CN1380416A - Novel recombinant tissue type plasminogen activating enzyme and its preparation method - Google Patents
Novel recombinant tissue type plasminogen activating enzyme and its preparation method Download PDFInfo
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- CN1380416A CN1380416A CN 02111322 CN02111322A CN1380416A CN 1380416 A CN1380416 A CN 1380416A CN 02111322 CN02111322 CN 02111322 CN 02111322 A CN02111322 A CN 02111322A CN 1380416 A CN1380416 A CN 1380416A
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Abstract
The present invention utilizes PCR method to constitute the gene of new-type tissue plasminogen activating enzyme, i.e, remove digitaliform zone, epidermal growth factor zone and most of K1 trigone zone from coded natural tissue plasminogen activator molecule structure, but remain part of K1 trigone zone, k2 trigone zone and light chain zone, then uses yeast to make secretion, expression, separation and purification so as to obtain the invented new-type recombinant tissue plasminogen activator. The biological activity test shows that it possesses obvious fibrinolysis activity, can be further used for preparing new thrombolytics medicine.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of novel recombinant tissue type plasminogen activating enzyme and preparation method thereof.
Background technology
(Tissue typeplasminogen activator t-PA), is made up of 527 amino acids natural (also claiming wild-type) human histiotype plasminogen activator.Be the physiological activator of fibrinolytic system in the blood, plasminogen activation changes plasmin into specifically, the scleroproein in the plasmin energy thrombus, thereby make thrombolysis revascularization, therefore t-PA is used for the treatment of Acute Myocardial Infarction clinically, cerebral infarction, lung infraction, the thromboembolism treatment of multiple thrombus diseases such as Renal vein infraction, be thrombolytic drug preferably at present, but it exist the transformation period short, needs a large amount of at short notice administrations, cost an arm and a leg, and can cause the shortcoming of intracranialing hemorrhage.In order to overcome the shortcoming of wild-type t-PA, all be devoted to the R and D of novel thrombolytic drug both at home and abroad.The existing at present tissue-type plasminogen activator mutant that several obtain based on transforming on the architecture basics of wild-type t-PA.As reteplase (r-PA), TNK-tPA, n-PA etc.R-PA is spliced by 1～3 amino acids among the t-PA and 176～527 amino acids, utilizes genetic engineering technique synthetic in intestinal bacteria.TNK-tPA is the multisite mutant of wild-type t-PA molecule, T103N, and N117Q, KHRR (296-299) AAAA, this mutant is expressed in Chinese hamster ovary celI, and n-PA has lacked the F district and the E district of wild-type t-PA, N117Q, K
1K
2P, this mutant is also expressed in Chinese hamster ovary celI.Adopt following two kinds of approach production rt-PA mutant both at home and abroad at present, express in Chinese hamster ovary cell CHO (1) more, and express in intestinal bacteria Ecoli (2).In Chinese hamster ovary celI, express, the production cost height, valuable product, and expressing with intestinal bacteria Ecoli, expression product does not have activity when just synthetic, just can be activated make peptide chain folding again by sex change and renaturation process after external, yields poorly and the production cost height
Summary of the invention
The purpose of this invention is to provide a kind of novel recombinant tissue type plasminogen activating enzyme (Nr-PA), it is with domestic and international rt-PA's mutant of reporting is all different on gene structure at present.In addition, the present invention also provides the method for a kind of production novel recombinant tissue type plasminogen activating enzyme of the present invention.
The present invention has designed a kind of novel recombinant tissue type plasminogen activating enzyme molecule, and by the yeast secreting, expressing, separation and purification, thus novel recombinant tissue type plasminogen activating enzyme of the present invention obtained, can further obtain novel thrombolytic drug.
The present invention utilizes methyl alcohol nutritional type yeast (commercially available) (Pichia pastoris, also claim pichia spp) gene expression system, this system is a promotor with alcohol oxidase (AOX1), novel recombinant tissue type plasminogen activating enzyme (Nr-PA) gene stably is integrated on the yeast chromosomal through transforming, this yeast can be with activated expression product direct secretion in the culture supernatant of extracellular, and the protein excretion of self but seldom, thereby help the purifying preparation in downstream.
