CN1057125C - Five urokinase variant gene and its expression in colibacillus - Google Patents
Five urokinase variant gene and its expression in colibacillus Download PDFInfo
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- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention belongs to the technical field of producing polypeptide medicines by means of gene engineering in a biological pharmaceutical industry and aims at using site-directed mutagenesis introduced by oligonucleotide to reform human urokinase genes and obtain 5 kinds of urokinase variant genes, namely (Lys 151-Glu) UK, (Lys 151-Glu, Arg 154-Gly) UK, (Lys 151-Glu, Arg 154-Glu) UK, (Arg 154-Gly) UK and (Arg 154-Glu) UK which can be efficiently expressed in colibacillus. Through separation and purification, 5 kinds of urokinase variant protein with good thrombus-dissolving effect and high selectivity to fibrin in thrombus are created to solve the problem of hemorrhagic side effect generated when thrombotic diseases are clinically cured and improve the safety of medication.
Description
The genetically engineered that the invention belongs in the biological-pharmacy is produced the polypeptide drugs technical field.
Urokinase (UK) is a kind of important plasminogen activator, is mainly secreted by kidney cell in human body.Its precursor uPA promptly becomes double-stranded polymer urokinase after activation such as plasmin, but activatory urokinase plasminogen activation produces plasmin, and plasmin can a large amount of hydrolysis of fibrin.This series reaction plays crucial effect in the body fibrinolytic system, can keep normal blood clotting-fibrinolytic balance in the body, guarantees the normal physiological function of body blood circulation and histoorgan.But under pathological conditions, the body fibrinolytic system is not enough to eliminate thrombus, and is essential by venous perfusion or local injection thrombolytic drug-plasminogen activator, and effectively the scleroproein in the thrombus is removed thrombus.Clinically, urokinase has been widely used in treating Acute Myocardial Infarction, cerebral thrombosis, myocardial ischemia necrosis, diabetes complicated multiple vessel embolism such as phlebothrombosis.
Because urokinase does not have selectivity to scleroproein, therefore when the treatment thrombus disease, also can activate the Profibrinolysin in the circulation of blood non-specificly, produce a large amount of free plasmins, cause Profibrinolysin in the circulation of blood and Fibrinogen equal size to reduce, side effect such as cause bleeding
For this reason, European and American countries is all making great efforts the development plasminogen activator, to solve the hemorrhage side effect of thrombolytic therapy.For urokinase, their work mostly concentrates in the heterozygote research between the close scleroproein structural domain of urokinase activity structural domain and other plasma proteins, really transform as number seldom, the rarely seen example that the success of purpose transformation is arranged to what urokinase itself carried out.
At home, Nanjing University just carried out the research of the protein engineering of country's " 863 " task-uPA since 1987.By computer assisted molecular designing, our mould has been built the three-dimensional structure of urokinase, and the positive charge amino-acid residue of finding to be comprised in the 149-158 fragment in the urokinase molecule is to kringle structural domain and the fibrinous potential restraining effect that is combined with; Competition shows also that in conjunction with test this fragment of synthetic or separation and purification has had strong inhibitory effects to t-PA really with fibrinous the combination, and part is replaced the positive charge amino-acid residue in this fragment, and competitive effect obviously reduces.Therefore, we think that the existence of the positive charge amino-acid residue in this fragment is to cause urokinase scleroproein not to be had one of architecture basics of affinity, remove these positive charge amino-acid residues or into the negative charge amino-acid residue of changing, urokinase is obtained fibrinous affinity.
The theoretical foundation and the chemical test result that provide according to protein engineering is provided, utilize oligonucleotide mediated rite-directed mutagenesis method, transform the urokinase gene, obtain five kinds of urokinase variant genes, that is: change the 151st Methionin (Lys) codon AAG in the urokinase gene into L-glutamic acid (Glu) codon GAG constructed urokinase variant gene (Lys
151→ Glu) UK; Change the 151st Lys codon AAG in the urokinase gene into Glu codon GAG and the 154th arginine (Arg) codon AGG changes the constructed urokinase variant gene (Lys of glycine (Gly) codon (GGG) into
151→ Glu, Arg
154→ Gly) UK; All change the 151st Lys codon AAG and the 154th Arg codon AGG in the urokinase gene into Glu codon GAG constructed urokinase variant gene (Lys
151→ Glu, Arg
154→ Glu) UK; Change the 154th Arg codon AGG in the urokinase gene into Gly codon GGG constructed urokinase variant gene (Arg
154→ Gly) UK; Change the 154th Arg codon AGG in the urokinase gene into Glu codon GAG constructed urokinase variant gene (Arg
154→ Glu) UK, and be implemented in efficiently expressing in the intestinal bacteria, through separation and purification, it is effective to create five kinds of thrombolysis, and scleroproein in the thrombus is had higher optionally urokinase variant albumen, to solve the clinical treatment Acute Myocardial Infarction, cerebral thrombosis, the myocardial ischemia necrosis, the hemorrhage side effect during multiple vessel embolism such as diabetes complicated phlebothrombosis improves drug safety.
