CN1384199A - Recombinant expression vector expressing human pancreatic tissue kallikrein gene and prepn of human pancreatic tissue kallikrein - Google Patents
Recombinant expression vector expressing human pancreatic tissue kallikrein gene and prepn of human pancreatic tissue kallikrein Download PDFInfo
- Publication number
- CN1384199A CN1384199A CN 02103538 CN02103538A CN1384199A CN 1384199 A CN1384199 A CN 1384199A CN 02103538 CN02103538 CN 02103538 CN 02103538 A CN02103538 A CN 02103538A CN 1384199 A CN1384199 A CN 1384199A
- Authority
- CN
- China
- Prior art keywords
- kallikrein
- gene
- expression vector
- human pancreatic
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to secretion or non-secretion recombinant vector carrying mature protein gene coding human pancreatic kallikrein and its transformed host cell. The present invnetion also relates to the gene engineering technology method of preparing recombinant human pancreatic kallikrein. The expression vector of the present invention can express effectively protein product with pancreatic kallikrein activity and has fused His affinity sequence and the expression product containing the affinity sequence. During separation and purification, His affinity resin may be used in chromatographic affinity purification. The present invention lays the foundation for developing humanized tissue kallikrein gene engineering product and medicine.
Description
Technical field
The present invention relates to a kind of recombinant expression vector of carrier's pancreatic kallikrein gene, relate in particular to the recombinant expression vector of carrier's pancreatic tissue kallikrein mature protein gene.
The invention still further relates to by this recombinant expression vector transformed host cells.
The invention still further relates to using gene engineering technique and prepare the method for the kallikrein of recombinating.
Background technology
Human kallikrein is divided into two big class, i.e. plasma kallikrein and tissue kallikreins.Tissue kallikrein is the compound of cluster similar performance, can be synthetic by liver, pancreas, cranial nerve etc.The tissue kallikrein gene encoded protein is at first expressed and is generated the precursor intact proteins, and this precursor protein is in vivo through becoming the maturation protein of biologically active after the proteolytic enzyme cutting processing.Tissue kallikrein gene coding tissue kallikrein (E.C.3.4.21.35), it is a kind of serine protease, generates by the low-molecular-weight prokinin of cracking to have vasoactive kassinin kinin, the latter participates in a series of physiology of body and pathologic process.
One, the physiological action of pancreatic kallikrein
1. the relation of pancreatic kallikrein and body blood pressure regulation and renal function
Kallikrein and kinin system (KKS) are to keep an important component part of depressurizing system in the blood pressure balance, with natriuretic peptide system, EDRF/NO, PGE
2/ PDI
2Diastole Deng the fellowship blood pressure is regulated.The functional defect of KKS or inhibition have participated in hypertensive generation probably and have kept.(Kallikrein KK) is the important member of body kallikrein kinin system in tissue kallikrein.KK is in many positions of human body wide expression, by with the interaction of other vaso-active substances, participate in the adjusting of renal function.Tissue kallikrein can cracking low molecular weight kininogen (kininogen) generates have vasoactive kassinin kinin (kininpeptide), the latter can with bradykinin (bradykinin) B
2Acceptor combines and produces physiological effect widely, comprises arteriole in the nephrectasia, and renal blood flow increasing suppresses juxtaglomerular cell secretion feritin and promotes medullary epithelium secretion PGE
2Suppress the heavily absorption of proximal convoluted tubule to sodium, water; After entering circulation of blood, the expansion peripheral blood vessel increases vascular permeability, makes blood pressure drops, and promotes the transhipment of ionogen and glucose.Studies show that when nephridial tissue was impaired, the kallikrein that is produced by tubule reduced, kidney kassinin kinin level descends.In the nephrotic syndrome rat model due to the anti-GBM, the UK level obviously reduces, and is inversely proportional to the proteinuria degree.Because the kallikrein in the urine derives from kidney, show self balance of the adjusting of organizing KK to participate in renal function and blood pressure.With the KK of rat tissue behind the purifying to raising the Dah of high salt diet
1The responsive rat of-salt carries out long-time perfusion to be found, the uriniferous tubules sclerosis of rat alleviates.Show, impose on and organize KK that the blood pressure regulation and the renal function of body are recovered all to play an important role.
2. the relation of pancreatic kallikrein and cardiovascular and cerebrovascular diseases
Pancreatic kallikrein can the low-molecular-weight prokinin generation of cracking have vasoactive kassinin kinin, and the expansion capillary vessel increases vascular permeability, microcirculation improvement.Pancreatic kallikrein is again a kind of activation factor simultaneously, can make the fiber activation of zymogen become cellulase, make insoluble Taka-proteinase be hydrolyzed into the little peptide of solubility, thereby diseases such as cerebral infarction, atherosclerosis played therapeutic action, and can treat thrombus, pre-preventing thrombosis forms again.
3. pancreatic kallikrein is to the influence of reproduction
Studies show that pancreatic kallikrein can improve fibrinolytic, the expansion surrounding blood vessel promotes the androgone hyperplasia, improves motility of sperm, promotes seminal fluid liquefaction.Behind rat injection pancreatic kallikrein, not only can increase the generation of sperm, and can promote sperm in external mobility.Someone utilizes pancreatic kallikrein treatment male infertility, obtains obvious curative effects.
4. pancreatic kallikrein is to the effect of other diseases
Studies show that pancreatic kallikrein all has better curative effect to diabetic nephropathy, retinal vein occlusion etc.
Two, present Research
The pancreatic kallikrein that uses all is the product by the extraction of pig internal organs, purifying at present.Still the source pancreatic kallikrein product of having no talent utilizes the research of genetic engineering technique production recombinant human pancreatic tissue kallikrein not appear in the newspapers as yet.
Summary of the invention
At the defective of prior art, an order of the present invention is to provide the recombinant expression vector that carries coding people source pancreatic kallikrein mature protein gene.
Another purpose of the present invention is to provide by this recombinant vectors transformed host cells.
A further object of the present invention is to provide a kind of method of utilizing genetic engineering technique to prepare the recombinant human pancreatic tissue kallikrein.
According to an aspect of the present invention, gene order according to the GeneBank report, utilize computer aided system to design the PCR primer, from Chinese's pancreatic tissue, extract RNA, the laggard performing PCR amplification of reverse transcription, the DNA that clone's acquisition has SEQ ID NO:1 sequence, its encoded polypeptides sequence such as SEQ ID NO:2.