The present invention adopts following technical proposal and step: (1) design novel recombinant tissue type plasminogen activating enzyme molecular structure, remove natural tissues type plasminogen activator (human tissue type plasminogen activator, t-PA) 1-173 amino acids in the structure, keep the 174-527 amino acids, promptly remove the finger-type district (F district) of t-PA, epidermal growth factor subarea (E district) and most K
1The triangular space, retained part K
1The triangular space, K
2Triangular space and light chain district (P district).Utilize round pcr, with plasmid pcDNA3-tPA is template, design upstream and downstream primer carries out pcr amplification, obtain the Nr-PA gene, recombinate with plasmid pUC19 (commercially available) then, whether enzymolysis screening positive clone, nucleotide sequence analysis checking have obtained and have designed the Nr-PA gene that conforms in advance.The Nr-PA gene is recombinated with Yeast expression carrier pPIC9K (commercially available) again, forms expression plasmid pSTPA-G, and expression plasmid transforms the transformed yeast bacterium by electricity, and yeast is through screening the engineering strain that obtains high expression level.(2) fermentation and abduction delivering: with yeast culture base amplification engineering bacteria, carry out feed supplement with glycerine solution before inducing, arrive certain cell concentration (OD
600Be about 200) then carry out abduction delivering with methanol solution.Fermentation parameter is: temperature 30-32 ℃, oxygen capacity is controlled at 35 ± 5%, pH=5, stirring velocity and DO interlock.(3) with methanol induction yeast expression novel recombinant tissue type plasminogen activating enzyme (Nr-PA), expression product is present in soluble form expresses in the supernatant, centrifugal collection supernatant behind the engineering bacterium fermentation, pass through ultrafiltration and concentration again, gel-filtration, ion-exchange, the three-step approach purifying obtains product.(4) product has significant fibrinolytic through Determination of biological activity.
Embodiment
Embodiment:
1.Nr-PA the clone of gene and the structure of expression plasmid of yeast:
With plasmid pcDNA3-tPA (containing wild-type t-PA molecule full length sequence) is template, the upstream and downstream primer that design contains NotI restriction endonuclease site carries out pcr amplification, obtain the Nr-PA gene, amplified fragments reclaims through agarose gel electrophoresis, behind the purifying, product is recombinated with plasmid pUC19 behind NotI restriction endonuclease enzymolysis, enzymolysis screening positive clone, nucleotide sequence analysis confirm to have obtained and design novel recombinant tissue type plasminogen activating enzyme (Nr-PA) gene that conforms in advance.With NotI the Nr-PA gene is cut out then, connect into the corresponding NotI site of Yeast expression carrier pPIC9K, goal gene and expression vector reorganization, obtain expression plasmid pSTPA-G, expression plasmid transformed into escherichia coli JM109 extracts plasmid, identifies with corresponding enzyme enzymolysis, obtain distinctive fragment, confirm to have obtained positive colony.
2. the screening height copies the engineering strain that efficiently expresses.
Expression plasmid transforms methyl alcohol nutritional type yeast GS115 by electricity, make the reorganization of goal gene and yeast chromosomal, screen the engineering strain that efficiently expresses with G418, and with the method testing goal gene of PCR, examine and determine positive recombinant clone bacterium, the above-mentioned positive colony bacterium of picking is inoculated in the low nutrient medium BMG of 10ml and (contains 13.4g/L yeast nitrogen base (YNB), 4 * 10
-4The g/L vitamin H, 10g/L glycerine, the 100mmol/L potassiumphosphate, pH6.0) in, 30 ℃ of quick jolting 250r/min, optical density(OD) OD to the nutrient solution
600During=2-6, centrifugal reject BMG training liquid, sterilized water washing 1 time adds BMM training liquid again and (contains 13.4g/L yeast nitrogen base (YNB), 4 * 10
-4The g/L vitamin H, 5g/L methyl alcohol, the 100mmol/L potassiumphosphate pH6.0), is diluted to OD
600=1, add percent by volume every day and be 0.5% methyl alcohol, continued inducing culture 3 days, every 12 hours sampling 1ml, centrifugal reject precipitation was got culture supernatant 10 μ l, is added in the hole of accomplishing fluently in advance on the casein plate, measures its fibrinolytic.The result shows that expression product has significant fibrinolytic.
3. the fermentation expression of engineering bacteria
Height by last method screening copies the engineering strain of high expression level as engineering bacteria, carry out high density fermentation with the 5L fermentor tank then, from-70 ℃ of profound hypothermia refrigerators, take out kind of a daughter bacteria, at room temperature thaw, at following stroke of YPD flat board of 100 grades of cleanliness factor conditions of kind of daughter bacteria culturing room, cultivated 2-3 days in 30 ℃ of incubators, picking list bacterium colony from the YPD flat board, under 100 grades of cleanliness factor conditions, be inoculated in the 10ml BMG nutrient solution equally, 30 ℃ of overnight incubation, this is a primary seed solution, primary seed solution is added in the 90mlBMG training liquid again, and 30 ℃ of cultivations are until OD
600≈ 6, and this is a secondary seed solution, again with secondary seed solution, accrue in the access 5L fermentor tank, treat OD in the fermentor tank
600Reach about 200, beginning methanol induction, methanol induction stopped fermentation after 30 hours.The centrifugation thalline is collected supernatant and is carried out purifying.Fermentation parameter is: temperature 30-32 ℃, oxygen capacity is controlled at 35 ± 5%, pH=5, stirring velocity and DO interlock.