Technology contents of the present invention comprises:
Pass through rite-directed mutagenesis, make up five kinds scleroproein had higher optionally urokinase variant gene, that is: change the 151st Lys and the 154th these two positive charge amino-acid residues of Arg in the urokinase into uncharged Gly or electronegative Glu respectively or simultaneously, the competitive inhibition when urokinase being combined with scleroproein with the positive charge in the 149-158 fragment in the attenuating urokinase.The present invention adopts synthesis method at random according to above-mentioned design philosophy, has synthesized a pair of complementary mutant primer, makes original Lys and Arg codon sport Glu and Glu/Gly codon respectively.Two primers through annealing, phosphorylation, 14 the amino acid whose dna fragmentations between the urokinase variant 150-163 that obtain encoding, its length is 41/45 base.Simultaneously, natural urokinase gene BalI and EcoRI double digestion, remove the amino acid whose encode fragment of 150-163, link to each other with above-mentioned annealing primer, the variant gene that obtains is through sequential analysis, filter out five kinds of different urokinase variant genes respectively, utilize BspHI and the NcoI characteristics of isocaudarner each other again, get variant gene with BspHI and HindIII double digestion, insert between the NcoI and HindIII point of contact of expression vector pKK233-2, transformed into escherichia coli JA221 obtains positive colony through screening, induce through 37 ℃ of cultivations and 0.1mM IPTG, make five kinds of urokinase variant protein expressions, detect, show that five kinds of proteic expression amounts of urokinase variant all account for more than 10% of bacterial protein through SDS-PAGE, molecular weight is 43kD, conforms to calculated value.
After a large amount of cultivation of engineering bacteria and expressing the urokinase variant protein expression, centrifugal collection thalline, carrying out ultrasonic bacteria breaking, centrifugal collection inclusion body, through external change renaturation, last CM-cellulose chromatography and antibody affinity column chromatography are collected the wash-out honeybee, measure the fibrin plate lytic activity, active part is merged, and with the SDS-PAGE detection, after the activation of immobilization plasmin obtains the urokinase variant gene that height ratio is lived.
Advantage of the present invention is that to have created five kinds of thrombolysis effective, scleroproein is had than highly selective, the novel thrombolytic drug that hemorrhage side effect is little, its key is to change the 151st and 154 two amino acid whose charge property in the urokinase respectively or simultaneously, make urokinase obtain preferably fibrinous selectivity be can be used as the treatment Acute Myocardial Infarction, cerebral thrombosis, the myocardial ischemia ring is dead, diabetes complicated multiple vessel embolism such as phlebothrombosis.
Be embodiments of the invention below:
1. five kinds of structures that scleroproein had higher optionally urokinase variant gene.
Synthetic a pair of complementary mutant primer, its sequence is as follows respectively: 5 '--C CAA AAG ACT CTG AGG CCC CGC TTT AAG ATT ATT GGG GGA G--3 '
G GA5′--AA?TTC?TCC?CCC?AAT?AAT?CTT?AAA?GCG?GGG?CCT?CAG?AGT?CTTTTG?G--3′
TC C anneals through phosphorylation, and 14 amino acid whose dna fragmentations between the urokinase 150-163 position obtain encoding.With urokinase gene BalI and the EcoRI double digestion that is cloned among the pUC9, remove amino acid whose encode fragment between the 150-163 position, link to each other with above-mentioned annealing fragment, Transformed E .coli, through screening by hybridization (is probe with first primer), select all positive colonies, refabrication DNA, with and urokinase 175-180 position between the sub-complementary primer of amino acid code carry out the double chain DNA sequence analysis, select five kinds of urokinase variant genes, all sport the urokinase variant gene (Lys of Glu with the 151st and 154 two amino acid
151→ Glu, Arg
154→ Glu) UK is an example, express.