The gene of the present invention clone's coding pancreatic tissue kallikrein has a base different with the Human Pancreas Kallikrein gene of GenBank report.With translation initiation codon ATG is 1, and the 340th bit base of the present invention clone's kallikrein gene is A, and that Genbank reports is G.The codon Genbank of coded amino acid is GAC, and Chinese people is AAC, and amino acids coding Genbank is that (Asp, Genbank), China is N (Asn) artificially for aspartic acid.Aspartic acid and N molecular weight are approaching, and Asp is 133.1, and Asn is 132.1, and structure is also close, but both side-chain radical differences.Asp is an acidic amino acid, and Asn is meta-alkalescence then.The amino acid whose variation in this position may exert an influence to the space structure and the activity of kallikrein.
In the process of synthetic pancreatic tissue kallikrein, will further be cut and be processed into sophisticated kallikrein with biologic activity by proteolytic enzyme by prekallikrein (by the kallikrein intact proteins of kallikrein full-length gene expression).Therefore, the activity of the kallikrein intact proteins of being expressed by clone's full-length gene is significantly less than the activity of kallikrein maturation protein.The present invention utilizes the computer software analysis to compare the aminoacid sequence of people (pancreatic tissue), pig, rat, mouse kallikrein, infer that prekallikrein (kallikrein intact proteins) is converted to the possible albumen cleavage site of kallikrein (kallikrein maturation protein) and the aminoacid sequence of kallikrein maturation protein, the synthetic pcr primer thing, from clone's human pancreatic tissue kallikrein gene complete sequence, access the gene of coding kallikrein maturation protein, sequence shown in SEQ ID NO.3.
According to a further aspect in the invention, provide the recombinant vectors that comprises SEQ ID NO:3 sequence.Wherein carrier can be with forms such as plasmid, virion and phages, and the recombinant vectors of structure can be expressed the human pancreatic tissue kallikrein maturation protein with high biologic activity.In one embodiment of the invention, make up the carrier of plasmid form, in another embodiment of the invention, made up the transfer vector of baculovirus form.
Can target DNA be inserted in the carrier by several different methods, generally speaking, dna sequence dna of the present invention can be inserted on the suitable restriction endonuclease sites by certain methods well known by persons skilled in the art.DNA in the expression vector operationally is connected with a suitable expression regulation sequence (promotor) to instruct the synthetic of mRNA, and expression vector also contains the ribosome bind site and the transcription terminator that are used for translation initiation.Expression vector contains the phenotypic character gene that is useful on the screening transformed host cell simultaneously.The plasmid or the virus that are used for construction of expression vector can obtain by commercial sources.
In the preferred embodiment of the present invention, by human pancreatic tissue kallikrein mature protein gene (SEQ ID NO.3) being inserted restriction enzyme EcoR1, the Xho1 site of plasmid pET20b, be built into secreted expression carrier, this carrier can go out to have the human tissue kallikrein maturation protein of high biologic activity at expression in escherichia coli, and, make the target protein of expressing be distributed in the cytoplasm at N end fusion signal peptide sequence; In another preferred embodiment of the present invention, by expressing human pancreatic tissue kallikrein mature protein gene (SEQ ID NO.3) being inserted restriction enzyme EcoR1, the Xho1 site of plasmid pE28, be built into the nonsecreting type expression vector, this carrier can go out to have tissue kallikrein's maturation protein of high biologic activity at expression in escherichia coli.More particularly, the present invention is further merged His Tag sequence at this gene C end, makes the target protein of expressing be easy to carry out affinity chromatography purification.In another preferred embodiment of the present invention,, construct baculovirus transfer vector with EcoR1, the Xho1 site that DNA of the present invention inserts virus vector pBacPAK9.
According to another aspect of the invention, further provide by carrier transformed host cells of the present invention, host cell can comprise prokaryotic cell prokaryocyte and eukaryotic cell, the prokaryotic cell prokaryocyte that wherein is used to transform can comprise various bacteriums, in a preferred embodiment of the invention, with the host cell of intestinal bacteria as carrier of the present invention.The eukaryotic cell host who is used to transform can be higher eucaryotic cells (as mammalian cell) or the low eukaryotic cell (as yeast cell) that waits, a preferred embodiment of the present invention with the insect cell of cultivation as host cell.
The conversion of host cell can be passed through the whole bag of tricks, methods such as calcium phosphate precipitation, the mediation of DEAE-dextran, electroporation for example, in one embodiment of the invention, the baculovirus transfer vector that adopts the present invention to make up imports DNA of the present invention the insect cell of cultivating by cotransfection, by high glycosylation, it has more near the structure of native protein and the enzyme activity of Geng Gao at people's pancreas tissue kallikrein protein of expression in escherichia coli at the albumen of expressed in insect cells.
According to another aspect of the invention, provide the method for preparing the kallikrein of recombinating.Method of the present invention comprises: the exercisable expression regulation sequence that is connected in of the gene of the pancreas tissue kallikrein maturation protein of 1) will encoding (SEQ IDNO.3), construction of expression vector; 2) transform protokaryon or eukaryotic cell with this expression vector, form host cell; 3) under suitable culture condition, cultivate host cell of the present invention; 4) from nutrient solution, isolate and have the active protein of kallikrein; 5) obtain the reorganization kallikrein with affinitive layer purification.
The present invention has successfully cloned coding and has had the active proteic new gene of pancreatic kallikrein from Chinese's pancreatic tissue, and further from this gene, accessed the gene fragment of coding human pancreatic tissue kallikrein maturation protein, made up secretor type, nonsecreting type expression vector and the rhabdovirus expression vector that carries coding human pancreatic tissue kallikrein mature protein gene with this gene fragment, and then obtained protokaryon and eukaryotic cell host by expression vector conversion of the present invention.Expression vector of the present invention can efficiently express the pancreas kallikrein with high biologic activity, the enzymatic spectrophotofluorimetry that adopts people such as improved potentiometric titration of International Pharmaceutical Federation (FIP), Nat'l Pharmaceutical ﹠ Biological Products Control Institute and Abe M to use has detected the activity of expression vector expressed proteins product of the present invention, and the expression vector that makes up with the gene (SEQ ID NO.1) that adopts coding human pancreatic tissue kallikrein intact proteins relatively simultaneously.The result shows that secretor type, nonsecreting type prokaryotic expression carrier and rhabdovirus expression vector expressed products that the present invention makes up all have higher pancreas kallikrein vigor, further animal experiment study can be used for, and the preclinical study of diseases such as hypertension can be further used for treating.Simultaneously, also merge the affine sequence of His in the expression vector of the present invention, comprised the expression product of this affine sequence, when separation and purification, can utilize the affine resin of His to carry out affinitive layer purification.The present invention is an exploit person source tissue kallikrein gene engineering product, and the genomic medicine of exploit person source tissue kallikrein lays the foundation.
