV phase) approximately, even operation at once, the postoperative five year survival rate is also only less than 15%.Because the pathogeny of ovarian cancer is not still asked Chu so far, therefore be difficult to take proper prophylactic methods.In order to reduce the harm of ovarian cancer, key is still found in early days, early treatment.At present clinically mostly with the diagnosis index of carbohydrate antigen 125 (CA125) single index as ovarian cancer.Because CA125 also is used for the detection of other malignant tumour, be not specific index therefore to ovarian cancer; And CA125 only has rising in the serous ovarian cancer patients serum, to the then not reaction of non-serous ovarian cancer, especially mucous ovarian cancer.CA125 is also insensitive to early ovarian cancer.
A kind of protein marker TN (Tetranectin is called for short TN) can obviously change when ovarian cancer takes place, and can solve and use the deficiency of individual event CA125 as the ovarian cancer diagnosis index at present clinically.TN is the tetramer protein that a molecular weight is 68KD, and iso-electric point is 5.8.The ovarian cancer that is reduced in of ovarian cancer patients serum TN level is in I phase or II and just occurs during the phase, and the classification of this reduction and ovarian cancer is proportionate.The reduction of ovarian cancer patients serum TN level not only comprises serous ovarian cancer, also comprises mucous ovarian cancer.The key of preparation TN detection kit is to prepare the pure product of TN earlier, can not buy yet still have the pure product of TN on the present domestic and international market.Purpose of the present invention in order to fill up this blank, provides a kind of purification process of oophoroma early diagnosis protein marker just.Obtaining to carry out the making of TN Antibody Preparation and detection kit behind the pure product of TN, be applied to improve clinically the rate that picks of ovarian cancer, the recall rate of the recall rate of early ovarian cancer and mucous ovarian cancer particularly, so that can take effective comprehensive therapeutic plan as early as possible, reach the purpose that improves survival rate, reduces mortality ratio.
Therefore, the purification process that the purpose of this invention is to provide a kind of ovarian cancer protein mark TN (Tetranectin, i.e. TN).
The pure product of TN that use the inventive method to obtain can be used for TN polyclonal antibody or MONOCLONAL ANTIBODIES SPECIFIC FOR, and the preparation of plurality of reagents boxes such as ELISA (enzyme linked immunoassay mensuration) test kit, RIA (radioimmunoassay) test kit, chemical luminescence reagent kit.These detection kit can be used for good, the virulent diagnosis of ovarian tumor clinically, and the particularly detection of the detection of early ovarian cancer and mucous ovarian cancer also can be used for the monitoring and the prognosis evaluation of ovarian cancer post operation chemotherapy.
Above-mentioned purpose of the present invention is implemented by following technical proposal:
Be used to prepare a kind of separation purification method of oophoroma early diagnosis protein marker TN, it is characterized in that: it is made up of the proteic roughing out of TN, moderate purifying and three key steps of polishing purification; It has also comprised the authentication method of the pure product of TN.
1.TN proteic roughing out process using saturated ammonium sulphate method, it mainly comprises following two steps: a) saturated ammonium sulphate: add the physiological saline of equivalent in blood plasma, add solid ammonium sulfate to 50% saturation ratio more in proportion; B) the pH value is adjusted: treat after protein is separated out the pH of solution to be transferred to 5.8; Treat to carry out after precipitation process finishes centrifugal, collecting precipitation, dialysis desalination promptly obtain TN albumen crude product.
2.TN proteic moderate purifying process mainly comprises ion exchange chromatography and two steps of affinity chromatography, the order of two kinds of chromatographic step can be changed.
A) ion exchange chromatography:, select the weakly-basic anion displacement chromatography for use according to the charged characteristic of TN albumen.The optional free radical of anionite group is that dextran (Sephadex), agarose (Sepharose) or cellulosic commodity are (such as Pharmacia company, Carl-Schloicher ﹠amp for diethylamino ethyl (DEAE), medium; Schuell company or Shanghai organic chemistry institute etc.).Adopting model in the inventive method is the ion-exchange gel (Pharmacia company product) of DEAE Sepharose Fast Flow.Irritate post according to a conventional method, link with protein purification instrument (Pharmacia company product or other company's analogous products).Adopting model in the inventive method is AKDA Series P urifier 100 protein purification instrument (Pharmacia company products).Step 1 gained TN albumen crude product is carried out chromatography with an amount of chromatography damping fluid dissolving back upper prop, and the chromatography damping fluid can be three carboxymethylamino methane-hydrochloric acid (Tris-HCl) damping fluids, phosphoric acid salt (PBS) damping fluid or other damping fluid of different concns, different PH; The chromatography damping fluid of selecting for use in the inventive method is a 0.02M Tris-HCl damping fluid (PH7.2).Elution process can adopt the NaCl linear gradient elution method or adopt NaCl linear elution method; Select for use the NaCl gradient to wash method in the inventive method.Flow velocity is generally 1～3ml/min; Select flow velocity 3ml/min in the inventive method for use.Monitor with ultraviolet absorption method at the eluted protein peak, and the antigen-antibody reaction method is adopted in the determination of activity of each protein peak.Collection has active target protein peak, and is standby through desalination, after concentrating.
