CN1288245C - Long-noded pit viper dissolving fiber protein No.4 gene and use thereof - Google Patents
Long-noded pit viper dissolving fiber protein No.4 gene and use thereof Download PDFInfo
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- CN1288245C CN1288245C CN 03140212 CN03140212A CN1288245C CN 1288245 C CN1288245 C CN 1288245C CN 03140212 CN03140212 CN 03140212 CN 03140212 A CN03140212 A CN 03140212A CN 1288245 C CN1288245 C CN 1288245C
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- agkistrodon acutus
- acutus venom
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to the technical field of gene engineering, particularly to a deinagkistrodon acutus (five-paced pit viper) fibrinotytic(fibrinogenolytic) gene No. 4, a carrier containing the gene, a host cell for gene engineering by the carrier and a fibrinotytic(fibrinogenolytic) medicine made of the gene to resist embolism disease caused by thrombosis. Fibrinolysis factors FII are extracted from the coarse poison of a five-paced pit viper. Fibrinolysis factor FII genes are sieved and cloned. The fibrinolysis factors FII are expressed in yeast cells. The expressed products are purified, and fibrinolytic activity is measured. The present invention expressing the fibrinolysis factors by a gene engineering yeast system has the advantages of high activity, large yield, stable quality and low cost, and the present invention can become a novel potent thrombolytic drug and have great social benefits and economic benefits.
Description
[technical field]
The present invention relates to gene engineering technology field, relate in particular to agkistrodon acutus (agkistrodon acutus venom) solution fibrin (former) No.4 gene, contain this gene carrier, utilize this carrier to carry out genetically engineered host cell and this gene be used to prepare the medicine of solution fibrin (former), with the caused embolism class diseases of antagonism thrombosis.
[background technology]
Thrombosis caused embolism class diseases, especially myocardial infarction and cerebral apoplexy in the blood vessel, in the world to the illness of human health risk maximum, its sickness rate and case fatality rate all occupy first of each disease at present.In China, along with generally prolonging and the development of demographic structure aging of the significantly improving of most of communicable disease Be Controlled, people's living standard, crowd's life-span, the sickness rate of the heart, cerebrovascular thrombus disease has risen to first and second in various diseases, more than annual morbidity 3,000,000 people.And have reason to expect that the sickness rate of this type of disease still can further increase from now on.
At present, the treatment of embolism class diseases mainly relies on antiplatelet drug (as Asprin), anticoagulant (as heparin) and plasminogen activator.From the principle, Asprin and heparin all are anticoagulant mechanism, make blood lose the effect that prevention thrombosis and thrombus enlarge of playing with fixed attention, and be then invalid to the thrombus that has existed, and can not use as treating especially first aid.Plasminogen activator is the medicine of at present clinical treatment thrombus disease commonly used, comprises streptokinase, urokinase and tissue plasminogen activator (tPA).Their common mechanism is endogenic non-activity Profibrinolysin to be activated be plasmin, the scleroproein in latter's hydrolysis thrombus and thrombolysis.Therefore, the common feature of this class medicine is, its molten fine effect is indirect, and onset is slower, and a little less than the effect, especially all the more so to bigger thrombus.And under the situation of the heart, cerebrovascular generation embolism, cardiac muscle and neurone are after blood flow is cut off, and minute i.e. generation of anoxic number is dead.Because the action principle of its indirect thrombolysis, also make above-mentioned three kinds of medicines that pair thrombus poor selectivity, easily side effect such as cause bleeding are arranged.In addition, anaphylaxis also can take place in streptokinase, and tPA costs an arm and a leg and limited it and used widely.
What especially need to emphatically point out is, the thrombolysis medicine that uses now, and 25% patient is invalid even combined utilization is also had an appointment." vascular reocclusion " takes place after using existing thrombolysis medicine in the patient of about 5-30% in addition.And this kind is inaccessible more invalid to continuing use thrombolysis medicine.
In sum, existing thrombolysis medicine can not satisfy clinical service requirements fully, presses for higher, the single-minded more thrombolytic drug rapidly of curative effect of development usefulness.
The research report Agkistrodon contortrix snake venom of external relevant snake venom contains direct acting plasmin, Crotalus atrox (western diamondback rattlesnake) snake venom also contains scleroproein (former) lytic enzyme, but plasminogen activation not, at rat vein thrombus model intravenously administrable, show thrombolytic effect with shadowgraph technique.Find no kidney, liver, the heart, lung tissue necrosis and hemorrhage reaction with histological method inspection.
Venine product that uses clinically in China such as defibrase, embolism-resisting enzyme, its mechanism are that the Fibrinogen with blood plasma changes scleroproein into as zymoplasm, reduce the viscosity of blood plasma, in the hope of reaching anti-bolt effect.Its curative effect and mechanism at all can not be comparable with the thrombolysis medicine.
Contain direct fibrinolytic composition in the exclusive agkistrodon acutus venom (Agkistrodon acutus venom) of China, and tool intensive biological activity.With column chromatography method separation and purification method, from 15 components, obtain several protein with molten fine effect.Wherein component II (fibrinolytic factor) has the biological nature of high using value: (1) is thrombolysis directly: after destroying Profibrinolysin with hot plate method, fibrinolytic factor still can solution fibrin; (2) effect fast: in experiment in vitro, the fibrinolytic factor effect is more fast again than urokinase; (3) usefulness height: biological activity ratio's urokinase of fibrinolytic factor stronger (130 μ g are equivalent to urokinase 450u); (4) side effect is low: although not purified agkistrodon acutus venom has tangible hemorrhage effect, fibrinolytic factor is not also seen hemorrhage reaction under 500 μ g/ml concentration.Above-mentioned these characteristics, the strong medicine of supporting may become the treatment thrombotic diseases of a new generation from the fibrinolytic factor in the agkistrodon acutus venom.
But, the agkistrodon acutus venom of natural source, its composition and biological activity all exist provincialism, seasonal variation, in fact are difficult to obtain stable clinical efficacy; In addition, because composition complexity in the raw product snake venom, though purified processing, the real single Chemical Composition of still difficult acquisition makes clinical application imply some toxic side effect; Nature snake venom output is limited, and cost is higher, and it is low that ability is occupied in market; Its molecular structure and structure and active relation still can not be illustrated with the protein technology.Therefore, address the above problem, requisite beyond doubt to the scientific basic of the clinical application of real establishment fibrinolytic factor.