The accompanying drawing summary
Below, in conjunction with the accompanying drawings,, describe in detail but do not limit the present invention by description to preferred embodiment of the present invention.
Fig. 1 is the agarose gel electrophoresis result of pcr amplification product
Swimming lane 1:100bp DNA ladder;
Swimming lane 2: Human Pancreas Kallikrein gene;
Fig. 2 is the order-checking collection of illustrative plates of the present invention clone's new gene;
Fig. 3 behaviour (pancreatic tissue), pig, rat, the comparative analysis of mouse kallikrein aminoacid sequence;
Fig. 4 expression vector pE20mK of the present invention structural representation;
Fig. 5 is the structural representation of another secreted expression carrier of the present invention pE28pK;
Fig. 6 is the structural representation of invention nonsecreting type expression vector pE28mK;
Fig. 7 is the structural representation of baculovirus transfer vector Pbac-KK of the present invention;
Fig. 8 analyzes the curve of expression product vigor for adopting potentiometric titration.
The embodiment of invention
The clone and the order-checking of embodiment 1 people's pancreas tissue kallikrein gene
1 materials and methods
1.1 material
The have drawn from part flesh tissue of a customary pancreatectomy patient postoperative of pancreatic tissue is cleaned blood, extracts total RNA immediately.
1.2 plasmid and bacterial strain
Plasmid pBluescript II KS (+), intestinal bacteria XL1-Blue purchase respectively in STRATAGENE (brilliant U.S. company).
1.3 toolenzyme and other reagent
RNA separating kit, reverse transcription test kit are purchased in STRATAGENE (brilliant U.S. company), and restriction enzyme, modifying enzyme, pfu high-fidelity Taq archaeal dna polymerase etc. are purchased respectively in promega, gene company and Shanghai and given birth to the worker.
1.4 PCR design of primers and synthetic
According to the gene order of GENBANK report, utilize computer aided system design one couple of PCR primers, give birth to the synthetic and polyacrylate hydrogel electrophoresis purifying of worker by Shanghai.
Primer sequence is: upstream primer is sequence shown in SEQ ID NO.4;
Downstream primer is sequence shown in SEQ ID NO.5.
1.5 RNA extracts and pcr amplification
Behind the preparation tissue homogenate, total RNA is extracted in the explanation of reference reagent box.The sex change agarose electrophoresis is confirmed RNA quality, determined by ultraviolet spectrophotometry rna content.Adopt oligo (dT) primer to carry out reverse transcription.Use eppendorf grads PCR instrument, 95 ℃, 40s; 60 ℃, 1min; 72 ℃, 1min; 10 ℃ of amplifications of total temperature gradient, 30 circulations, 72 ℃ are extended 10min and finish.Get an amount of product after the affirmation top condition and carry out pcr amplification.Directly the big fragment of an amount of Klenow is added in the amplification solution, 25 ℃ of reaction 30min mend the thing of showing no increases in output.
1.6 construction of recombinant plasmid
Adopt LMP agarose method to reclaim the PCR product, insert plasmid KS.Connect the back and transform the XL1-Blue recipient bacterium, utilize X-gal to carry out Lan-Bai screening, enzyme is cut evaluation.
1.7 dna sequence analysis alkaline lysis method of extracting plasmid is also used the PEG-8000 purifying, uses the two-way order-checking of universal primer.
2 results
2.1 the amplification of kallikrein gene is PCR product 1% agarose gel electrophoresis, ethidium bromide staining, and visible specific DNA band between 800-900bp and is estimated size～830bp conform to (Fig. 1).
2.2 gene clone is carried out in the clone of kallikrein gene and evaluation according to a conventional method, uses restriction enzyme styI, PvuII to identify recombinant plasmid respectively, the result is correct.
2.3 Shanghai order-checking portion of Sangon bio-engineering corporation carried out sequential analysis after the sequential analysis of kallikrein gene prepared recombinant plasmid.The KK of The sequencing results and GenBank report carries out sequence relatively, and both have a Nucleotide difference as a result.Fig. 2 is kallikrein gene order-checking collection of illustrative plates, and The sequencing results obtains the sequence of SEQ ID NO.1, its for aminoacid sequence be SEQ ID NO.2.
The clone of embodiment 2 human pancreatic tissue kallikrein mature protein genes
Utilize the aminoacid sequence (Fig. 3) of computer software comparative analysis people (pancreatic tissue), pig, rat, mouse kallikrein, infer that prekallikrein (kallikrein intact proteins) is converted to the possible proteolytic enzyme cutting site of kallikrein (kallikrein maturation protein) and the aminoacid sequence of kallikrein maturation protein, the PCR primer of synthetic following sequence:
Upstream (eukaryotic cell): sequence shown in the SEQ ID NO.6;
Upstream (prokaryotic cell prokaryocyte): sequence shown in the SEQ ID NO.7;
Downstream: sequence shown in the SEQ ID NO.8;
In above-mentioned design of primers:
1. the maturation protein front increases an ATG, is used for eukaryotic expression;
2. the maturation protein front does not have ATG, is used for prokaryotic expression;
3. shared downstream primer;
4. upstream and downstream all increases restriction enzyme site and is beneficial to the clone.
The 72nd base of the initial SEQ ID of encoding sequence NO 1 sequence of the maturation protein of amplification has the sequence shown in the SEQ ID NO.3.
The structure of embodiment 3 expression vectors
1. the structure of human pancreatic tissue kallikrein maturation protein secreted expression carrier pE20mK
Figure 4 shows that Chinese's pancreatic tissue kallikrein gene Procaryon secreted expression carrier pE20mK.Concrete grammar is to utilize ripe kallikrein gene (the SEQ ID NO.3 of high-fidelity Taq enzymatic amplification from plasmid KSKK, mKK), after utilizing restriction enzyme EcoR I, XhoI digestion, be inserted into EcoR I, the XhoI site of plasmid pET20b, guarantee that kallikrein gene and His frame are correct, construct secreted expression carrier pE20pK.This carrier can go out tissue kallikrein's maturation protein at expression in escherichia coli, and merges signal peptide sequence at the N end, makes the target protein of expressing tend to be distributed in the tenuigenin (periplasmic localization).