4. ultrafiltration and concentration desalination
With the centrifugal institute supernatant that obtains through the ultra-filtration equipment ultrafiltration and concentration and slough inorganic salt.
5.Sephadex G-75 and Q-Sepharose FF column chromatography
Earlier with Sephadex G-75 gel-filtration, then with 10 times of column volume PB damping fluid balance Q-Sepharose FF chromatographic columns, the direct upper prop of solution after the gel-filtration, Waters chromatographic instrument are controlled flow velocity and are detected protein peak after the ultrafiltration and concentration desalination.Behind the end of the sample, be washed till baseline with the PB damping fluid, the 0-1mol/lNaCl gradient elution is collected elution fraction, SDS-PAGE method analysis purposes albumen.
6. purity is identified and molecular weight determination
Sample carries out 15%SDS-PAGE, after the Coomassie brilliant blue R-250 dyeing, and PharmaciaImagemaster VDS sweep measuring purity, molecular weight.Target protein purity＞95%, molecular weight is about 39KD.
7. biological activity is identified: (1) casein plate solusphere method:
The learn from else's experience yeast culture supernatant 10 μ l of methanol induction directly are added on the casein plate in the hole, and 37 ℃ of wet boxes spend the night, and observe the size of solusphere, compare with standard t-PA solusphere, measure the fibrinolytic of expression product.The result shows that expression product has fibrinolytic, and fibrinolytic is greater than 3000IU/ml in the training liquid.(2) returning face plate method:
SDS-PAGE analyzes expression product, after electrophoresis finishes, with the gel dimidiation, half carries out Coomassie brilliant blue R-250 dyeing, as seen induces back yeast culture supernatant to be about the 39KD place band that concentrates at molecular weight, behind second half flush away SDS, be attached on the casein gel slab, after 37 ℃ of wet boxes spent the night, being equivalent to the 39KD place had a tangible lucent area, the casein that is this position is degraded, shows that expression product has fibrinolytic.
Description of drawings: Fig. 1 is a pcDNA3-tPA plasmid map of the present invention.Fig. 2 is an expression plasmid pSTPA-G collection of illustrative plates of the present invention.Fig. 3 is that expression plasmid pSTPA-G of the present invention cuts the identification and analysis collection of illustrative plates through enzyme.
Wherein 1: standard nucleic acid molecular weight (λ DNA/Hind III+EcoR I.);
2:pSTPA-G cuts the fragment that is produced with Not I enzyme: (1.0kb, 9.3kb);
3:pSTPA-G cuts the fragment that is produced with EcoR I enzyme: (0.47kb, 9.8kb)
4:pSTPA-G cuts the sheet (2.5kb+3.0 that is produced with Xhol I and Sal I enzyme
Kb+4.8kb); Fig. 4 is that casein plate solusphere method of the present invention is measured the expression product fibrinolytic
The fibrinolytic of 1-5.t-PA standard substance (100IU/ml, 50IU/ml, 25 wherein
IU/ml，12.5IU/ml，6IU/ml)
6. negative control product
7. the expression supernatant liquor before the engineering bacteria yeast fermentation is induced
8-13. engineering bacterium fermentation is induced the fibrinolytic of 1-6 days the expression supernatant liquor in back
Novel recombinant tissue type plasminogen activating enzyme sequence table .txt
Sequence table＜110〉Fudan University＜120〉a kind of novel recombinant tissue type plasminogen activating enzyme and preparation method thereof＜130 without＜160〉1＜170〉PatentIn version 3.1＜210 1＜211〉354＜212〉PRT＜213〉recombinant protein＜220＜221〉CHAIN＜222〉(1) .. (354)＜223〉＜400〉1Ser Glu Gly Asn Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg1,5 10 15Gly Thr His Ser Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn
20 25 30Ser?Met?Ile?Leu?Ile?Gly?Lys?Val?Tyr?Thr?Ala?Gln?Asn?Pro?Ser?Ala
35 40 45Gln?Ala?Leu?Gly?Leu?Gly?Lys?His?Asn?Tyr?Cys?Arg?Asn?Pro?Asp?Gly
50 55 60Asp?Ala?Lys?Pro?Trp?Cys?His?Val?Leu?Lys?Asn?Arg?Arg?Leu?Thr?Trp65 70 75 80Glu?Tyr?Cys?Asp?Val?Pro?Ser?Cys?Ser?Thr?Cys?Gly?Leu?Arg?Gln?Tyr
85 90 95Ser?Gln?Pro?Gln?Phe?Arg?Ile?Lys?Gly?Gly?Leu?Phe?Ala?Asp?Ile?Ala
Novel recombinant tissue type plasminogen activating enzyme sequence table .txt
100 105 110Ser?His?Pro?Trp?Gln?Ala?Ala?Ile?Phe?Ala?Lys?His?Arg?Arg?Ser?Pro
115 120 125Gly?Glu?Arg?Phe?Leu?Cys?Gly?Gly?Ile?Leu?Ile?Ser?Ser?Cys?Trp?Ile
130 135 140Leu?Ser?Ala?Ala?His?Cys?Phe?Gln?Glu?Arg?Phe?Pro?Pro?His?His?Leu145 150 155 160Thr?Val?Ile?Leu?Gly?Arg?Thr?Tyr?Arg?Val?Val?Pro?Gly?Glu?Glu?Glu
165 170 175Gln?Lys?Phe?Glu?Val?Glu?Lys?Tyr?Ile?Val?His?Lys?Glu?Phe?Asp?Asp
180 185 190Asp?Thr?Tyr?Asp?Asn?Asp?Ile?Ala?Leu?Leu?Gln?Leu?Lys?Ser?Asp?Ser
195 200 205Ser?Arg?Cys?Ala?Gln?Glu?Ser?Ser?Val?Val?Arg?Thr?Val?Cys?Leu?Pro
210 215 220Pro?Ala?Asp?Leu?Gln?Leu?Pro?Asp?Trp?Thr?Glu?Cys?Glu?Leu?Ser?Gly225 230 235 240Tyr?Gly?Lys?His?Glu?Ala?Leu?Ser?Pro?Phe?Tyr?Ser?Glu?Arg?Leu?Lys
245 250 255Glu?Ala?His?Val?Arg?Leu?Tyr?Pro?Ser?Ser?Arg?Cys?Thr?Ser?Gln?His
260 265 270Leu?Leu?Asn?Arg?Thr?Val?Thr?Asp?Asn?Met?Leu?Cys?Ala?Gly?Asp?Thr
275 280 285Arg?Ser?Gly?Gly?Pro?Gln?Ala?Asn?Leu?His?Asp?Ala?Cys?Gln?Gly?Asp
290 295 300
Novel recombinant tissue type plasminogen activating enzyme sequence table .txtSer Gly Gly Pro Leu Val Cys Leu Asn Asp Gly Arg Met Thr Leu Val305 310 315 320Gly Ile Ile Ser Trp Gly Leu Gly Cys Gly Gln Lys Asp Val Pro Gly
325 330 335Val?Tyr?Thr?Lys?Val?Thr?Asn?Tyr?Leu?Asp?Trp?Ile?Arg?Asp?Asn?Met
340 345 350Arg?Pro
Nucleotide primer sequence table .txt
Nucleotides sequence tabulation＜110〉Fudan University＜120〉a kind of novel recombinant tissue type plasminogen activating enzyme and preparation method thereof＜130〉nothing＜160〉2＜170〉PatentIn version 3.1＜210〉1＜211〉24＜212〉DNA＜213〉nucleotides＜400〉1gcggccgcct ctgagggaaa cagt 24＜210〉2＜211〉35＜212〉DNA＜213〉nucleotides＜400〉2atagcggccg ctcacggtcg catgttgtca cgaat 35
Claims (2)
1. a novel recombinant tissue type plasminogen activating enzyme is characterized in that removing 1-173 amino acids in the natural t-PA polypeptide chain, keeps the 174-527 amino acids, the finger-type district (F district) of promptly removing natural t-PA, epidermal growth factor subarea (E district) and most K
1Triangular space, and retained part K
1The triangular space, K
2Triangular space and light chain district (P district).
2. the preparation method of a novel recombinant tissue type plasminogen activating enzyme, it is characterized in that adopting the following step: (1) is template with plasmid pcDN3-tPA, design upstream and downstream primer makes up the novel recombinant tissue type plasminogen activating enzyme gene by pcr amplification, recombinate with plasmid pUC19, the enzymolysis screening positive clone, the nucleotide sequence analysis checking; (2) novel recombinant tissue type plasminogen activating enzyme gene and yeast expressed plasmid vector reorganization, construction expression plasmid pSTPA-G, transformed yeast bacterium, the engineering bacteria of screening high expression level; (3) fermentation and abduction delivering: with low salt culture medium amplification engineering bacteria, cell concentration reaches OD
600Be that abduction delivering is carried out with methanol solution in 200 backs, fermentation parameter is: temperature 30-32 ℃, oxygen capacity is controlled at 35 ± 5%, pH=5, stirring velocity and DO interlock; (4) the yeast expressed novel recombinant tissue type plasminogen activating enzyme of methanol induction is behind the engineering bacterium fermentation, through centrifugal, collect supernatant, ultrafiltration and concentration, gel-filtration and ion-exchange three-step approach purifying obtain the end product novel recombinant tissue type plasminogen activating enzyme.
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| CN1172000C CN1172000C (en) | 2004-10-20 |
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