Yeast does not need special substratum just energy ramp and large scale fermentation; Zymic DNA is simple and colony screening is convenient; Yeast is that fungal organism has than the complete gene expression regulation of intestinal bacteria mechanism with to the processing of expression product and modifies, and as can glycosylation, can form correct disulfide linkage or the like, has the precedent of echidnotoxin in the yeast expression success.This problem is made probe with the antibody of agkistrodon acutus venom fibrinolytic factor FII, from Agkistrodon poison gland cDNA library, filter out the gene of FII, be cloned into Yeast expression carrier PPIC9K, transformed yeast cell, realize agkistrodon acutus venom fibrinolytic factor FII efficiently expressing in yeast, with purification by chromatography reorganization agkistrodon acutus venom fibrinolytic factor FII and measure its fibrinolytic activity.This research obtains the agkistrodon acutus venom fibrinolytic factor FII of a large amount of genetically engineered preparations, for the pharmacodynamic study and the evaluation of follow-up a kind new medicine are laid a good foundation.
The research of snake venom once play a part crucial to Biomedical Development, as DNA isolation restriction endonuclease from snake venom, greatly promoted molecular biological research, found toxin from bungarotoxin, for the purifying of neurotransmitter receptor has played conclusive effect.The agkistrodon acutus venom fibrinolytic factor FII that the present invention prepares with genetically engineered, compare with the multiple snake venom biochemical preparation of present clinical application, can overcome bioactive regional disparity and chemical unhomogeneity, improve curative effect and output, become the clinical new drug that has great market value and have independent intellectual property right.
[summary of the invention]
Agkistrodon acutus venom fibrinolytic factor FII is solution fibrin directly, is not effectively seeing hemorrhage reaction under the fibrinolytic dosage.Raw product snake venom composition complexity, being difficult to purifying is single composition; Snake venom output is limited, is unfavorable for scale operation, and using gene engineering technique is expressed fibrinolytic factor and can be addressed the above problem.The present invention separates fibrinolytic factor FII from the thick poison of Agkistrodon; Finish screening, the clone of fibrinolytic factor FII gene; In yeast cell, express fibrinolytic factor FII; The purifying expression product is also measured fibrinolytic.
Method: adopt ion exchange chromatography and gel-filtration separating natural agkistrodon acutus venom fibrinolytic factor FII from agkistrodon acutus venom, measure its activity as substrate, observe it at fibrinogenic action site by SDS-PAGE with Fibrinogen, scleroproein; Preparation polyclonal antibody, monoclonal antibody and Agkistrodon poison gland cDNA library are made probe with antibody and screen fibrinolytic factor FII gene from the libraries, and cut method such as connection with enzyme it is cloned among the expression plasmid of yeast PPIC9K; Obtain the high expression level yeast strain by transforming and screening; With ion exchange chromatography and hydrophobic chromatography purification of Recombinant agkistrodon acutus venom fibrinolytic factor FII, SDS-PAGE detects purity, and the fibrin plate method is measured its fibrinolytic; Observe it to IgG and albuminous effect by SDS-PAGE; Measure fibrinolytic in its body with dog lung thrombus model.
The result: by three chromatographies from thick poison purifying fibrinolytic factor FII, SDS-PAGE is shown as single band, molecular weight is 25,500.A α, B β chain that agkistrodon acutus venom fibrinolytic factor FII fibrin degradation is former almost do not have Degradation to the γ chain; Can rely on the ground solution fibrin by dosage.Successfully prepare polyclonal antibody, monoclonal antibody and Agkistrodon poison gland cDNA library, from the library, screened a fibrinolytic factor FII gene, made up recombinant plasmid PPIC9K-FII.Behind the transformed yeast cell, PCR confirms that recombinant plasmid is integrated on the yeast chromosomal.With the former activity of fibrin degradation is index, screens a high expression level bacterial strain, peaks in the 3rd day activity of expression.Reorganization agkistrodon acutus venom fibrinolytic factor FII by the twice chromatographic purifying is single district band at SDS-PAGE, and scleroproein is had stronger solvency action, and IgG and albumin are not had Degradation.Fine to the dissolving of bottom right pulmonary thrombosis, administration is recanalization rate average out to 83.3% after 1 hour.Conclusion: efficiently expressed agkistrodon acutus venom fibrinolytic factor FII in yeast, reorganization fibrinolytic factor FII has higher fibrinolytic.
The isolating cellulolytic activity factor of natural agkistrodon acutus venom FII, output is lower, the quality instability.The present invention's molten fine factor of gene engineering yeast system expression, active high, output is big, steady quality, cost is low.At the whole animal thrombus model, thrombolysis is respond well, and hemorrhage reaction is few.Point out it can become novel potent thrombolysis medicine consumingly, social benefit and economic benefit are huge.
[description of drawings]
Fig. 1: the DEAE-SephadexA-50 ion exchange chromatography of agkistrodon acutus venom;
The Sephadex G-75 gel filtration chromatography of Fig. 2: component I I;
Fig. 3: the double-layer of component I I is analysed with Sephadex G-75;
Fig. 4: it is quantitative with SDS-PAGE component I I to be carried out molecular weight;
Fig. 5: the typical curve of protein marker among the SDS-PAGE;
Fig. 6: with the isolating Fibrinogen hydrolysate of SDS-PAGE;
The scleroproein activity of Fig. 7: component I I;
Fig. 8: the formaldehyde agarose electrophoresis of whole RNA of Agkistrodon poison gland;
Fig. 9: the CDNA library structure of agkistrodon acutus venom;
Figure 10: the positive findings of component I I in the antibody screening agkistrodon acutus venom CDNA library;
Figure 11: the sequence of the insertion dna fragmentation of nine positive colonies;
The deduced amino acid of the ORF dna sequence dna of Figure 12: component I I;
Figure 13: the SDS-PAGE at the component I I of expression in escherichia coli fusion rotein analyzes;
Figure 14: two steps that in yeast, make up the expression vector of component I I target dna;
Figure 15: the dual restriction of recombinant plasmid pPIC9K-F II;
Figure 16: the scleroproein activity of analyzing culture supernatant with SDS-PAGE;
Figure 17: the DEAE-Sepharose ion exchange chromatography of culture supernatant;
Figure 18: the Buty-Toyopearl chromatography of culture supernatant;
Figure 19: with the analysis of SDS-PAGE to reorganization component I I purity;
Figure 20: the direct scleroproein activity of reorganization component I I;
Figure 21: analyze reorganization component I I to IgG and albuminous effect with SDS-PAGE;
Figure 22: reorganization component I I is to the thrombolytic effect of arterial thrombus model under the dog right lung.