2. the structure of human pancreatic tissue kallikrein intact proteins nonsecreting type expression vector pE28pK
Figure 5 shows that Chinese's pancreatic tissue kallikrein gene protokaryon nonsecreting type expression vector pE28pK.Concrete grammar is to utilize the complete kallikrein gene of high-fidelity Taq enzymatic amplification (SEQ ID NO.1 from plasmid KSKK, pKK), after utilizing restriction enzyme EcoR I, XhoI digestion, be inserted into EcoR I, the XhoI site of plasmid pET28, guarantee that kallikrein gene and His frame are correct, construct nonsecreting type expression vector pE28pK.This carrier can go out the complete preceding leach protein of tissue kallikrein at expression in escherichia coli, and merges His Tag sequence at the C end, makes the target protein of expressing be easy to carry out affinity chromatography purification.
3. the structure of human pancreatic tissue kallikrein maturation protein nonsecreting type expression vector pE28mK
Figure 6 shows that Chinese's pancreatic tissue kallikrein gene protokaryon nonsecreting type expression vector pE28mK.Concrete grammar is to utilize ripe kallikrein gene (the SEQ ID NO.3 of high-fidelity Taq enzymatic amplification from plasmid KSKK, mKK), after utilizing restriction enzyme EcoRI, XhoI digestion, be inserted into EcoR I, the XhoI site of plasmid pET28, guarantee that kallikrein gene and His frame are correct, construct nonsecreting type expression vector pE28mK.This carrier can go out tissue kallikrein's maturation protein at expression in escherichia coli, and merges His Tag sequence at the C end, makes the target protein of expressing be easy to carry out affinity chromatography purification.
5. the structure of baculovirus transfer vector PBac-KK
Figure 7 shows that the baculovirus expression transfer vector of Chinese's pancreatic tissue kallikrein gene.Concrete grammar is to utilize restriction enzyme EcoR I, XhoI to cut out kallikrein gene (SEQ ID NO.3) from plasmid KSKK, is inserted into EcoR I, the XhoI site of transfer vector pBacPAK9, constructs baculovirus transfer vector PBac-KK.
The expression of embodiment 4 human pancreatic tissue kallikrein genes in intestinal bacteria
1. the preparation of competent escherichia coli cell and conversion
The calcium chloride preparation method is adopted in the competent escherichia coli cell preparation, and concrete grammar is translated " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., Science Press (1992), the 55th～56 page.Just adopting by 0.75mmol/L Tris-HCl replacement water is solvent preparation 0.1mol/L CaCl
2The method for transformation of competent cell carries out with reference to above-mentioned reference.
2. (except that specified otherwise was arranged, reagent was prepared all with reference to " molecular cloning experiment guide " second edition, Science Press (1992) in a large amount of preparations of plasmid
Translate " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., Science Press (1992), adopts the alkaline lysis preparation, with PEG precipitator method purifying by the 26th～28 page.Concrete operation method is as follows, and the thalline of centrifugal results after plasmid transforms, cultivates suspends, washs thalline once with STE, uses special TE (10mmol/L Tris, 50mmol/L EDTApH8.0) then, and precipitation suspends.Add 2 times to the solution II cracking of TE volume, add the solution III neutralization of 1.5 times of volumes, get supernatant after centrifugal, add the isopropanol precipitating of 0.7 times of volume.The centrifugal supernatant of abandoning.With TE (pH8.0) dissolution precipitation, add and wait doubly to the 5mol/LLiCl of TE volume centrifugal removal macromole RNA.Shift out in the new centrifuge tube of supernatant to, add 2 times to the dehydrated alcohol of supernatant volume, deposit D NA.Add an amount of 70% washing with alcohol precipitation once, airing.Use the TE dissolution precipitation, the RNA enzyme final concentration that wherein contains no DNA enzyme is 20Mg/ml, to remove RNA.Add to wait doubly to the PEG of TE volume solution (20%PEG 6000,2.5mol/L NaCl) mixing, it is centrifugal to put 4 ℃ of backs of spending the night, and abandons supernatant.Use the TE dissolution precipitation, with waiting doubly to full phenol, the phenol of closing of TE volume: chloroform, each extracting is once used the dehydrated alcohol deposit D NA of 2 times of volumes then in order, and with 70% washing with alcohol precipitation once, airing is dissolved among the TE (pH8.0) standby at last.
3. the recovery of the digestion with restriction enzyme of plasmid and dna fragmentation
The digestion with restriction enzyme of plasmid all carries out with reference to following reaction conditions, every microgram plasmid DNA digests with 5 unit limit restriction endonucleases, the restriction enzyme reaction damping fluid that adds 1/10 volume, adding sterilization two, to heat up in a steamer water adjustment reaction cumulative volume be 10 times of used restriction enzyme volume, the temperature of reaction reaction of recommending by the supplier of various restriction enzymes 2～3 hours.Plasmid with digestion with restriction enzyme after, DEAE-cellulose membrane electrophoresis absorption method is adopted in the recovery of dna fragmentation, concrete grammar is translated " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., Science Press (1992), the 319th～321 page.This research adopts DE-81 ion exchange paper (Whatman company product) to replace the DEAE-cellulose membrane.
4. the terminal dephosphorylation of linear plasmid and being connected of dna fragmentation
Enzyme is cut the terminal dephosphorylation of back linear plasmid, adopts calf intestinal alkaline phosphatase (CIP), and concrete operations are translated " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., Science Press (1992), the 40th～41 page.After the dephosphorylation reaction, the deactivation of CIP adopts 5mmol/L EDTA (PH8.0) to exist down, 75 ℃ of heating 10min.Use phenol then: CIP is removed in the chloroform extracting, and the linear plasmid behind the ethanol sedimentation dephosphorylation is dissolved in two the heating up in a steamer in the water of sterilizing, and is used for ligation.