B) affinity chromatography: all have the characteristic of good affinity according to TN albumen and heparin (Heparin) and Methionin (Lysine), can select heparin affinity chromatography or Methionin affinity chromatography for use.The affinity chromatography gel can be bought (such as Pharmacia company, Bio-Rad company etc.).The chromatography damping fluid can be Tris-HCl damping fluid, PBS damping fluid or other damping fluid of different concns, different PH.Use HeparinSepharose 6 Fast Flow or Lysine Sepharose 4B affinity chromatography gel (being Pharmacia company product) in the inventive method, the chromatography damping fluid is respectively 0.02M Tris-HCl damping fluid or the PBS damping fluid of PH7.2, and flow velocity is 1ml/min; The collection at the monitoring of elution process, protein peak and determination of activity, target protein peak and treatment process all with step 2a) identical.
3.TN proteic polishing purification process using polyacrylamide gel electrophoresis, it comprises that mainly protein denaturation, electrophoresis, target protein reclaim three steps:
A) protein denaturation: will carry out sex change routinely from the TN albumen that step 2 obtains the moderate purifying, be the monomer that the tetramer of 68KD is reduced to 17KD with TN albumen by molecular weight.Working method is as follows: add the phosphoric acid buffer contain 1%SDS (sodium laurylsulfonate), 1% mercaptoethanol in right amount (0.01M, PH7.2), standby after 2～5 minutes 100 ℃ of heating;
B) electrophoresis: can select the slab electrophoresis groove of home-made or import and supporting glue mould for use, use the slab electrophoresis groove of Bio-Rad company and supporting glue mould in the inventive method.Select the polyacrylamide gel of 7.5% or 10% concentration for use according to the proteic molecular weight of TN; Prepare gel buffer liquid, electrode buffer and sample buffer routinely, and the glue program is carried out glue, application of sample, electrophoresis routinely; Electrophoresis finishes the back to be got glue, fixes, dyes and decolouring according to routine, and a visible tangible band is the pure product of TN albumen in the position of 17KD;
C) target protein reclaims: the adhesive tape at 17KD place is carefully downcut, carry out the albumen wash-out, use 422 Electro-Eluter electroelution instrument (Bio-Rad company) in the inventive method with the electroelution instrument of home-made or import.The electroelution damping fluid is the carbonate buffer solution (concentration is 50mM) that contains 0.1%SDS; Steps such as schedule of operation comprises broken glue, the pre-treatment of film cap, add gel particle and damping fluid in the wash-out pipe, electroelution.After treating the electroelution EP (end of program), the solution in the collection membrane cap promptly obtains the pure product of TN.
4.TN the evaluation of pure product comprises following content:
A) protein content identify to adopt the Bradford method, and key step comprises: the preparation ultimate density is 0.01% to examine the protein reagent of Ma Shi light blue, 4.7% ethanol and 8.5% phosphoric acid, and get an amount of this protein reagent with in right amount by step 3c) the pure product of gained TN mix; Prepare standard protein solution, promptly prepare bovine serum albumin (BSA) solution of 5 different concns, add an amount of protein reagent and mixing respectively; Above-mentioned all solution are respectively got each hole of 0.2ml adding microwell plate, and reaction finishes the back and is worth in 595nm place mensuration photoabsorption (OD) on the uv-spectrophotometric instrument; With the different concns value of BSA solution as X-coordinate, corresponding OD value as ordinate zou drawing standard curve; By the measured OD value of the pure product solution of TN to be measured, find its protein content from typical curve.The protein content of the pure product of TN that obtained by the inventive method separation and purification is greater than 2.5mg/ml.
B) purity and molecular weight identification adopt the SDS-PAGE gel electrophoresis, and key step comprises: prepare the gel liquid storage routinely and prepare 10% gel; Prepare sample buffer routinely, and prepare by step 3c with this sample buffer) the pure product of gained TN, and the protein standard substance; According to the proteic molecular weight of TN, molecular weight protein matter standard substance in using in the inventive method, its molecular weight ranges is 14～97KD; Carry out electrophoresis according to a conventional method, comprise the preparation electrode buffer, prepared gel is put into electrophoresis chamber, protein sample sex change, added sample, electrophoresis; Electrophoresis process is got glue, dyeing, decolouring after finishing routinely.Measure the migration distance of each sample on gel, and calculate relative mobility according to formula; With each proteinic molecular weight in the protein standard substance is that the logarithm of ordinate zou, corresponding relative mobility is an X-coordinate drawing standard curve; According to the resulting relative mobility of TN albumen to be measured, find its molecular weight from typical curve.The molecular weight of the pure product of reduced form TN that obtained by the inventive method separation and purification is 17KD, conforms to document.With gel imaging instrument gel is scanned and analyzes simultaneously, can obtain the purity of the pure product of TN to be measured.The purity of protein of the pure product of TN that obtained by the inventive method separation and purification is greater than 95%.
C) iso-electric point (PI) is identified the polyacrylamide isoelectric focusing electrophoresis method that adopts, key step comprises: handle sheet glass, preparation glue-filling slot, preparation gelating soln, encapsulating, application of sample (comprising the protein standard substance of known PI value, the pure product of TN to be measured), electrophoresis with silicone oil, and fixing, the dyeing of gel and decolouring; Albumen with known PI value is standard, according to they residing positions behind electrophoresis, with the PI value be ordinate zou, respective distance on gel is that X-coordinate is drawn the PI gradient curve; According to TN albumen to be measured residing position behind electrophoresis, find its iso-electric point from gradient curve.The iso-electric point of the pure product of TN that obtained by the inventive method separation and purification is 5.8, conforms to document.