[embodiment]
One, the separation of agkistrodon acutus venom fibrinolytic factor FII
1.DEAE-Sephadex A-50 ion exchange chromatography
Method: DEAE-Sephadex A-50 ion-exchange gel is rinsing and adorn post (gel column diameter 2.6cm, high 100cm) after soaking about 24 hours at 25 ℃ repeatedly in the ammonium acetate buffer of 0.05M PH 8.0.Treat that effluent liquid pH value finishes near 8.0 o'clock balances, the thick poison of 3g agkistrodon acutus venom is dissolved in the ammonium acetate buffer of 10ml 0.05M PH8.0, and 1500rpm 15 minutes is centrifugal to remove upper prop behind the sediment.Make the straight line gradient elution with the ammonium acetate buffer of 0.05M PH8.0 and each 750ml of ammonium acetate buffer of 1M PH5.0, the elutriant gathering speed is 4tube/h, 3ml/tube.Under the 280nm wavelength, measure absorbance value.Measure each peak fibrinolytic, the component with fibrinolytic with the mini dialysis desalting, concentrate after, the vacuum freeze drier freeze-drying is standby.
Fibrinolytic is measured with reference to Astrup and Mullerti method, detects fibrinolytic with the fibrin plate method.Getting 0.2% calf fibrinogen solution 20ml is in the culture dish of 9.5cm in diameter, and the culture dish horizontal positioned adds the zymoplasm (100U/ml) of 80 μ l, mixes to shake even placement and form dull and stereotyped grumeleuse.Add testing sample 20 μ l in the surface of flat board, each sample adds 3 points, and 37 ℃ of insulations are after 12 hours, to dissolve dull and stereotyped cartographic represenation of area fibrinolytic vigor.
The result: the thick poisons DEAE-Sephadex A-50 of agkistrodon acutus venom ion exchange chromatography obtains 10 protein peak (see figure 1)s, and wherein the 2nd peak (FII) has higher fibrinolytic, and the fibrinolytic that records with fibrin plate is 60.23 ± 16.47mm
2/ μ g.
2.Sephadex G-75 gel chromatography
Method: Sephadex G-75 gel is with the rinsing and adorn post (gel column diameter 1.1cm, high 100cm) after soaking about 24 hours at 25 ℃ repeatedly of the ammonium acetate buffer of 0.05M PH8.0.After balance is finished, the fibrinolytic composition 85mg that DEAE-Sephadex A-50 ion exchange chromatography is obtained is dissolved in the ammonium acetate buffer of 2ml 0.05M PH8.0, upper prop, ammonium acetate buffer wash-out with 0.05M PH8.0, elutriant is measured absorbance value under the 280nm wavelength, after pressing preceding method mensuration activity, collecting needs the activated protein peak.The result: further through Sephadex G-75 gel permeation chromatography, obtain 2 protein peaks ((see figure 2), wherein first peak has higher fibrinolytic, its fibrinolytic is 87.51 ± 24.95mm
2/ μ g.
3.Sephadex the G-75 gel is chromatography once more
Method: the fibrinolytic composition 60mg that Sephadex G-75 gel-filtration for the first time obtains is by method 2 chromatography once more.
The result: obtain a protein peak (see figure 3) with Sephadex G-75 gel permeation chromatography once more, its fibrinolytic is 90.49 ± 12.41mm
2/ μ g.
Two, the purity of agkistrodon acutus venom fibrinolytic factor FII and determination of activity
1. the purity detecting of agkistrodon acutus venom fibrinolytic factor FII
Method: adopt the vertical flat electrophoresis of SDS-PAGE, resolving gel concentration 12%, PH8.8 concentrates gum concentration 4%, PH6.8, electrode buffer adopts the Tris-glycine, and voltage 200V, electrophoresis time are 45 minutes.Trichoroacetic acid(TCA) with 20% behind the electrophoresis is fixed, Coomassie brilliant blue R250 dyeing, 45% methyl alcohol, 7% acetate decolouring.Sample on agkistrodon acutus venom fibrinolytic factor FII (1mg/ml) the 20 μ l.Molecular weight standard adopts MBP-β-galactosidase (175,000), and MBP-paramyosin (83,000), Glutamic dehydrogenase (62,000), Aldolase (47,500), Triosephosphate isomerase (32,500), β-LactoglobulinA (25,000), Lysozyme (16,500).
Result: obtain Yi Tiao district band (see figure 4) through SDS-PAGE, relative mobility Rf (X) by standard protein makes the regression curve (see figure 5) to molecular weight logarithm 1gMW, get equation 1gMW=-1.55X+5.5l, r=0.95, to get the molecular weight of agkistrodon acutus venom fibrinolytic factor FII be 25,550 to Equation for Calculating thus.
2. the former determination of activity of agkistrodon acutus venom fibrinolytic factor FII solution fibrin
Method: get 0.2% bovine fibrinogen solution 75 μ l and be added in the Ependoff pipe, add agkistrodon acutus venom fibrinolytic factor FII (2mg/ml) 25ull again 37 ℃ of insulations.Insulation 1h adds electrophoresis protein example treatment solution 50 μ l after the time, get 20 μ l and be SDS-PAGE.25 μ l make positive control with Fibrinolysin (human) (0.05U/ml).