The T that the ligation of linear plasmid and dna fragmentation provides by GIBCO-BRL company
4The condition that dna ligase is recommended is carried out.Plasmid vector is pressed in the connection of sticky end: the mol ratio of target DNA fragment=1: 2,25 ℃ connect 1h.Plasmid vector is adopted in flat terminal connection: the ratio of target DNA fragment mol ratio=1: 5, and 14～16 ℃, connect 17～24h, will connect product transformed into escherichia coli competent cell then.
5. the screening of recombinant plasmid and evaluation
With ligation thing transformed competence colibacillus bacterium, be applied to and contain on the antibiotic flat board, select single bacterium colony that incubated overnight grows, be inoculated in the 2ml LB liquid nutrient medium, cultivate 16～20h.Translate " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc. then, Science Press (1992), the alkaline lysis of the 19th～22 page of introduction prepares plasmid in a small amount, compare the agarose gel electrophoresis that carries out plasmid with the empty carrier plasmid, select recombinant plasmid, make digestion with restriction enzyme, agarose gel electrophoresis is identified goal gene and the direction of insertion thereof inserted.
The expression of embodiment 5 goal gene
Experiment guide with reference to Novagen company carries out.Recombinant plasmid is transformed the expression strain of pET expression system respectively, as BL21 (DE3), BL21 (DE3) plysS, HMS174 (DE3) etc. and non-expression (contrast) bacterial strain BL21, select single bacterium colony that incubated overnight grows, be inoculated in the 2ml LB liquid nutrient medium, be cultured to OD in 37 ℃
600Reach 0.6-1.0,4 ℃ of preservations are spent the night.Collect thalline next day, be resuspended in the 2ml fresh culture, be inoculated in the 50ml substratum then, be cultured to OD in 37 ℃
600Reach 0.6-1.0, add IPTG and induce, continue to cultivate 3hr.Collect thalline, carry out proteic purifying, analysis after the cracking.
The separation and purification and the determination of activity of embodiment 5 recombinant proteins
1. affinitive layer purification
Merge the proteic purifying of His Tag: the Ni that uses Qiageen company to produce
2+The affine resin of-NTA metal, behind the thalline ultrasonic degradation, last Ni
2+-NTA affinity column uses different damping fluids to carry out the tonsure wash-out successively.Collect elutriant respectively and carry out the SDS-PAGE analysis.To contain target protein elutriant dialysis back freeze-drying preserves.
2. conventional purifying: the experimental procedure of reference reagent box specification sheets and " fine works molecular biology experiment guide " the 10th chapter part is carried out chromatography purification.
3. the activation analysis of kallikrein
The biological activity of kallikrein can be measured by its catalysis activity.Pig kallikrein (the Sigma product is available from Shenzhen brilliant U.S. company) with purifying is standard substance, adopts two kinds of methods to detect recombinant human pancreatic kallikrein vigor.The result shows that the preliminary purification product of the kallikrein that this experiment is expressed has the kallikrein catalytic activity.
Adopt two kinds of methods to detect recombinant human pancreatic kallikrein vigor
(1) recommends (Ruyssen Rand Lauwers A.Pharmaceutical Enzymes Properties and Assay Methods:Kallikrein.1978.147-152), the improved potentiometric titration (Yang Huaxin of Nat'l Pharmaceutical ﹠ Biological Products Control Institute with International Pharmaceutical Federation (FIP), Shen Jia, Liu Jinxiu.Pharmaceutical analysis magazine 1994; 14 (2): 9-12) detect recombinant human pancreatic kallikrein vigor.
(2) adopt (Abe M such as Abe M, Nakamura F, Tan F, et al.Expression of ratkallikrein and epithelikrein polarity in transfected Madin-Darby caninekidney cells.Hypertension 1995; 26:891-898) the enzymatic spectrophotofluorimetry that uses detects recombinant human pancreatic kallikrein vigor.
The expression of embodiment 7 human pancreatic tissue kallikrein genes in insect cell (sf9)
1. cotransfection
(1) inoculation 1.0 * 10
6The sf9 insect cell adds the 1.5ml perfect medium in culture dish, cultivates 4hr for 27 ℃;
(2) with transfer vector and linearizing wild-type baculovirus DNA cotransfection;
A. use the perfect medium that contains 10% foetal calf serum to cultivate the sf9 insect cell;
B. grow when cell and change fresh culture, re-suspended cell when in blocks;
C. counting cells;
D. inoculate 2 * 10
6The sf9 insect cell is in the 60mm culture dish;
E. dropwise add the Lipofectin-DNA mixture, mixing was cultivated 4-5 hour for 27 ℃;
(3) the laggard line space spot screening of homologous recombination filters out recombinant baculovirus under the opticmicroscope.
2. express
Express (experimental procedure of reference reagent box specification sheets and " fine works molecular biology experiment guide " the 16th chapter part is expressed) behind the large scale culturing recombinant baculovirus in the inoculation insect cell.
3. the preliminary purification of expressing protein
The experimental procedure of reference reagent box specification sheets and " fine works molecular biology experiment guide " the 16th chapter part is carried out chromatography purification.
4. the activity of expressing protein detects
Carry out with reference to prokaryotic expression its lytic activity detection method.
The human pancreatic tissue kallikrein maturation protein of embodiment 8 escherichia coli expressions and the biological activity of intact proteins are relatively
Make up kallikrein intact proteins, maturation protein carrier (expression vector pE28pK of the present invention and expression vector pE28mK) respectively and in intestinal bacteria, express, preliminary purification, renaturation, the amino acid that utilizes the zymoplasm excision to merge, adopt potentiometric titration to detect the product vigor, the result shows: the vigor of the smart ammonia amino acid of pE28mK expression product hydrolysis benzoyl ethyl ester is apparently higher than the expression product (Fig. 8) of pE28pK.Illustrate that expression vector pE28mK can express the ripe kallikrein with biologos.The prekallikrein that expression vector pE28pK expresses does not obtain the cutting processing after the translation in intestinal bacteria.
Above embodiment explanation, the present invention successfully made up can carrier's pancreatic tissue kallikrein mature protein gene recombinant expression vector, the recombinant human pancreatic tissue kallikrein that this carrier is expressed in host cell has higher biologic activity, and can utilize affinity chromatography that recombinant protein is carried out purifying.
The invention is not restricted to upper type, those skilled in the art can make various changes and distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to scope of the present invention.