D) activity identification adopts the antigen-antibody reaction method, and key step comprises: with TN antibody sandwich micropore enzyme plate, antibody concentration is the 2ug/ hole; The pure product solution of TN albumen to be measured that adds different concns was 37 ℃ of water bath heat preservations 60 minutes; After treating that antigen-antibody reaction is finished, add an amount of TN antibody again and carry out integrated enzyme reaction with horseradish peroxidase (HRP) mark; TN antibody and HRP enzyme mark TN antibody can be bought (such as, DAKO company); And then develop the color, colorimetric and interpretation of result; The OD that records at the 450nm place according to the pure product of TN albumen to be measured of different concns is worth the activity of the pure product of TN albumen to be measured.The activity of the pure product of TN that obtained by the inventive method separation and purification is: concentration is that the pure product of the TN of 2.4ng/ml promptly have the activity that obvious antigen-antibody reaction takes place with 2ug TN antibody.
E) specificity is identified and is adopted the Western-Blot method, and key step comprises: the elementary operation of SDS-PAGE gel electrophoresis and step 4b) basic identical, on same clotting glue, do a application of sample that repeats when noting application of sample by the application of sample order; Preparation electrotransfer damping fluid is that nitrocellulose filter (NC film), Whatman Lu paper (specification 3mm) of 0.45um is made into " gel sandwich " with running gel with the aperture; " gel sandwich " inserted carry out electrophoresis in the electrophoresis chamber that fills the electrotransfer damping fluid; Electrophoresis finishes half NC film substantive dyeing to show zone of protein; Second half NC film that has the repetition application of sample places the TN antibody-solutions of dilution slowly to shake 2 hours after damping fluid flushing, the sealing of bovine serum albumin (BSA) solution; Add an amount of HRP enzyme mark second antibody again, continue reaction 2 hours; The NC film contrasts through half NC film of flushing, colour developing back and substantive dyeing, can learn the specificity of TN sample to be measured.Identify tangible specific combination district band is arranged by the pure product of TN that the inventive method separation and purification obtains through the Western-Blot method in 17KD position and TN antibody.
The inventive method is compared with existent method and is had the following advantages:
1. in the saturated ammonium sulphate technology that when TN albumen crude product separates, adopts, increased the operation steps that pH value is adjusted, made target protein TN when its iso-electric point (PH5.8), have minimum solubleness, therefore can more fully precipitate.In the foreign literature, also be only consider in the general saturated ammonium sulphate operation steps add the per-cent of saturated ammonium sulphate, and often ignored the pH value of reaction system and the gap between the target protein iso-electric point.The inventive method has overcome this shortcoming, has prevented losing of target protein effectively, has the higher rate of recovery;
2. the inventive method is more convenient than method of the prior art.Adopted heparin affinity chromatography and Methionin affinity chromatography in the affinity chromatography technology of TN albumen moderate purifying process, what replaced that the former K4 ring of the blood fibrinolytic protein lyase that adopts in existing document affinity chromatography brings is loaded down with trivial details and inconvenient.Because the former K4 ring of blood fibrinolytic protein lyase affinity chromatography medium must prepare voluntarily, and heparin affinity chromatography and Methionin affinity chromatography medium can be bought, (such as Phamacia company);
3. the present invention has increased the polishing purification step, improved the purity of the pure product of TN by polyacrylamide gel electrophoresis, overcome the not high enough shortcoming of purity of the pure product of TN that obtain with prior art, make and to guarantee when using the pure product of this TN to go to prepare antibody, can obtain the high antibody of specificity, thereby reduce the influence of the nonspecific reaction that may exist in the ELISA detection method, improve the specificity and the accuracy of detection kit.
Further specify the present invention with embodiment below.It should be understood that embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.Except as otherwise noted, the percentage ratio among the present invention is weight percentage.
Embodiment one: a kind of ovarian cancer protein mark TN purification process
Be used to prepare a kind of separation purification method of oophoroma early diagnosis protein marker TN, it is characterized in that: it is made up of the proteic roughing out of TN, moderate purifying and three key steps of polishing purification.
1.TN proteic one step of roughing out process using saturated ammonium sulphate method, it mainly comprises following two steps:
(1) saturated ammonium sulphate: get the 50ml human plasma, nylon wire adds equivalent physiological saline, mixing after crossing the Lu; Add 31.3g solid ammonium sulfate to 50% saturation ratio again; Rotated 1 hour on magnetic stirring apparatus in 4 ℃;
(2) pH value adjustment: treat that protein separates out the back and with 4N hydrochloric acid the PH of solution is transferred to 5.8, continue at 4 ℃ and on magnetic stirring apparatus, rotated 1 hour; Treat precipitation process finish the back in 4 ℃ at 12000rmp centrifugal 30 minutes, collecting precipitation; With 0.02M phosphoric acid buffer (PH7.2) dissolution precipitation, the dress dialysis tubing promptly obtains TN albumen crude product in 4 ℃ of dialysis desalinations.
2.TN proteic moderate purifying process mainly comprises DEAE ion exchange chromatography and two steps of Methionin (Lysine) affinity chromatography, the instrument selection model is the protein purification instrument (Pharmacia company product) of " AKDA Series P urifier 100 ".
(1) DEAE ion exchange chromatography:
Select ion-exchange gel: selecting model for use is the weakly alkaline ion-exchange gel (Pharmacia company product) of DEAE Sepharose Fast Flow;
Chromatography column specification: 2.6 * 40cm;
Preparation chromatography damping fluid: 0.02M Tris-HCl damping fluid (PH7.2), 1M NaCl solution;
Elution process: adopt the NaCl linear gradient elution method;
Operation steps is as follows: a) irritate column method routinely the DEAE ion-exchange gel is poured into chromatography column; B) ion exchange column, sample divider and protein purification instrument are linked; C) with the TN albumen crude product of 0.02M Tris-HCl damping fluid dilution by step 1 (2) acquisition, go up sample on the quadrat method routinely, applied sample amount is 15ml; D) with UHICRN V400 software programming, control the chromatography process automatically, comprise the control of NaCl gradient elution and the uv-absorbing monitoring of protein peak, flow velocity is 3ml/min, sample collection 2ml/ pipe; E) measure the activity of each protein peak with the ELISA method, collect and have the active protein peak of TN, the dress dialysis tubing, dialysis desalting, to concentrate the back standby.