The result: the SDS-PAGE after bovine fibrinogen and the FII effect is (see figure 6) as a result.FII has hydrolytic action to fibrinogenic A α chain, B β chain, the γ chain does not almost have hydrolysis, the degradation fragment that produces is mainly 45,000Da, and plasmin all has hydrolytic action to fibrinogenic A α chain, B β chain, γ chain, the degradation fragment molecular weight that produces is 47,000Da, 44,000Da and 23,000Da, all the energy fibrin degradation is former to show FII and plasmin, but the action site difference.
3. agkistrodon acutus venom fibrinolytic factor FII solution fibrin determination of activity
Method: getting 0.2% bovine fibrinogen 20ml is in the culture dish of 9.5cm in diameter, the culture dish horizontal positioned, add zymoplasm (100U/ml) 80 μ l, after mixing the fibrin plate grumeleuse that shakes even placement formation, add testing sample 20 μ l in the surface of flat board, each sample adds 3 points.37 ℃ of insulations are after 12 hours, to dissolve dull and stereotyped cartographic represenation of area fibrinolytic vigor.
The result: the fibrin plate method is measured the agkistrodon acutus venom fibrinolytic factor FII fibrinolytic activity of 0.25mg/ml, 0.5mg/ml, 1mg/ml, four concentration of 2mg/ml, makes the positive and negative control respectively with urokinase and the physiological saline of 500u/ml.The fibrinolysis vigor (seeing Table 1) of different concns agkistrodon acutus venom fibrinolytic factor FII shows that agkistrodon acutus venom fibrinolytic factor FII can dose-dependently ground solution fibrin (see figure 7).
The scleroproein activity of table 1. component I I. (n=3, mean ± SD)
| Concentration (mg/ml) | Lysed area (mm 2) |
| 0.25 0.5 1 2 | 31.33±3.40 60.00±3.27 105.00±4.08 137.67±5.31 |
Three, the preparation of anti-agkistrodon acutus venom fibrinolytic factor FII antibody
1. the sero-fast preparation of the anti-agkistrodon acutus venom fibrinolytic factor of rabbit FII
Method: after the agkistrodon acutus venom fibrinolytic factor FII 1ml of (1) animal immune 3mg/ml and the emulsification of 1ml complete Freund's adjuvant thorough mixing, the subcutaneous multi-point injection of rabbit back, booster immunization once is total to immune 4 weeks weekly.
(2) immunizing rabbit is with after the 3% vetanarcol anesthesia for Antiserum Preparation, and the carotid artery intubate is got blood, and after at room temperature solidifying, centrifugal receipts supernatant is put in 4 ℃ serum is separated out.
(3) ELISA measures antibody titer and gets the agkistrodon acutus venom fibrinolytic factor FII of 0.1mg/ml respectively and (be dissolved in pH9.5, NaHCO
3/ Na
2CO
3Damping fluid) 100 μ l are in each hole of enzyme plate, and 4 ℃ are spent the night coated elisa plate.Next day, remove the liquid in the hole, wash twice with PBST (0.05%Tween-20).With 37 ℃ of sealings of confining liquid [1 * PBS, 1%BSA (bovine serum albumin)] 1h, (one is anti-for the rabbit anti-serum of removal confining liquid adding 2 * serial dilution, with 1 * PBS, the 1%BSA dilution), hatches 1h for 37 ℃, use PBST detersive enzyme target 3 times, (two is anti-to add goat-anti rabbit HRP-IgG, dilution in 1: 1000), hatches 1h for 37 ℃, after PBST washing 3 times, every hole adds each one of substrate solution A, B respectively, determines sero-fast tiring according to color.
The result: the agkistrodon acutus venom agkistrodon acutus venom fibrinolytic factor FII that uses purifying is as 1, No. 2 two rabbit of antigen immune, and after immune 4 weeks, the preparation antiserum(antisera) is with the anti-agkistrodon acutus venom fibrinolytic factor FII serum antibody titer of ELISA method mensuration rabbit.The result shows: the serum antibody titer of two anti-agkistrodon acutus venom Thrombin-like enzymes of rabbit is 1: 20,000.
2. agkistrodon acutus venom fibrinolytic factor FII MONOCLONAL ANTIBODIES SPECIFIC FOR
Method: (1) immunity and cytogamy and colony screening give the Balb/C mouse subcutaneous injection in 7 ages in week, per injection 0.2ml with after agkistrodon acutus venom fibrinolytic factor FII (1mg/ml) and the emulsification of equal-volume complete Freund's adjuvant thorough mixing.2 week immunity 1 time, immunity is 3 times altogether.3d is with 0.2ml agkistrodon acutus venom fibrinolytic factor FII (1mg/ml) tail vein injection before merging.Get immune mouse spleen cell and prepare cell suspension and mouse SP2/10 myeloma cell, down after the fusion, be seeded in 5 96 porocyte culture plates respectively, place =5%CO in =50%PEG (u 3700) effect by 10: 1 mixed
2Incubator is cultivated.The 1st day to the 4th big HAT selection substratum that contains =15% foetal calf serum, the HT substratum of using instead in the 4th day to the 10th day used.Use the RPMI-1640 that contains =10% foetal calf serum after the 10th day instead.
(2) measure antibody titer by preceding method with ELISA
Result: screen the anti-fibrinolytic factor FII cell strain of 20 strains by 2 fusions, two strain specificity height (called after C1, C4) wherein, show that with the detection of IgG classification agent box two strain hybridoma excretory monoclonal antibodies are IgG I, two strain monoclonal antibody height are tired.
Four, the structure in Agkistrodon poison gland expression type cDNA library
1. the extraction of sample and total RNA
Method: (1) gets poison gland immediately with the Agkistrodon sacrificed by decapitation, puts into the Eppendorf pipe after the taking-up, uses the dry ice quick-frozen immediately.
(2) extract total RNA with Promega company test kit.Take by weighing the about 1.7g of bilateral poison gland, add the 20ml denaturing soln after the homogenate, homogenate is 20 seconds again, add the abundant mixing of 2M sodium acetate 2ml, add 1ml phenol-chloroform-Virahol mixing again, vibrated 10 seconds, ice bath 90 seconds, centrifugal (4 ℃, 12,000rpm 20min), moves to supernatant in the centrifuge tube of 50ml, add the equal-volume Virahol, mixing is placed 20min, centrifugal (4 ℃ at-30 ℃, 12,000rpm, 15min) precipitated rna.Abandon supernatant, precipitation is with 75% ice-cold washing with alcohol, centrifugal remove supernatant after, vacuum-drying, with the water dissolution that DEPC (diethylpyrocarbonate) handles, content and the purity of mensuration RNA.