SEQUENCE LISTING＜110〉Shenzhen people's hospital＜120〉a kind of preparation of expressing recombinant expression carrier and the recombined human pancreatic tissue kallikrein of human pancreatic tissue kallikrein gene＜130〉PI02943＜150〉CN01104112.9＜151〉2001-02-20＜160〉8＜170〉PatentIn version 3.1＜210〉1＜211〉818＜212〉DNA＜213〉Homo sapiens＜220〉＜221〉CDS＜222〉(4) .. (789)＜223〉＜400〉1acc atg tgg ttc ctg gtt ctg tgc ctc gcc ctg tcc ctg ggg ggg act48
Met?Trp?Phe?Leu?Val?Leu?Cys?Leu?Ala?Leu?Ser?Leu?Gly?Gly?Thr
1 5 10 15ggt?gct?gcg?ccc?ccg?att?cag?tcc?cgg?att?gtg?gga?ggc?tgg?gag?tgt96Gly?Ala?Ala?Pro?Pro?Ile?Gln?Ser?Arg?Ile?Val?Gly?Gly?Trp?Glu?Cys
20 25 30gag?cag?cat?tcc?cag?ccc?tgg?cag?gcg?gct?ctg?tac?cat?ttc?agc?act144Glu?Gln?His?Ser?Gln?Pro?Trp?Gln?Ala?Ala?Leu?Tyr?His?Phe?Ser?Thr
35 40 45ttc?cag?tgt?ggg?ggc?atc?ctg?gtg?cac?cgc?cag?tgg?gtg?ctc?aca?gct192Phe?Gln?Cys?Gly?Gly?Ile?Leu?Val?His?Arg?Gln?Trp?Val?Leu?Thr?Ala
50 55 60gct?cat?tgc?atc?agc?gac?aat?tac?cag?ctc?tgg?ctg?ggt?cgc?cac?aac240Ala?His?Cys?Ile?Ser?Asp?Asn?Tyr?Gln?Leu?Trp?Leu?Gly?Arg?His?Asn
65 70 75ttg?ttt?gac?gac?gaa?aac?aca?gcc?cag?ttt?gtt?cat?gtc?agt?gag?agc288Leu?Phe?Asp?Asp?Glu?Asn?Thr?Ala?Gln?Phe?Val?His?Val?Ser?Glu?Ser80 85 90 95ttc?cca?cac?cct?ggc?ttc?aac?atg?agc?ctc?ctg?gag?aac?cac?acc?cgc336Phe?Pro?His?Pro?Gly?Phe?Asn?Met?Ser?Leu?Leu?Glu?Asn?His?Thr?Arg
100 105 110caa?gca?aac?gag?gac?tac?agc?cac?gac?ctc?atg?ctg?ctc?cgc?ctg?aca384Gln?Ala?Asn?Glu?Asp?Tyr?Ser?His?Asp?Leu?Met?Leu?Leu?Arg?Leu?Thr
115 120 125gag?cct?gct?gat?acc?atc?aca?gac?gct?gtg?aag?gtc?gtg?gag?ttg?ccc432Glu?Pro?Ala?Asp?Thr?Ile?Thr?Asp?Ala?Val?Lys?Val?Val?Glu?Leu?Pro
130 135 140acc?cag?gaa?ccc?gaa?gtg?ggg?agc?acc?tgt?ttg?gct?tcc?ggc?tgg?ggc480Thr?Gln?Glu?Pro?Glu?Val?Gly?Ser?Thr?Cys?Leu?Ala?Ser?Gly?Trp?Gly
145 150 155agc?atc?gaa?cca?gag?aat?ttc?tca?ttt?cca?gat?gat?ctc?cag?tgt?gtg528Ser?Ile?Glu?Pro?Glu?Asn?Phe?Ser?Phe?Pro?Asp?Asp?Leu?Gln?Cys?Val160 165 170 175gac?ctc?aaa?atc?ctg?cct?aat?gat?gag?tgc?gaa?aaa?gcc?cac?gtc?cag576Asp?Leu?Lys?Ile?Leu?Pro?Asn?Asp?Glu?Cys?Glu?Lys?Ala?His?Val?Gln
180 185 190aag?gtg?aca?gac?ttc?atg?ctg?tgt?gtc?gga?cac?ctg?gaa?ggt?ggc?aaa624Lys?Val?Thr?Asp?Phe?Met?Leu?Cys?Val?Gly?His?Leu?Glu?Gly?Gly?Lys
195 200 205gac?acc?tgt?gtg?ggt?gat?tca?ggg?ggc?ccg?ctg?atg?tgt?gat?ggt?gtg672Asp?Thr?Cys?Val?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Met?Cys?Asp?Gly?Val
210 215 220ctc?caa?ggt?gtc?aca?tca?tgg?ggc?tac?gtc?cct?tgt?ggc?acc?ccc?aat720Leu?Gln?Gly?Val?Thr?Ser?Trp?Gly?Tyr?Val?Pro?Cys?Gly?Thr?Pro?Asn
225 230 235aag?cct?tct?gtc?gcc?gtc?aga?gtg?ctg?tct?tat?gtg?aag?tgg?atc?gag768Lys?Pro?Ser?Val?Ala?Val?Arg?Val?Leu?Ser?Tyr?Val?Lys?Trp?Ile?Glu240 245 250 255gac?acc?ata?gcg?gag?aac?tcc?tgaacgccca?gccctgtccc?ctaccccca818Asp?Thr?Ile?Ala?Glu?Asn?Ser
260<210>2<211>262<212>PRT<213>Homo?sapiens<400>2Met?Trp?Phe?Leu?Val?Leu?Cys?Leu?Ala?Leu?Ser?Leu?Gly?Gly?Thr?Gly1 5 10 15Ala?Ala?Pro?Pro?Ile?Gln?Ser?Arg?Ile?Val?Gly?Gly?Trp?Glu?Cys?Glu
20 25 30Gln?His?Ser?Gln?Pro?Trp?Gln?Ala?Ala?Leu?Tyr?His?Phe?Ser?Thr?Phe
35 40 45Gln?Cys?Gly?Gly?Ile?Leu?Val?His?Arg?Gln?Trp?Val?Leu?Thr?Ala?Ala