(2) Methionin affinity chromatography:
Select affinity gel: selecting model for use is Lysine Sepharose 4B d1 affinity chromatography gel (Pharmacia company product);
Chromatography column specification: 1.6 * 40cm;
Preparation chromatography damping fluid: 0.02M phosphate buffered saline buffer (PH7.2), 1M NaCl solution;
Elution process: adopt the NaCl linear gradient elution method;
Operation steps is as follows: a) irritate column method routinely Lysine affinity chromatography gel is poured into chromatography column; B) affinity column, sample divider and protein purification instrument are linked; C) dilute by the TN protein product of step 2 (1) through the acquisition of DEAE ion exchange column purifying with the 0.02M phosphate buffered saline buffer, add sample on the quadrat method routinely, applied sample amount is 5ml; D) with UHICRN V400 software programming, control the chromatography process automatically, comprise the control of NaCl gradient elution and the uv-absorbing monitoring of protein peak, flow velocity is 1ml/min, sample collection 1ml/ pipe; E) with step 2 (1) e).
3.TN proteic polishing purification process using polyacrylamide gel electrophoresis, it comprises that mainly protein denaturation, electrophoresis, target protein reclaim three steps:
(1) protein denaturation: the preparation contain 2%SDS, 2% mercaptoethanol phosphoric acid buffer (0.02M, PH7.2); Get this damping fluid 0.5ml, add the moderate purifying TN albumen 0.5ml that is obtained from step 2 (2), mixing; Heating is standby after 3 minutes in 100 ℃ of water-baths, and the proteic concentration of TN this moment is 5ug/ul;
(2) electrophoresis: electrophoresis chamber is the slab electrophoresis groove, and instrument uses Bio-Rad company electrophoresis apparatus, and operation steps is as follows:
A. prepare phosphoric acid buffer (0.2M, PH7.2): get 0.2M sodium dihydrogen phosphate 280ml, add 0.2M disodium phosphate soln 720ml, behind the mixing PH is transferred to 7.2;
B. prepare the gel liquid storage: acrylamide (Acr) 30g, methylene-bisacrylamide (Bis) 0.8g, distilled water 100ml;
C. prepare sample buffer: get sodium lauryl sulphate (SDS) 100mg, mercaptoethanol 0.1ml, glycerine 1ml, bromine phenol basket 2mg, phosphoric acid buffer 0.5ml, adding distil water is to 10ml;
D. prepare gel buffer liquid: take by weighing SDS 0.2g, add phosphoric acid buffer 100ml;
E. prepare electrode buffer: take by weighing SDS 1.0g, add phosphoric acid buffer 500ml, adding distil water is to 1000ml;
F. prepare staining fluid: take by weighing 0.25g Xylene Brilliant Cyanine G R-250, add 454ml 50% methyl alcohol and 46ml glacial acetic acid;
G. prepare destainer: 50ml methyl alcohol, 75ml glacial acetic acid are mixed with 875ml distilled water.
H. the selection of protein molecular weight standard product: molecular weight protein matter standard substance (Bio-Rad product) in selecting for use, molecular weight ranges 14～97KD;
I. sample well template (comb) specification is 2 * 10mm;
J. operation steps is as follows: a) 10% preparing gel: get gel liquid storage 6.67ml, add gel buffer liquid 10ml, 1%TENED (Tetramethyl Ethylene Diamine) 2ml, distilled water 1.23ml, this mixed solution need outgas 10 minutes; Add freshly prepared 10% ammonium persulphate 0.1ml; Be added to immediately behind the mixing in the gel slab mould, polymerization needed finish in 30 minutes approximately; B) preparation of sample: TN protein product after the sex change and protein standard substance are mixed with the equivalent sample buffer respectively, standby; C) application of sample: take out comb, with electrode buffer flushing gel sample hole, every hole application of sample 20ul; D) electrophoresis: connect power supply, electric current is transferred to 50～80mA, treat that the blue dyes in the sample migrates to apart from the about 1cm in gel lower end place, stop electrophoresis.E) dyeing and decolouring: get glue, fix, dye and decolouring according to routine, a visible tangible band is the pure product of TN albumen in the position of 17KD;
(3) target protein reclaims: the adhesive tape at 17KD place is carefully downcut, and the wash-out that carries out target protein with 422 Electro-Eluter electroelution instrument (Bio-Rad company) reclaims.Main operational steps is as follows: a) preparation electroelution damping fluid: (50mM, the carbonate buffer solution of PH9.4): get 0.1mM sodium carbonate solution 10ml, add 0.1mM sodium hydrogen carbonate solution 40ml, with distilled water 50ml mixing; B) broken glue: use ophthalmology point tweezers that the careful folder of the adhesive tape of downcutting is broken, make it to become the gel particle of 1mm size; C) film cap pre-treatment: need before the film cap uses in the electroelution damping fluid, to soak 1 hour; D) the wash-out pipe is installed: fill it up with the electroelution damping fluid in the film cap, and the translation of wash-out pipe is installed on the film cap; E) electroelution: gel particle and an amount of electroelution damping fluid are put into the wash-out pipe, and wash-out pipe jack is put into electrophoresis chamber with the wash-out device, connects power supply and carries out electroelution, constant current 5～6 hours.F) albumen reclaims: after treating the electroelution EP (end of program), reclaim electroelution solution and can obtain the pure product of TN.