The result: this experiment is extracted total RNA426 μ g, OD with a step phenol method
260/ OD
280Ratio be 20.Show that through formaldehyde agarose gel electrophoresis (see figure 8) it is clear that two rRNA of 18S and 28S are with, and the fluorescence intensity of 28S is about two times of 18S, shows that total DNA of extraction has kept molecule integrity preferably.
2.RNA separation and purification
Method: Oligo (dT) fibre columns is with chromatography sample loading buffer thorough washing, will be dissolved in RNA (3mg) in the DEPC water then in 65 ℃ of insulation 5min, and the ice bath prompt drop is to room temperature.Add upper prop behind isopyknic sample-loading buffer, wash 3 times, use elution buffer wash-out mRNA then with sample loading buffer.Under the 260nm wavelength, measure the absorbance value of mRNA, calculate the amount of the mRNA of purifying.
The result: it is 10 μ g that total RNA is separated the amount that obtains mRNA through Oligo (dT) Mierocrystalline cellulose affinity column.
3.cDNA the structure in library
Method: (according to the explanation of the cDNA of Promega company synthetic agent box, schema (see figure 9)
(1) cDNA first chain is synthetic.
(2) cDNA second chain is synthetic.
(3) end at double-stranded cDNA connects Not I, Sal I joint.
(4) connection of double-stranded cDNA and the conversion double-stranded cDNA that contains Not I, Sal I joint is connected at 16 ℃ with the T4 ligase enzyme with linearized vector pSport I 1 μ g and spends the night.Connect product 10 μ l and add in the 50 μ l competence bacillus coli DH 5 alphas, 4 ℃ successively, 30min; 42 ℃, 120s; 4 ℃, 5min; 37 ℃, 10min; 37 ℃ of wave and culture 1h.Get 2 μ l and dilute 10,100,1000 times respectively, coat on the LB agar plate that contains penbritin 50 μ g/ml, be inverted for 37 ℃ and cultivate 12h to single bacterium colony formation, the counting bacterial plaque is also calculated the titre in cDNA library, library liquid-80 ℃ preservation.
The result: with library bacterium liquid dilution back coated plate counting bacterium colony, the titre that calculates library bacterium liquid is 5 * 10
7Pfu/ml.
Five, the acquisition of agkistrodon acutus venom fibrinolytic factor FII gene
1. antibody screening cDNA library
Method: (1) gets library bacterium liquid suitably after the dilution, gets 100 μ l, evenly coats on the LB culture plate that contains penbritin 50 μ g/ml, is inverted for 37 ℃ and cultivates 12-16h and grow to the needle point size to bacterium colony.
(2) will soaking into also with IPTG (10mmol/ml) solution, air dried NC film (nitrocellulose filter) be attached to the LB culture plate that covers with bacterium colony, on 3 asymmetric positions, perform mark with syringe needle, taking the NC film off places on the agarose plate and (has one of bacterium colony to face up), put 37 ℃ and cultivate 6-8h again, former LB culture plate is in 4 ℃ of preservations.
(3) take out the NC film, fixedly behind the 30min, place confining liquid (4 μ g/ml N,O-Diacetylmuramidases, 1 μ g/ml DNA enzyme, 1%BSA) jog 30min with chloroform, subsequently with PBST (the each 15ml that washes film 3 times, 5min), (1 * PBS, 1%BSA in 10ml rabbit anti-serum diluent, one anti-thinning ratio 1: 1000) jog NC film 3h, with PBST wash film 3 times (each 15ml, 5min), (two resist for NC film and 10ml goat-anti rabbit HRP-IgG, dilution in 1: 1000) jog 3h washes film 3 times again.
(4) NC film jog 30min in DAB colour developing liquid uses the distilled water rinsing, observes.Positive colony is brown, contrasts former LB culture plate, finds out positive bacterium colony.
(5) with polyclone positive colony coated plate respectively, with monoclonal antibody screen once more, method is the same.
The result: with polyclonal antibody (rabbit anti-serum) is that the 9cm flat board contains 5 * 10 approximately with the diameter
3The density of individual bacterium colony is screened, and has screened about 200,000 bacterium colonies altogether, obtains 15 primary dcreening operation positive colonies, and each clone is multiple again to sieve once, and the clone who still is positive has 11.(positive reaction is cloned in and presents brown spot on the NC film, sees Figure 10).
Positive colony difference coated plate with above-mentioned 11 immune serums screen screens once more with monoclonal antibody, and 9 and C are arranged
1, C
4Two strain monoclonal antibodies all play positive reaction.
2. extracting plasmid cDNA sequencing
Method: the plasmid extraction kit specification sheets according to QIAGEN company carries out.The positive single bacterium colony of picking, 37 ℃ of shaking culture 24h in 5ml LB substratum.
(1) gets 1.5ml incubated overnight bacterium, centrifugal 30s (4 ℃ 12000rpm), are reclaimed bacterium.
(2) abandon supernatant, precipitate resuspendedly with 250 μ l solution P1, thermal agitation is up to cannot see block bacterium.
(3) add 250 μ l solution P2, put upside down centrifuge tube fast 5 times.
(4) add 350 μ l solution N3, will manage inversion immediately, put upside down 5 times, solution is uniformly dispersed in the heavy-gravity bacterial lysate.
(5) centrifugal 10min (4 ℃ 12000rpm), as seen precipitate in vain.
(6) drawing supernatant liquor moves in the centrifugal post of QIA.Centrifugal 30-60s abandons centrifugate.
(7) add 0.5ml solution PB, centrifugal 30-60s abandons centrifugate.
(8) add 0.75ml solution PE, centrifugal 30-60s abandons centrifugate.
(9) empty centrifugal 1min.
(10) add 50 μ l solution E B or H2O, leave standstill 1min, centrifugal 1min.
Extractive plasmid is made sequencing primer with T7 and SP6, adopts the terminal cessation method of the two deoxidations of Sanger to measure and inserts fragments sequence.