2. utilize BspHI and the NcoI characteristics of isocaudarner each other, get variant gene with BspHI and HindIII double digestion, insert between the NcoI and HindIII point of contact of expression vector pKK233-2, transformed into escherichia coli JA221, obtain positive colony through screening, the picking positive colony is seeded in 2mlLB/Amp (the 100 μ g/ml) nutrient solution, 37 ℃ of shaking culture 8 hours, be seeded in 40ml LB/Amp (the 50 μ g/ml) nutrient solution in 2% ratio, 37 ℃ of shaking culture 8 hours are seeded in 200ml LB/Amp (the 50 μ g/ml) nutrient solution in 20% ratio again, 37 ℃ of shaking culture 1 hour, the IPTG that adds preheating is to final concentration 0.1mM, 37 ℃ of shaking culture 1.5 hours.
3. with medium centrifugal, collect thalline, be suspended in pH7.5 100mM and contain in the PBS solution of 0.5M Nacl, carrying out ultrasonic bacteria breaking 10 minutes (ultrasonic 15 seconds, intermittently 15 seconds), centrifugal 20 minutes of 3000rpm, collecting precipitation suspends with the pH7.5 100mM Tris-Cl solution that contains 5M urea, places 30 minutes for 4 ℃, centrifugal 20 minutes of 3000rpm, collecting precipitation dissolved centrifugal 20 minutes of 15000rpm with the pH7.5 100mM Tris-Cl solution that contains 8M urea and 50mM 2 mercapto ethanol 16 hours in 4 ℃, discard precipitation, supernatant is to containing 8M urea, and pH7.5 100mM Tris-Cl solution was dialysed 2 hours, splashes into to contain 2M urea again, 5mMEDTA, 0.005%Tween, 1.25mMGSH, 0.25mMGSSG, in the 0.05M Tris-Cl solution of pH11.0, in 4 ℃ of renaturation 24 hours.Renaturation solution after 16 hours, transfers to 4.0 with pH value of solution, last CM-cellulose chromatography to the 0.01MPBS dialysis, use pH5.0, contain the PBS eluant solution of 0.5M NaCl, elutriant is gone up the antibody affinity column chromatography after dialysing, with the PBS flush away foreign protein that contains 0.5M NaCl, again with containing 3.5M MgCl
2The PBS wash-out, collect the wash-out honeybee, measure the fibrin plate lytic activity, active part is merged, detect with SDS-PAGE, through coomassie brilliant blue staining, sample is a band, the working sample protein concentration mixes sample than the ratio of the dried glue of 1g with the 1mM sample with plasmin-sepharose CL, 37 ℃ were slowly stirred 1 hour, centrifugal 20 minutes of 5000rpm, get supernatant, freeze-drying concentrates, and promptly gets than living to be the pure product of urokinase variant albumen of 110000IU/mg.
Claims (2)
1 one kinds have higher optionally urokinase variant gene to scleroproein, it is characterized in that, it is selected from a kind of in the following urokinase variant gene: by oligonucleotide mediated rite-directed mutagenesis, change the 151st Methionin (Lys) codon AAG in the urokinase gene into the constructed urokinase variant gene of L-glutamic acid (Glu) codon GAG (UK of Lys151 → Glu); Change the 151st Lys codon AAG in the urokinase gene into Glu codon GAG and the 154th arginine (Arg) codon AGG and change the constructed urokinase variant gene of glycine (Gly) codon (GGG) (Lys151 → Glu, the UK of Arg154 → Gly) into; All change the 151st Lys codon AAG in the urokinase gene and the 154th Arg codon AGG into the constructed urokinase variant gene of Glu codon GAG (Lys151 → Glu, the UK of Arg154 → Glu); With the 154th Arg codon AGG in the urokinase gene is the constructed urokinase variant gene of the Gly numeral GGG (UK of Arg154 → Gly); Change the 154th Arg codon AGG in the urokinase gene into the constructed urokinase variant gene of the Glu codon GAG (UK of Arg154 → Glu).
2 according to the described application that scleroproein is had higher optionally urokinase variant gene of claim 1, it is characterized in that gained urokinase variant albumen can be used as preparation treatment Acute Myocardial Infarction, cerebral thrombosis, the myocardial ischemia necrosis, the medicine of diabetes complicated multiple vessel embolism such as phlebothrombosis.
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| CN1102438A (en) * | 1993-07-15 | 1995-05-10 | 格吕伦塔尔有限公司 | Bifunctional variants of urokinase with improved fibrinolytic properties and thrombin-inhibiting activity |
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| CN1102438A (en) * | 1993-07-15 | 1995-05-10 | 格吕伦塔尔有限公司 | Bifunctional variants of urokinase with improved fibrinolytic properties and thrombin-inhibiting activity |
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