Embodiment two: a kind of ovarian cancer protein mark (Tetranectin, TN, TN) purification process
(separation purification method TN) is characterized in that for Tetranectin, TN: it is made up of the proteic roughing out of TN, moderate purifying and three key steps of polishing purification to be used to prepare a kind of oophoroma early diagnosis protein marker.
1.TN proteic two step of roughing out process using saturated ammonium sulphate method, it mainly comprises following two steps:
(1) two step saturated ammonium sulphate: a) the first step: get the 50ml human plasma, nylon wire adds equivalent physiological saline after crossing the Lu; Add 22.8g solid ammonium sulfate to 30% saturation ratio behind the mixing earlier; Rotated 1 hour on magnetic stirring apparatus in 4 ℃; Leave the heart 30 minutes in 4 ℃ of per minutes 12000, collect supernatant liquor; B) second step: add 13.1g solid ammonium sulfate to 50% saturation ratio again; Rotate half an hour on magnetic stirring apparatus in 4 ℃;
(2) pH value adjustment: identical with embodiment one step 1 (2).
2.TN proteic moderate purifying process mainly comprises heparin (Heparin) affinity chromatography and two steps of DEAE ion exchange chromatography.Instrument selection " AKDA Series P urifier 100 " protein purification instrument (Pharmacia company).
(1) heparin affinity chromatography:
Select affinity gel: select Heparin Sepharose 6 Fast Flow affinity chromatography gels (Pharmacia company product) for use;
Chromatography column specification: 1.6 * 40cm;
Preparation chromatography damping fluid: 0.02M Tris-HCl damping fluid (PH7.2), 1M NaCl solution;
Elution process: adopt the NaCl linear gradient elution method;
Operation steps is as follows: a) irritate column method routinely Heparin affinity chromatography gel is poured into chromatography column; B) affinity column, sample divider and protein purification instrument are linked; C) with the TN albumen crude product of 0.02M Tris-HCl damping fluid dilution by step 1 (2) acquisition, go up sample on the quadrat method routinely, applied sample amount is 5ml; D) identical with embodiment one step 2 (2) d; E) with embodiment one step 2 (2) e) identical.
(2) DEAE ion exchange chromatography:
Select ion-exchange gel: selecting model for use is the ion-exchange gel (Pharmacia company product) of DEAE Sepharose Fast Flow;
Chromatography column specification: 2.6 * 40cm;
Preparation chromatography damping fluid: 0.02M Tris-HCl damping fluid (PH7.2), 1M NaCl solution;
Elution process: adopt the NaCl linear gradient elution method;
Operation steps is as follows: a) irritate column method routinely the DEAE ion-exchange gel is poured into chromatography column; B) ion exchange column, sample divider and protein purification instrument are linked; C) with the TN protein product of 0.02M Tris-HCl damping fluid dilution by the acquisition of step 2 (1) process Heparin affinity chromatography column purification, go up sample on the quadrat method routinely, applied sample amount is 15ml; D) with embodiment one step 2 (1) d) identical; E) with embodiment one step 2 (1) e) identical.
3.TN proteic polishing purification process using polyacrylamide gel electrophoresis, it comprises that mainly protein denaturation, electrophoresis, target protein reclaim three steps: electrophoresis chamber adopts slab electrophoresis groove instrument, uses Bio-Rad company electrophoresis apparatus.
(1) protein denaturation: identical with embodiment one step 3 (1);
(2) electrophoresis: electrophoresis chamber adopts slab electrophoresis groove instrument, uses Bio-Rad company electrophoresis apparatus.
A. prepare Tutofusin tris-hydrochloric acid (Tris-HCl) damping fluid (0.1M, PH7.2): get 0.2M Tris solution 50ml, add 0.2M hydrochloric acid soln 22.5ml, adding distil water transfers to 7.2 with PH to 100ml behind the mixing;
B. prepare sample buffer: get sodium laurylsulfonate (SDS) 100mg, mercaptoethanol 0.1ml, glycerine 1ml, tetrabromophenol sulfonphthalein 2mg, Tris-HCl damping fluid 0.5ml, adding distil water is to 10ml;
C. prepare gel buffer liquid: take by weighing SDS 0.2g, add Tris-HCl damping fluid 100ml;
D. prepare electrode buffer: take by weighing SDS 1.0g, add Tris-HCl damping fluid 500ml, adding distil water is to 1000ml;
E. the prescription of preparing gel liquid storage, staining fluid and destainer respectively with embodiment one step 3 (2) b), 3 (2) f), 3 (2) g) identical;
F. the selection of protein molecular weight standard product: H is identical with embodiment one step 3 (2);
G. sample well template (comb) specification is 4 * 10mm;
H. operation steps is as follows: a) 7.5% preparing gel: get gel liquid storage 5.0ml, add gel buffer liquid 10ml, 1%TENED (Tetramethyl Ethylene Diamine) 2ml, distilled water 3.0ml, this mixed solution need outgas 10 minutes; Add freshly prepared 10% ammonium persulphate 0.1ml; Be added to immediately behind the mixing in the gel slab mould, polymerization needed finish in 40 minutes approximately; B) preparation of sample: with embodiment one step 3 (2) Jb) identical; C) application of sample: take out comb, with electrode buffer flushing gel sample hole, every hole application of sample 40ul; D) electrophoresis process and dyeing, decolouring program respectively with embodiment one 3 (2) Jd) and 3 (2) Je) identical.: get glue, fix, dye and decolouring according to routine, a visible tangible band is the pure product of TN albumen in the position of 17KD;
(3) target protein reclaims: operate identical with embodiment one step 3 (3).