The result: above-mentioned 9 the single bacterium colonies of the positive of difference picking, after overnight shaking is cultivated in the LB substratum that contains penbritin, get 1.5ml extracting plasmid.Universal primer T7, SP6 with plasmid multiple clone site two ends do primer, carry out the PCR reaction, and 9 positive colonies all amplify specific band.
Adopt the terminal cessation method of the two deoxidations of Sanger, above-mentioned 9 positive colonies are inserted the cDNA segment all check order result's (seeing Figure 11).
3. the homology analysis of sequence
Method: enter NCBI (The National Center of BiotechnologyInformation) website by the internet, utilization BLASTn and BLASTp program are carried out the homology analysis of Nucleotide and derivation probable protein.Determine to contain the FII plasmid of agkistrodon acutus venom fibrinolytic factor FII gene.
Result: 9 cDNA sequences that obtain are carried out homology search in NCBI-BlasTn and BLASTP database, find that these several sequences belong to SVMPs family (amino acid sequence homology reaches as high as 85%) mostly, and FII-2 wherein and FII-4 are more conservative on sequence, belong to the ADAM family relevant with SVMPs family.It is big that functional experiment shows that FII-4 has a molten fibre, and the little characteristics of hemorrhagic so in 9 cDNA sequences that obtain, are preferentially carried out protein expression to FII-2 (plasmid that will contain this insertion sequence is decided to be the FII plasmid, this sequence is following be about to it be called the FII gene).Its possible aminoacid sequence (seeing Figure 12).Six, the expression of agkistrodon acutus venom fibrinolytic factor FII gene in intestinal bacteria
Method: get the 0.2ml bacterium (containing the bacterium colony of FII plasmid through extracting) that spends the night and add 20ml and contain in the SOB substratum of ammonia benzyl, 37 ℃ of joltings 3 hours are to OD
600About=0.5, adding the IPTG final concentration is 1 μ m, jolting 4 hours, and 1500rpm, centrifugal 20min removes supernatant, and precipitation adds 0.5ml water and 0.5ml SDS sample-loading buffer, 100 ℃ of water-bath 5min, 10,000rpm is centrifugal, gets on the supernatant 20 μ l sample and makes SDS-PAGE.
Result: through the intestinal bacteria lysate that contain FII plasmid of IPTG after inducing, on the SDS-PAGE collection of illustrative plates, exist a size to be about the band of 40KD, do not inducing and empty intestinal bacteria do not have this band (seeing Figure 13).Results suggest FII gene can be with the form of fusion rotein at expression in escherichia coli.
Seven, agkistrodon acutus venom fibrinolytic factor FII gene clone
Method: (seeing schema Figure 14)
1.PCR amplification FII gene
With the two-step pcr method sequence of signal is introduced 5 ' of goal gene and hold, introduce restriction site simultaneously.
Primer 1:5 ' AGA GAG GCT GAA GCT AAT CTT ACT CCT GAA C3 '
Primer 2: 5 ' CT CTC GAG AAA AGA GAG GCT GAA GCT AAT C3 '
Reverse primer
Primer 3:5 ' GAG CGG CCG CCT CAC GCC TCC AAA AGT TC3 '
Step:
(1) first round primer 1 is a forward, and primer 3 is that oppositely the FII plasmid is a template
(2) second take turns to primer 2 is a forward, and primer 3 is for being template with first round PCR product oppositely, and reaction conditions is identical with the first round.
2. recombinant plasmid PPIC9K-FII makes up (goal gene is cloned among the PPIC9)
Second takes turns the PCR product, after 1% agarose electrophoresis, cuts glue and reclaims the purpose fragment.The purpose fragment and the plasmid PPIC9 that reclaim are cut with XhoI, NotI enzyme respectively, electrophoresis, recovery, then in the T4 linked system, 16 ℃ connect 12 hours, transform and experience bacterium Top 10F, and coating contains the LB culture plate of ammonia benzyl, picking colony, the extracting plasmid, enzyme is cut evaluation, will contain the segmental plasmid purification of purpose.
3. recombinant expression plasmid PPIC9K-FII makes up (goal gene is cloned among the PPIC9K)
Have segmental plasmid PPIC9 of purpose and plasmid PPIC9K with Sac I and Not I double digestion, connect then, transform, the picking positive colony is identified a large amount of PPIC9K-FII plasmids of extracting.
Result: be amplimer with primer 1, primer 3 earlier, the FII plasmid is that template is carried out the PCR reaction, and again with primer 2, primer 3 is made amplimer, aforementioned PCR product is that template is carried out the PCR reaction, obtains a size and is about the consistent product of gene size 700bp and agkistrodon acutus venom fibrinolytic factor FII.Product is reclaimed the back use XhoI, NotI double digestion respectively, connect construction recombination plasmid PPIC9-FII then, identify through the double digestion reaction with plasmid PPIC-9.
SacI, NotI double digestion recombinant plasmid PPIC9-FII with downcutting than the small segment subclone to go in the same PPIC9K plasmid with SacI, NotI double digestion, set up expression of recombinant yeast plasmid PPIC9K-FII.Identify the product that has size to be, consistent with theoretical value (seeing Figure 15) for 700bp through the double digestion reaction.Check order with the terminal cessation method of the two deoxidations of Sanger, the result proves the dna sequence dna that contains agkistrodon acutus venom fibrinolytic factor FII among the recombinant plasmid PPIC9K-FII.
Eight, the expression of agkistrodon acutus venom fibrinolytic factor FII in yeast expression system
1. the conversion of yeast cell and screening
Method: with the PPIC9K-FII plasmid 50ug SacI linearization for enzyme restriction that extracts, with phenol, chloroform extracting, ethanol sedimentation, TE dissolving ,-30 ℃ frozen.3% polyoxyethylene glycol is adopted in the competent preparation of yeast.With linearizing plasmid, add the salmon sperm dna mixing, add in the yeast recipient cell, be uniformly coated on two RD flat boards, be inverted flat 96 hours for 30 ℃, bacterial strain on the RD plate is washed with distilled water, be coated on contain different concns G418 (0.5,1,2,3, on the YPD flat board of 4mg/ml, be inverted dull and stereotyped 30 ℃ of cultivations, lower concentration G418 flat board had bacterium colony since the 4th day, bacterium colony on the 6th day picking 3mg/ml G418 flat board, extracting DNA, PCR identifies.