50 55 60His?Cys?Ile?Ser?Asp?Asn?Tyr?Gln?Leu?Trp?Leu?Gly?Arg?His?Asn?Leu65 70 75 80Phe?Asp?Asp?Glu?Asn?Thr?Ala?Gln?Phe?Val?His?Val?Ser?Glu?Ser?Phe
85 90 95Pro?His?Pro?Gly?Phe?Asn?Met?Ser?Leu?Leu?Glu?Asn?His?Thr?Arg?Gln
100 105 110Ala?Asn?Glu?Asp?Tyr?Ser?His?Asp?Leu?Met?Leu?Leu?Arg?Leu?Thr?Glu
115 120 125Pro?Ala?Asp?Thr?Ile?Thr?Asp?Ala?Val?Lys?Val?Val?Glu?Leu?Pro?Thr
130 135 140Gln?Glu?Pro?Glu?Val?Gly?Ser?Thr?Cys?Leu?Ala?Ser?Gly?Trp?Gly?Ser145 150 155 160Ile?Glu?Pro?Glu?Asn?Phe?Ser?Phe?Pro?Asp?Asp?Leu?Gln?Cys?Val?Asp
165 170 175Leu?Lys?Ile?Leu?Pro?Asn?Asp?Glu?Cys?Glu?Lys?Ala?His?Val?Gln?Lys
180 185 190Val?Thr?Asp?Phe?Met?Leu?Cys?Val?Gly?His?Leu?Glu?Gly?Gly?Lys?Asp
195 200 205Thr?Cys?Val?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Met?Cys?Asp?Gly?Val?Leu
210 215 220Gln?Gly?Val?Thr?Ser?Trp?Gly?Tyr?Val?Pro?Cys?Gly?Thr?Pro?Asn?Lys225 230 235 240Pro?Ser?Val?Ala?Val?Arg?Val?Leu?Ser?Tyr?Val?Lys?Trp?Ile?Glu?Asp
245 250 255Thr?Ile?Ala?Glu?Asn?Ser
260＜210〉3＜211〉726＜212〉DNA＜213〉Homo sapiens＜400〉3attgtgggag gctgggagtg tgagcagcat tcccagccct ggcaggcggc tctgtaccat60ttcagcactt tccagtgtgg gggcatcctg gtgcaccgcc agtgggtgct cacagctgct120cattgcatca gcgacaatta ccagctctgg ctgggtcgcc acaacttgtt tgacgacgaa180aacacagccc agtttgttca tgtcagtgag agcttcccac accctggctt caacatgagc240ctcctggaga accacacccg ccaagcagac gaggactaca gccacgacct catgctgctc300cgcctgacag agcctgctga taccatcaca gacgctgtga aggtcgtgga gttgcccacc360caggaacccg aagtggggag cacctgtttg gcttccggct ggggcagcat cgaaccagag420aatttctcat ttccagatga tctccagtgt gtggacctca aaatcctgcc taatgatgag480tgcgaaaaag cccacgtcca gaaggtgaca gacttcatgc tgtgtgtcgg acacctggaa540ggtggcaaag acacctgtgt gggtgattca gggggcccgc tgatgtgtga tggtgtgctc600caaggtgtca catcatgggg ctacgtccct tgtggcaccc ccaataagcc ttctgtcgcc660gtcagagtgc tgtcttatgt gaagtggatc gaggacacca tagcggagaa ctcctgaacg720cccagc726＜210〉4＜211〉20＜212〉DNA＜213〉＜220〉＜221〉＜222〉 ( 1 ) .. ( 20 )＜223〉＜400〉4ctggcccctg gacacctctg20＜210〉5＜211〉20＜212〉DNA＜213〉＜220〉＜221〉＜222〉 ( 1 ) .. ( 20 )＜223〉＜400〉5gtaggggaca gggctgggcg20＜210〉6＜211〉29＜212〉DNA＜213〉＜220〉＜221〉＜222〉 ( 1 ) .. ( 29 )＜223〉＜400〉6cgggatccat gattgtggga ggctgggag29＜210〉7＜211〉26＜212〉DNA＜213〉＜220〉＜221〉＜222〉 ( 1 ) .. ( 26 )＜223〉＜400〉7cgggatccat tgtgggaggc tgggag26＜210〉8＜211〉29＜212〉DNA＜213〉＜220〉＜221〉＜222〉 ( 1 ) .. ( 29 )＜223〉＜400〉8cagacaagct tcaggagttc tccgctatg29
Claims (9)
1. the recombinant vectors that comprises nucleotide sequence shown in the SEQ ID NO.3.
2. recombinant vectors according to claim 1 is characterized in that described carrier is a plasmid vector.
3. recombinant vectors according to claim 2 is characterized in that merging His Tag sequence at the C of described carrier end, makes the target protein of expressing be easy to carry out affinity chromatography purification.
4. recombinant vectors according to claim 1 is characterized in that described carrier is a baculovirus transfer vector.
5. described carrier transformed host cells of claim 2.
6. host cell according to claim 5 is characterized in that described cell is intestinal bacteria.
7. described carrier transformed host cells of claim 4.
8. host cell according to claim 7 is characterized in that described cell is an insect cell.