Embodiment three: a kind of ovarian cancer protein mark (Tetranectin, TN, TN) pure product evaluation method is used a kind of oophoroma early diagnosis protein marker (Tetranectin that the inventive method prepares from human plasma, TN, TN) pure product, its authentication step comprises following content:
1. protein content is identified and is adopted the Bradford method, and key step comprises:
(1) preparation protein reagent: take by weighing 100mg and examine Ma Shi light blue G-250, be dissolved in 50ml 95% ethanol, add 100ml 85% phosphoric acid, with distilled water with solution dilution to 1000ml, be mixed with ultimate density and be 0.01% and examine the protein reagent of Ma Shi light blue, 4.7% (w/v) ethanol and 8.5% (w/v) phosphoric acid;
(2) the pure product sample of preparation TN to be measured: get 10ul through using the pure product of TN that the inventive method obtains, be diluted to 1ml with protein reagent, standby;
(3) preparation standard protein solution: promptly prepare bovine serum albumin (BSA) solution (the BSA standard substance are available from Bio-Rad company) of 5 different concns, their concentration is 80ug/ml, 40ug/ml, 20ug/ml, 10ug/ml, 5ug/ml;
(4) ultraviolet detection: above-mentioned all solution are respectively got 0.2ml add each hole of microwell plate, every kind of solution capable diplopore of all making even is measured photoabsorption (OD) in 595nm place and is worth on the uv-spectrophotometric instrument;
(5) calculation result: with the different concns value of BSA solution as X-coordinate, corresponding diplopore OD mean value as ordinate zou drawing standard curve: obtain the measured diplopore OD mean value of the pure product solution of TN to be measured, the protein content of finding it from typical curve is 28.5ug/ml; Multiply by extension rate 100, calculation result is: the protein content of the pure product of TN is 2.85mg/ml.
2. purity and molecular weight identification adopt the SDS-PAGE gel electrophoresis, instrument selection Bio-Rad company electrophoresis apparatus, and electrophoresis chamber is the slab electrophoresis groove.Key step comprises:
(1) the preparation phosphoric acid buffer (0.2M, PH7.2): get 0.2M sodium dihydrogen phosphate 280ml, add 0.2M disodium phosphate soln 720ml, behind the mixing PH is transferred to 7.2;
(2) preparation gel liquid storage: acrylamide (Acr) 30g, methylene-bisacrylamide (Bis) 0.8g, distilled water 100ml;
(3) prepare following solution with phosphoric acid buffer:
A) sample buffer: get sodium laurylsulfonate (SDS) 100mg, mercaptoethanol 0.1ml, glycerine 1ml, tetrabromophenol sulfonphthalein 2mg, phosphoric acid buffer 0.5ml, adding distil water is to 10ml;
B) gel buffer liquid: take by weighing SDS 0.2g, add phosphoric acid buffer 100ml;
C) electrode buffer: take by weighing SDS 1.0g, add phosphoric acid buffer 500ml, adding distil water is to 1000ml;
D) preparation staining fluid: take by weighing 0.25g Xylene Brilliant Cyanine G R-250, add 454ml 50% methyl alcohol and 46ml glacial acetic acid;
E) preparation destainer: 50ml methyl alcohol, 75ml glacial acetic acid are mixed with 875ml distilled water.
Sample well template (comb) specification is 0.5 * 0.75mm;
(4) working method is as follows: a) 10% preparing gel: get gel liquid storage 6.67ml, add gel buffer liquid 10ml, 1%TENED (Tetramethyl Ethylene Diamine) 2ml, distilled water 1.23ml; This mixed solution degassing added freshly prepared 10% ammonium persulphate 0.1ml after 10 minutes; Be added to immediately behind the mixing in the gel slab mould, finished gel polymerisation approximately through 30 minutes; B) preparation of testing sample: the pure product of TN albumen are diluted to the testing sample solution that concentration is about 2ug/ul with phosphoric acid buffer, get 25ul, add the 25ul sample buffer and mix, standby; C) preparation of protein standard substance: middle molecular weight protein matter standard substance are available from Bio-Rad company (molecular weight ranges 14～97KD); Get 10ul, add the 10ul sample buffer and mix, standby; D) protein denaturation: will all in 100 ℃ of water-baths, heat 3 minutes by the pure product of the TN albumen solution to be measured of step b) preparation and the protein standard solution of step c) preparation; E) application of sample: prepared gel is put into electrophoresis chamber, fill it up with electrode buffer, take out comb, behind electrode buffer flushing gel sample hole, every hole application of sample 20ul; F) electrophoresis: connect power supply, electric current is transferred to 50～80mA, treat that the blue dyes in the sample migrates to apart from the about 1cm in gel lower end place, stop electrophoresis: g) dyeing and decolouring: get glue, dyeing and decolouring according to routine, a visible tangible band is the pure product of TN albumen in the position of 17KD;
(5) estimating of molecular weight: measure the migration distance of each protein on gel in the protein standard substance, and calculate relative mobility according to formula; With each proteinic molecular weight in the protein standard substance is that the logarithm of ordinate zou, corresponding relative mobility is an X-coordinate drawing standard curve; Measure the relative mobility of the pure product of TN albumen to be measured, the molecular weight of finding it from typical curve is 17KD, conforms to document.
(6) calculating of purity: with gel imaging instrument gel is scanned and analyzes, the result who obtains is: the purity of the pure product of TN that obtained by the inventive method separation and purification is 96.5%.
3. iso-electric point (PI) is identified and is adopted polyacrylamide isoelectric focusing electrophoresis method, selects water-cooled disk electrophoresis groove for use, and key step comprises:
(1) sheet glass is handled: the side at preparing gel sheet glass (thickness 3mm) is coated with the very thin waterproof silicone oil of last layer with gauze, and placement is dried;
(2) preparation glue-filling slot: will cool off and poor transfer to level, and spreading Lu paper, and placing sheet glass, plastics interval mould frame successively, scribble the sheet glass of silicone oil; Use clamp then, promptly make 9 * 9 * 0.05cm
3Glue-filling slot;
(3) obtain solution: a) 30% monomer liquid storage: 29.1g acrylamide (Acr) and 0.9g methylene-bisacrylamide (Bis), be settled to 100ml with distilled water, standby with brown bottle 4 ℃ of preservations; B) electrode solution: 1M phosphoric acid is acid electrode solution, and the 1M sodium oxide is alkaline electrode solution; C) amphotericeledrolyte and standard protein liquid (the standard protein mixed solution of known PI, PI scope 3.5～9.3) are all available from Bio-Rad company; D) stationary liquid: distilled water is prepared 35% methyl alcohol-10% trichoroacetic acid(TCA)-3.5% sulphosalicylic acid; E) staining fluid: distilled water is prepared 0.1% coomassie brilliant blue R250-35% ethanol-10% acetate; F) destainer; Distilled water is prepared 25% ethanol-10% acetate; G) preserve liquid: distilled water is prepared 2% glycerine-35% ethanol-10% acetate;
(4) encapsulating: a) gel preparation: get 30% monomer liquid storage 2.0ml, amphotericeledrolyte 0.6ml, distilled water 5.4ml, mix the back degassing 10 minutes, add TEMED 8ul, 10% ammonium persulphate 50ul again, rapidly mixing;
B) encapsulating: the gelating soln with mixing pours into glue-filling slot at once, prevents that bubble from producing gel polymerisation after about 1 hour;
(5) application of sample (comprising the protein standard substance of known PI value, the pure product of TN to be measured): a) carefully take off sheet glass and plastics mould frame at interval; B) (1 * 10cm) soaks into acid electrode solution and alkaline electrode solution respectively, and is layered on positive and negative the two poles of the earth of gel respectively to get two Lu paper slips; C) (5 * 5mm) are placed on the centre of gel, and (5 * 5mm) are placed on inclined to one side anodal position with the application of sample paper that flooded the pure product of TN to be measured will to flood the application of sample paper of protein standard substance;
(6) electrophoresis: a) press battery lead plate, fill it up with electrode solution, cover safety screen, connect water of condensation, opening power; B) constant voltage (60V) changes constant current (8mA) into after 15 minutes, treats that voltage changes constant voltage (550V) 30 minutes into when rising to 550～580 (V); C) close power supply, open safety screen, take out application of sample paper, cover safety screen again, voltage is transferred to 580V continued electrophoresis about 120 minutes; D) treat that electric current descends when approaching zero, finishes electrophoresis;
(7) fixing, the dyeing of gel and decolouring: a) with gel with fixing about 4 hours of stationary liquid, b) cleaned 1 hour, c) with staining fluid dyeing 30 minutes, d) with destainer decolouring 2 hours, e) with preserving liquid rinsing 10 minutes with destainer;
(8) mensuration of protein PI: the albumen with known PI value is standard, according to they residing positions behind electrophoresis, is that ordinate zou, respective distance are that X-coordinate is drawn the PI gradient curve with the PI value; According to TN albumen to be measured residing position behind electrophoresis, find its iso-electric point from gradient curve.The iso-electric point of the pure product of TN that obtained by the inventive method separation and purification is 5.8, conforms to document.
4. activity identification adopts the antigen-antibody reaction method, and key step comprises:
(1) obtain solution: a) scavenging solution:, become the 1X scavenging solution with distilled water diluting during use with 0.2M PBS (PH7.4) preparation 0.05% polysorbas20 solution (20X); B) substrate solution: 3% superoxol of phosphorus phosphorus-citrate buffer solution (PH5.0) preparation; C) colour developing liquid: concentration is tetramethyl benzidine (TMB) methanol solution of 0.1mg/ml: d) reaction terminating liquid: 2M sulfuric acid;
(2) add antibody: add TN antibody 100ul in each hole of micropore enzyme plate, antibody concentration is the 2ug/ hole;
(3) add antigen: add pure each 100ul of product solution of TN albumen to be measured of different concns, 37 ℃ of water bath heat preservations 60 minutes;
(4) integrated enzyme reaction: after treating that antigen-antibody reaction is finished, wash plate 4 times, each 3 minutes with scavenging solution; The TN antibody 100ul that adds with horseradish peroxidase (HRP) mark carried out integrated enzyme reaction, 37 ℃ of water bath heat preservations 60 minutes; Repeat to wash plate 4 times with scavenging solution, each 3 minutes;
(5) color reaction: every hole added substrate solution, colour developing liquid each 50ul successively, 37 ℃ of water bath heat preservations 10 minutes; Add the 50ul reaction terminating liquid again and finish reaction;
(6) interpretation of result; The OD value that records at the 450nm place according to the pure product of TN albumen to be measured of different concns gets final product to such an extent that judge their activity.The activity of the pure product of TN that obtained by the inventive method separation and purification is: concentration is that the pure product of the TN of 2.4ng/ml promptly have the activity that obvious antigen-antibody reaction takes place with 2ug TN antibody.
5. specificity is identified and is adopted the Western-Blot method, and key step comprises:
(1) preparation of SDS-PAGE gel electrophoresis: elementary operation and step 2 are basic identical, do a application of sample that repeats by the application of sample order when noting application of sample on same clotting glue;
(2) obtain solution: a) electrotransfer damping fluid: 25mM Tris-192mM glycine-20% (V/V) methanol solution, PH8.3; B) TBS damping fluid: 25mM Tris-HCl, 0.5M sodium chloride solution, PH7.5;
C) TBST damping fluid: the TBS damping fluid adds 0.05% polysorbas20; D) CBS damping fluid: 0.1M sodium bicarbonate-1.0M magnesium chloride, PH9.8; E) substrate solution: 3mg tetrazolium chloride nitro indigo plant (NBT), 1.5mg bromine chloro-indole toluene amine salt (BCIP) is dissolved among the 170ul DMSO, thoroughly after the dissolving, is diluted to 10ml with the CBS damping fluid;
(3) make " gel sandwich ": is that nitrocellulose filter (NC film), the Whatman filter paper (specification 3mm) of 0.45um is made into " gel sandwich " with running gel with the aperture, notes preventing the bubble generation;
(4) electrophoresis: " gel sandwich " inserted in the electrophoresis chamber that fills the electrotransfer damping fluid, and NC face anode is carried out electrophoresis, 100mA constant current 3 hours;
(5) dyeing: electrophoresis finish with half NC film with 0.1% amino black dye, 7% acetate decolours, and can show zone of protein;
(6) antigen-antibody reaction: second half NC film that has the repetition application of sample was placed in the 3%BSA confining liquid through the flushing of TBS damping fluid in 10 minutes, slowly shook 60 minutes in 37 ℃; After the TBS damping fluid washes 3 times (each 5 minutes), place TN antibody-solutions again, slowly shook 2 hours in 37 ℃ with the dilution in 1: 50 of TBST damping fluid;
(7) integrated enzyme reaction: after using the TBST damping fluid to wash 3 times (each 5 minutes), add the second antibody with the HRP enzyme labelling of TBST damping fluid dilution in 1: 150, continuation was reacted 2 hours;
(8) color reaction: after using the TBST damping fluid to wash 3 times (each 5 minutes), washed 10 minutes through the TBS damping fluid again; Add substrate solution, reacted 5～10 minutes, present tangible brown stripe to the antigenic region band;
(9) termination reaction: use distilled water wash, termination reaction;
(10) protein-specific analysis: will contrast through the NC film after antigen-antibody reaction and the colour developing and half NC film of substantive dyeing, and can learn the specificity of TN sample to be measured.Identify tangible specific combination district band is arranged by the pure product of TN that the inventive method separation and purification obtains through the Western-Blot method in 17KD position and TN antibody.
Claims (4)
1. the separation purification method of an oophoroma early diagnosis protein marker TN is characterized in that it is made up of roughing out, moderate purifying and three key steps of polishing purification of TN, wherein:
A. the saturated ammonium sulphate method is adopted in the roughing out of TN, comprises two steps of saturated ammonium sulphate and pH value adjustment;
B. the moderate purifying process of TN adopts ion exchange chromatography, two chromatographic step of affinity chromatography;
C. the polishing purification process using polyacrylamide gel electrophoresis of TN comprises that protein denaturation, electrophoresis, target protein reclaim three steps.
2. according to the process of claim 1 wherein that the saturated ammonium sulphate among the step a is the physiological saline that adds equivalent in blood plasma, add solid ammonium sulfate to 50% saturation ratio more in proportion; It is to treat after protein is separated out the pH value of solution to be transferred to 5.8 that the pH value is adjusted.
3. according to the method for claim 2, wherein the order of ion exchange chromatography among the step b and affinity chromatography can be changed.
4. according to the method for claim 3, wherein ion exchange chromatography is selected the weakly alkaline ion exchange chromatography for use, and affinity chromatography is selected heparin affinity chromatography or Methionin affinity chromatography for use.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198690A (en) * | 2015-04-30 | 2016-12-07 | 嘉和生物药业有限公司 | A kind of internal standard indicant being applied to capillary gel electrophoresis analyzing proteins |
| CN106544421A (en) * | 2016-10-21 | 2017-03-29 | 武汉科技大学 | SPAG6 genes are used as diagnosis on ovarian tumors and the purposes for the treatment of mark |
| CN115112742A (en) * | 2022-07-01 | 2022-09-27 | 山东先声生物制药有限公司 | Method for correcting molecular weight isoelectric point by using physicochemical reference substance |
-
2003
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198690A (en) * | 2015-04-30 | 2016-12-07 | 嘉和生物药业有限公司 | A kind of internal standard indicant being applied to capillary gel electrophoresis analyzing proteins |
| CN106198690B (en) * | 2015-04-30 | 2018-11-06 | 嘉和生物药业有限公司 | A kind of internal standard indicant applied to capillary gel electrophoresis analysis albumen |
| CN106544421A (en) * | 2016-10-21 | 2017-03-29 | 武汉科技大学 | SPAG6 genes are used as diagnosis on ovarian tumors and the purposes for the treatment of mark |
| CN106544421B (en) * | 2016-10-21 | 2020-05-08 | 武汉科技大学 | Application of SPAG6 gene as ovarian tumor diagnosis and treatment marker |
| CN115112742A (en) * | 2022-07-01 | 2022-09-27 | 山东先声生物制药有限公司 | Method for correcting molecular weight isoelectric point by using physicochemical reference substance |
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