Result: extract the PPIC9K-FII plasmid, use the SacI linearization for enzyme restriction, use PEG method transformed competence colibacillus yeast cell behind the purifying.After conversion product was coated on the YPD plate that contains G418, lower concentration (0.5mg/ml) plate began to have bacterium colony to grow on the 4th day.The 6th day dull and stereotyped 6 bacterium colonies of picking 3mg/ml G418.
2. the great expression of the screening of high expression level bacterial strain and FII
Method: picking 6 strains will confirm the yeast strain of goal gene through PCR, be seeded in respectively in the MD substratum of 10ml, 30 ℃ of joltings are spent the night, centrifugal, heavy adding BMMY substratum, 30 ℃ of joltings, mend 1% methyl alcohol every day, sampling every day is after 37 ℃ of reactions of sample and Fibrinogen, be SDS-PAGE, observe fibrinogenic degrading activity.Wherein 3 pipes of the 3rd day sample thief are frozen.
Get one of frozen bacterium, add in the 100ml MD substratum, 30 ℃ of joltings are spent the night, and are extended to 25 liters big bottles then, each 2 liters, 30 ℃ of joltings added 100 liters of jar fermenters in 24 hours, and 30 ℃ of BMMY substratum added 1% methyl alcohol in 24 hours, add every day to 1%, fermented centrifuging and taking supernatant, ultrafiltration and concentration to 2 liter 96 hours.
The result: after the yeast 6 strains cultivation of FII gene will be arranged, every day, sampling was each 1 part, behind each sample supernatant and 37 ℃ of insulations of Fibrinogen 1h, was SDS-PAGE.Wherein the 1st, 2 and No. 3 bacterial strain is being cultivated the 3rd day institute's sample thief supernatant promptly to the original hydrolytic action of scleroproein (seeing Figure 16), make positive control with natural five nevin fibrinolytic enzyme FII), identical to the main fragment that produces after fibrinogenic hydrolysis method and the hydrolysis with natural fibrinolytic factor FII.This three saccharomycetes strain of results suggest can efficient secretory expression fibrinolytic factor FII.
Through behind the methanol induction, collected the expression supernatant in 96 hours in fermentation, obtain the FII of great expression.Nine, purifying and the determination of activity of reorganization agkistrodon acutus venom fibrinolytic factor FII
1.DEAE-Sepharose FF anion-exchange chromatography
Method: (1) ultrafiltration and concentration
Supernatant through the Millipore ultrafiltration and concentration, when being concentrated into 2L, is added 2 times of volume 50mM NH4Ac ultrafiltration.
(2) after DEAE-Sepharose FF uses sample-loading buffer (50mM NH4AC PH8.0) balance columns, column volume on 50 times the sample behind the ultrafiltration and concentration, then with sample-loading buffer to baseline, add 0.1M NH4ACPH6.5 and wash impurity, then with the elutriant wash-out target protein that contains 0.2M NH4AC PH5.2.
Result: FII collects and expresses supernatant through after a large amount of expression, and the sample 200ml after 50 times of the hyperconcentration goes up the DEAE-sepharose post, obtains 5 protein peaks (seeing Figure 17) through behind the wash-out, and the elution peak of 0.2M NH4Ac pH5.2 has higher fibrinolytic.
2. hydrophobic chromatography (Buty-Toyopearl)
Method: the sample that will cross after the ion-exchange adds Buty-Toyopearl on the 2M NaCl, with 4 times of volumes contain 1M NaCl, 20mM PBS liquid is washed impurity, reach baseline, use 0.1M NaCl then, 20mM PBS liquid is washed impurity albumen, washes binding purposes albumen with 2mM PBS liquid at last.
The result: after active peak concentrated, further through the Buty-Toyopearl hydrophobic chromatography, obtain 3 egg peaks (seeing Figure 18), wherein the 3rd peak has higher fibrinolytic.With the 3rd peak collection, desalination, be drying to obtain reorganization agkistrodon acutus venom fibrinolytic factor FII.
3. the purity detecting of reorganization agkistrodon acutus venom fibrinolytic factor FII
Method: adopt SDS-PAGE, concrete operations are the same.
The result: the reorganization agkistrodon acutus venom fibrinolytic factor FII behind the visible purifying of SDS-PAGE is a single district band (seeing Figure 19).
4. the direct fibrinolytic activity of reorganization agkistrodon acutus venom fibrinolytic factor FII is measured
Method: adopt heating fibrin plate method, the fibrin plate for preparing was heated cool to room temperature 30 minutes at 85 ℃.Add reorganization agkistrodon acutus venom fibrinolytic factor FII 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, natural FII0.5mg/ml, urokinase 5000IU/ml and physiological saline respectively on heating fibrin plate surface.37 ℃ of insulations are after 12 hours, to dissolve dull and stereotyped cartographic represenation of area fibrinolytic vigor.
The result: heating fibrin plate method is measured the direct fibrinolytic activity of reorganization agkistrodon acutus venom fibrinolytic factor FII, the result shows, reorganization agkistrodon acutus venom fibrinolytic factor FII is according to amount ground solution fibrin, and the more natural FII of activity is strong, and (the solusphere area that reorganization agkistrodon acutus venom fibrinolytic factor FII 0.5mg/ml produces is 50mm
2, and the solusphere area that natural FII 0.5mg/ml produces is 40mm
2See Figure 20, table 2).Heating scleroproein to 85 ℃ will be wherein the Profibrinolysin deactivation, so the direct solution fibrin and do not rely on the activation of Profibrinolysin of FII.
The direct scleroproein activity of table 2 reorganization component I I
| Concentration (mg/ml) | Lysed area (mm 2) |
| 2 1 0.5 0.25 0.125 FII 0.5 | 147 110 50 28 14 40 |
5. reorganization agkistrodon acutus venom fibrinolytic factor FII is to human IgG and albuminous effect
Method: the human IgG solution 10 μ l that get 10mg/ml add reorganization agkistrodon acutus venom fibrinolytic factor FII (0.5mg/ml) 10 μ l again 37 ℃ of insulations in the Ependoff pipe.Add electrophoresis protein example treatment solution 10 μ l behind the insulation 24h, get 20 μ l and be SDS-PAGE.Human albumin with same concentration replaces IgG to repeat this experiment.
Result: the SDS-PAGE result's (seeing Figure 21) after human IgG, albumin and the reorganization agkistrodon acutus venom fibrinolytic factor FII effect.Behind human IgG and the reorganization agkistrodon acutus venom fibrinolytic factor FII effect 24h, its heavy chain has certain hydrolysis, and light chain still is kept perfectly.Behind human albumin and the reorganization agkistrodon acutus venom fibrinolytic factor FII effect 24h, peptide chain still is kept perfectly, and prompting reorganization agkistrodon acutus venom fibrinolytic factor FII does not have hydrolytic action to human albumin.
Ten, reorganization agkistrodon acutus venom fibrinolytic factor FII is to the effect of dog pulmonary thrombosis
Method: the pulmonary thrombosis model with reference to the Nowk element carries out.Get Beagle dog blood 15ml before the experiment.External solidifying after 2 hours as the thrombus of testing usefulness.Divide two groups at random with the Beagle dog, one group is that administration group, another group are physiological saline group, 6 every group.Use vetanarcol 30mg%, 1ml/kg.Femoral vein is separated in dog anesthesia back, S=6.0Fcobra or Humterhead conduit are inserted femoral vein, through hypogastric vein, right atrium, right ventricle, pulmonary trunk, selectivity enters the bottom right pulmonary artery.Dog thrombus with 15ml injects the bottom right pulmonary artery through conduit then, manually forms the bottom right pulmonary thrombosis.Variation with Digital Subtraction pulmonary arteriography method record bottom right PTE.After the bottom right pulmonary thrombosis was through 2 hours, beginning iv administration, reorganization agkistrodon acutus venom fibrinolytic factor FII dosage is 0.24mg/kg.Control group administered physiological saline 1ml/kg.Before the administration, after the administration 15,30,60,90,120min, be connected with efficient again with Digital Subtraction pulmonary arteriography method record bottom right pulmonary thrombosis respectively.Estimate the thrombolytic effect of FII with the pulmonary artery recanalization rate.
The result:
The agkistrodon acutus venom fibrinolytic factor of recombinating FII 0.24mg/kg group, bottom right pulmonary thrombosis dissolving fine (seeing Figure 22), administration is recanalization rate average out to 83.3% (seeing Table 3) after 1 hour.And the physiological saline group, the bottom right pulmonary thrombosis changes seldom, to 1 hour recanalization rate average out to 0.6% behind the physiological saline.
Table 3 reorganization component I I is to the thrombolytic effect of arterial thrombus model under the dog right lung
| Beagle dog(number) | Time after injection | Recovery rate of pulmonary artery (％) |
| 3 2 1 6 | 1h 1h - NS | ＞90 ＞80 ＞70 0.6 |
Agkistrodon acutus venom solution fibrin (former) No.4 gene and application thereof
＜110〉Zhongshan University
＜120〉agkistrodon acutus venom solution fibrin (former) No.4 gene and application thereof
<160>1
<210>1
<211>666bp
<212>DNA
＜213〉agkistrodon acutus (Agkistrodon acutus snake)
<220>
<221>CDS
<222>(1)...(666)
<400>1
atggagattg ttattgttgt ggaccattca atgtacacta aatacaatgg cgattcagat 60
aagataaaag catgggtata tgaaatgatc aacactataa gtgagagtta cagatatttg 120
tatatcgata taatagtgtc tgccttggaa atgtggtccg agaaagattt gattaatgtg 180
gagacatcag cggaaaatac tttgaaatca tttggagaat ggagagcgaa agatttgatt 240
catcgcataa gtcatgataa tgctcagtta ctcacggcca ctgacttcga tggaccaact 300
ataggattgg cttacgtggc cagcatgtgt gacccaaagc gttctgtagg agttgttcag 360
gatcatagct cagtaaatca tttggttgca attacactgg cccatgaaga atttgctcat 420
aatctgggcg ttcatcatga cgaaggttcc tgttcttgtg gtagtggtta cacatgcatt 480
atgtctcctg tgataaactc tggagagtta tcaaatattt cagcgattgt agttatatcc 540
aatgtcggga gtatatatcc gaaggagaac ccaccttgca ttctcaataa acccttgaga 600
acagatactg tttcaactcc agtttctgga aatgaacttt tggaggcggg aaaagattat 660
gactga666
Claims (5)
1, a kind of isolating polynucleotide No.4 gene, its nucleotides sequence is classified as:
5’-atggagattgttattgttgtggaccattcaatgtacactaaatacaatggcgattcagataagataaaag
catgggtatatgaaatgatcaacactataagtgagagttacagatatttgtatatcgatataatagtgtctgc
cttggaaatgtggtccgagaaagatttgattaatgtggagacatcagcggaaaatactttgaaatcatttgg
agaatggagagcgaaagatttgattcatcgcataagtcatgataatgctcagttactcacggccactgact
tcgatggaccaactataggattggcttacgtggccagcatgtgtgacccaaagcgttctgtaggagttgtt
caggatcatagctcagtaaatcatttggttgcaattacactggcccatgaagaatttgctcataatctgggc
gttcatcatgacgaaggttcctgttcttgtggtagtggttacacatgcattatgtctcctgtgataaactctgg
agagttatcaaatatttcagcgattgtagttatatccaatgtcgggagtatatatccgaaggagaacccacc
ttgcattctcaataaacccttgagaacagatactgtttcaactccagtttctggaaatgaacttttggaggcg
ggaaaagattatgactga-3’。
2, the polynucleotide in the claim 1 is characterized in that these polynucleotide are DNA.
3, a kind of carrier that contains the DNA of claim 2.
4, a kind of host cell of using the vector gene through engineering approaches of claim 3.
5, be used for the pharmaceutical composition of solution fibrin (former) with the caused embolism class diseases of antagonism thrombosis, the polynucleotide that wherein comprise claim 1 or 2 are as activeconstituents.
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