9. a method for preparing the kallikrein of recombinating comprises the steps:
1) will the encode gene of pancreas tissue kallikrein maturation protein operationally connects
Be connected to expression regulation sequence, construction of expression vector;
2) transform protokaryon or eukaryotic cell with described expression vector, form host cell;
3) under suitable culture condition, cultivate described host cell;
4) from nutrient solution, isolate and have the active protein of kallikrein;
5) obtain the reorganization kallikrein with affinitive layer purification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02103538 CN1228447C (en) | 2001-02-20 | 2002-02-05 | Recombinant expression vector expressing human pancreatic tissue kallikrein gene and prepn of human pancreatic tissue kallikrein |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN01104112 | 2001-02-20 | ||
| CN01104112.9 | 2001-02-20 | ||
| CN 02103538 CN1228447C (en) | 2001-02-20 | 2002-02-05 | Recombinant expression vector expressing human pancreatic tissue kallikrein gene and prepn of human pancreatic tissue kallikrein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1384199A true CN1384199A (en) | 2002-12-11 |
| CN1228447C CN1228447C (en) | 2005-11-23 |
Family
ID=25740273
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02103538 Expired - Fee Related CN1228447C (en) | 2001-02-20 | 2002-02-05 | Recombinant expression vector expressing human pancreatic tissue kallikrein gene and prepn of human pancreatic tissue kallikrein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1228447C (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308035C (en) * | 2004-11-03 | 2007-04-04 | 广东天普生化医药股份有限公司 | Application of human urine kininogenase for preparing medicine to treat acute coronary syndromes |
| WO2009039704A1 (en) * | 2007-09-27 | 2009-04-02 | Shanghai Wanxing Biopharmaceuticals, Co., Ltd. | A method for producing human kallikrein 1 |
| CN102438648A (en) * | 2009-03-25 | 2012-05-02 | 代阿麦迪卡股份有限公司 | Tissue kallikrein for the treatment of pancreatic beta-cell dysfunction |
| CN103710367A (en) * | 2013-12-23 | 2014-04-09 | 江苏众红生物工程创药研究院有限公司 | Recombinant homo sapiens kallikrein 1 as well as coding agene and preparation method thereof |
| US9364521B2 (en) | 2012-06-04 | 2016-06-14 | Diamedica Inc. | Human tissue kallikrein 1 glycosylation isoforms |
| US9616015B2 (en) | 2012-05-25 | 2017-04-11 | Diamedica Inc. | Formulations of human tissue kallikrein-1 for parenteral delivery and related methods |
| CN111575316A (en) * | 2020-05-19 | 2020-08-25 | 东莞市松山湖中心医院(东莞市石龙人民医院、东莞市第三人民医院、东莞市心血管病研究所) | hKLK1 recombinant plasmid, high-expression hKLK1 lentiviral particle, and construction method and application thereof |
| CN113073092A (en) * | 2021-04-15 | 2021-07-06 | 宁波瑞林生物科技有限公司 | Recombinant human tissue kallikrein and preparation method thereof |
| US11857608B2 (en) | 2017-03-09 | 2024-01-02 | Diamedica Inc. | Dosage forms of tissue kallikrein 1 |
-
2002
- 2002-02-05 CN CN 02103538 patent/CN1228447C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308035C (en) * | 2004-11-03 | 2007-04-04 | 广东天普生化医药股份有限公司 | Application of human urine kininogenase for preparing medicine to treat acute coronary syndromes |
| WO2009039704A1 (en) * | 2007-09-27 | 2009-04-02 | Shanghai Wanxing Biopharmaceuticals, Co., Ltd. | A method for producing human kallikrein 1 |
| CN102438648A (en) * | 2009-03-25 | 2012-05-02 | 代阿麦迪卡股份有限公司 | Tissue kallikrein for the treatment of pancreatic beta-cell dysfunction |
| US9616015B2 (en) | 2012-05-25 | 2017-04-11 | Diamedica Inc. | Formulations of human tissue kallikrein-1 for parenteral delivery and related methods |
| US9364521B2 (en) | 2012-06-04 | 2016-06-14 | Diamedica Inc. | Human tissue kallikrein 1 glycosylation isoforms |
| US9839678B2 (en) | 2012-06-04 | 2017-12-12 | Diamedica Inc. | Human tissue kallikrein 1 glycosylation isoforms |
| CN103710367A (en) * | 2013-12-23 | 2014-04-09 | 江苏众红生物工程创药研究院有限公司 | Recombinant homo sapiens kallikrein 1 as well as coding agene and preparation method thereof |
| CN103710367B (en) * | 2013-12-23 | 2016-08-24 | 江苏众红生物工程创药研究院有限公司 | A kind of recombined human kallikrein 1 and encoding gene thereof and preparation method |
| US11857608B2 (en) | 2017-03-09 | 2024-01-02 | Diamedica Inc. | Dosage forms of tissue kallikrein 1 |
| CN111575316A (en) * | 2020-05-19 | 2020-08-25 | 东莞市松山湖中心医院(东莞市石龙人民医院、东莞市第三人民医院、东莞市心血管病研究所) | hKLK1 recombinant plasmid, high-expression hKLK1 lentiviral particle, and construction method and application thereof |
| CN113073092A (en) * | 2021-04-15 | 2021-07-06 | 宁波瑞林生物科技有限公司 | Recombinant human tissue kallikrein and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1228447C (en) | 2005-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1033810C (en) | Process for enzymatic preparation of basic fibroblast growth factor | |
| CN1174097C (en) | Macrophage activating factors derived from cloned vitamin D binding protein | |
| CN1045992A (en) | The clone of transforming grouth factor beta 2 and expression | |
| CN110845603A (en) | Human collagen 17-type polypeptide, production method and use thereof | |
| EP1309604B1 (en) | Phage-dependent superproduction of biologically active protein and peptides | |
| CN1384199A (en) | Recombinant expression vector expressing human pancreatic tissue kallikrein gene and prepn of human pancreatic tissue kallikrein | |
| CN1075148A (en) | New megalokaryocyte amplification gene | |
| WO2019004878A1 (en) | Preparation of wheat cysteine protease triticain-alpha produced in soluble form and method of producing same | |
| CN87104540A (en) | The production of human lysozyme | |
| CN1798838A (en) | Inhibitor proteins of a protease and use thereof | |
| CN1150324C (en) | Novel human lysozyme gene, its encoding polypeptide and the method preparing for them | |
| CN116478969A (en) | Collagenase mutant, method for promoting secretory expression of recombinant collagenase and application thereof | |
| RU2453604C1 (en) | Hybrid protein (versions), bacterial strain escherichia coli - hybrid protein producer (versions) and method for producing methionine-free human interferon alpha-2 | |
| CN1224710C (en) | Novel human lysozyme gene, its encoding polypeptide and the method for preparing them | |
| CN1786175A (en) | Plasmid for expressing recombination human tPA and its construction method | |
| CN1247794C (en) | Production process of human horny cell growth factor-2 | |
| CN105112382B (en) | A method of people's myeloperoxidase is prepared based on silkworm-rhabdovirus system | |
| CN1034589C (en) | Antitumor antibiotic kedarciden | |
| CN1137261C (en) | Punctiform aerogenic monad prolyl endopeptidase and its preparation method and application | |
| CN1149290C (en) | Novel human lysozyme gene, its encoding polypeptide and the method for preparing them | |
| CN1402783A (en) | Polypeptides and genes encoding same | |
| CN1194092C (en) | Medusa cystatin gene, and expression and use thereof | |
| CN1219057C (en) | Cell factor CKLF-HIA with functions of hematopoietic stimulation and immunoregulation and its variant CKLF-HIB | |
| CN1786152A (en) | Engineering bacteria strain for producing recombination buman tPA and its preparation method | |
| CN117143210A (en) | Signal peptide for promoting alpha-lactalbumin expression, recombinant strain and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |