CN100406558C

CN100406558C - Method for preparing recombinant aprotinin

Info

Publication number: CN100406558C
Application number: CNB2005100168713A
Authority: CN
Inventors: 颜炜群; 杨莉莉
Original assignee: JINLIN SHENGYUAN SCIENCE & TECHNOLOGY Co Ltd
Current assignee: JINLIN SHENGYUAN SCIENCE & TECHNOLOGY Co Ltd
Priority date: 2005-06-14
Filing date: 2005-06-14
Publication date: 2008-07-30
Anticipated expiration: 2025-06-14
Also published as: CN1880451A

Abstract

The present invention relates to enzyme-suppressing peptide. More specifically, the present invention relates to a method for producing enzyme-suppressing peptide in methyl nourishing yeast, particularly to a method for using albumin signal peptide to produce enzyme-suppressing peptide having a natural N terminal amino acid sequence (Arg-Pro-Asp) by a DNA recombination technique in Pasteur pichia yeast.

Description

The method for preparing recombinant aprotinin

Invention field

The present invention relates to press down the enzyme peptide, specifically, the present invention relates in the methylotrophy yeast, produce the method that presses down the enzyme peptide, particularly use human serum albumin signal peptide, in pichia pastoris phaff, produce the method that presses down the enzyme peptide with the DNA recombinant technology with natural N-terminal aminoacid sequence (Arg-Pro-Asp).

Background of invention

Press down the enzyme peptide and be called " bovine pancreatic trypsin inhibitor " again (BPTI), belong to Kunitz-type inhibitor family member, has the serine protease of inhibition such as trypsinase, Quimotrase, the basic biologic activity of Tryptase and plasma kallikrein (W.Gebhard, H.Tschesche and H.Fritz, ProteinaseInhibitors, Barrett and Salvesen (eds.), Elsevier Science Publ.BV 375-387,1986; Tschesche et al.U.S.Pat.No.4,595,674).

Press down the basic protein that the enzyme peptide is made up of 58 amino-acid residues.For a long time; press down the enzyme peptide and mainly be used to treat acute pancreatitis; after the nineties; beginning is particularly used in the thoracic surgery in surgical operation; be used to protect thrombocyte, reduce hemorrhage and oozing of blood, be used for the treatment of patient (the Bistrup et al.Lancet 1 that blood coagulation disorders is arranged; 366-367,1988).At present commercially available presses down the enzyme peptide mainly by extracting in the organs such as ox lung or pancreas, and employed complex process and product purity are not high.

The yeast bio body can produce synthetic in many cells and at the protein of extracellular performance function, such extracellular protein is commonly called secretory protein.For guaranteeing that expression product passes through endoplasmic reticulum, these secretory proteins exist with sequence precursor (presequence) or preceding proteic form at first.Presequence, promptly signal peptide generally is to fall cleaved from the required product between metaphase.Signal peptide can be signal peptide, allos signal peptide or the heterozygosis signal peptide of desired protein self.A problem using the allos signal peptide usually to run into is that the allos signal peptide can not guarantee transposition effectively and cleaved.

Pichia pastoris phaff (Pichia pastoris) system is a very fast eukaryotic expression system of development in recent years, now generally is used to express many activated protein.Pichia spp can be grown in the substratum that with methyl alcohol is sole carbon source when lacking inhibition carbon source such as glucose, glycerine.Because pichia yeast expression system is an eukaryotic expression system, so can process folding and modify expressed protein, and have fermentation process maturation, fermentation density height, cultivate cheap and simple, exogenous protein not only can intracellular accumulation but also can secrete and expression efficiency height, expression product are easy to advantages such as purifying.

Existing people has expressed recombinant aprotinin or its analogue or varient (for example referring to (Auerswald et at.Biol.Chem.Hoppe Seyler 369,27-35,1988 intestinal bacteria with in yeast; And European patent 0419878, United States Patent (USP) 4,894,436,5,164,482,5,258,302,5,618,915,5,707,831,5,770,568 and 6,482,798).Yet prior art uses Yeast system to express in the practice that presses down the enzyme peptide, and being connected to the signal peptide sequence that presses down enzyme peptide N-terminal in the expression vector nearly all is α-mating factor signal sequence.Generally, the processing of the preceding former-alpha factor that so produces/press down enzyme peptide fusion protein needs two kinds of different proteolytic ferments.Promptly first kind of proteolytic enzyme at first cuts between the 19-20 of fusion rotein amino acids, and then by of protein incision enzyme (Kerx2 proteolytic enzyme) cutting of specific recognition basic aminoacids to (Lys-Arg), thereby cause pressing down the release and the secretion (A.J.Brake of enzyme peptide, Methods in Enzymology185,408-421,1990; Das et at.Biotechn.Progress 3,43-48,1987).Yet, with regard to recombinant aprotinin and its varient, Kex2 proteolytic enzyme can not cut have a natural N-terminal sequence " Arg-Pro-Asp " press down the enzyme peptide so that almost can not express a kind of quilt and evenly, correctly process and secrete and press down the enzyme peptide.In addition, attempting to change the N-terminal sequence that presses down the enzyme peptide also is unpractiaca with the correct processing that realizes expression product in the Yeast system, because all will cause deleterious immune response to any modification that presses down the enzyme peptide.

The signal peptide part of α-mating factor has been widely used in synthetic and secretion heterogenous protein (referring to european patent application Nos.0116201A, 0123294A, 0123544A, 0163529A and 0123289A) in the cereuisiae fermentum.α-the course of processing of mating factor signal sequence in Yeast system is: at first finish the first cutting of signal peptide (at sequence Glu-Lys-Arg under the effect of Kex2 proteolytic enzyme ^*Between the Arg and Glu among the Glu-Ala-Glu-Ala); Then, under the effect of STE13 gene coded protein, the Glu-Ala tumor-necrosis factor glycoproteins is cut once more.As seen, the cutting characteristics of α-mating factor signal peptide is to cut away the non-basic aminoacids in two basic aminoacids downstreams.Because but the N of enzyme peptide holds first amino acid is basic aminoacids (Arg), so α-mating factor signal peptide can't carry out correct cutting to pressing down the enzyme peptide.

Thereby, use yeast expression have natural N-terminal sequence press down the enzyme peptide time, the secretion level of expression product often very low in addition do not secrete fully or proteolysis processing incorrect.Infer fully not exposed of these situations to occur in part because of the processing site that is present in the signal peptide C-terminal and press down between the enzyme peptide N-terminal so that can not with the hydrolase of proteolysis center fully near due to.

The inventor previously is being engaged on the basis of pichia spp and human serum albumin research for a long time, use existing pichia pastoris phaff (Pichia pastoris) system, and utilize the albumin signal/leading peptide that has added suitable restriction enzyme site to replace the α from the cereuisiae fermentum-mating factor signal leader sequence that uses in the ordinary method, made up new recombinant vectors of the present invention, and successfully secreting, expressing correctly processed press down enzyme peptide protein matter, thereby finished the present invention.

Goal of the invention

An object of the present invention is to provide a kind of method of using pichia pastoris phaff system secreting, expressing recombinant aprotinin, this method comprises cultivates the pichia pastoris phaff bacterial strain that comprises reproducible expression vector, said expression vector carries and presses down enzyme peptide gene and allow to express the dna sequence dna that presses down the enzyme peptide in suitable nutritional medium, from the culture supernatant, reclaim then and but purifying is required the enzyme peptide, be characterised in that in the said carrier that directly being connected the leading or signal sequence that presses down enzyme peptide gene upstream is the albumin signal sequence.

According to a preferred embodiment of the invention, be characterised in that said press down the enzyme peptide be have a correct natural N-terminal aminoacid sequence press down the enzyme peptide.

According to a preferred embodiment of the invention, be characterised in that the normal chain of said albumin signal sequence and minus strand have the nucleotide sequence shown in the SEQ ID NO:1 and SEQ ID NO:2 in the sequence table respectively.

Another object of the present invention provides by the pharmaceutical composition that presses down enzyme peptide and appropriate carriers or vehicle with natural N-terminal aminoacid sequence of expressing according to the method described above.

Summary of the invention

An object of the present invention is to provide a kind of method of using pichia pastoris phaff system secreting, expressing recombinant aprotinin, this method comprises cultivates the pichia pastoris phaff bacterial strain that comprises reproducible expression vector, said carrier carries coding to be pressed down the gene of enzyme peptide and allow to express the dna sequence dna that presses down the enzyme peptide in suitable nutritional medium, from the culture supernatant, reclaim then and but purifying is required the enzyme peptide, be characterised in that in the said carrier that directly being connected the leading or signal sequence that presses down enzyme peptide gene upstream is the albumin signal sequence.

According to a preferred embodiment of the invention, be characterised in that said albumin signal sequence normal chain and minus strand have the nucleotide sequence shown in the SEQ ID NO:1 and SEQ ID NO:2 in the sequence table respectively.

In yeast cell, express the protein that produces by the DNA recombinant technology and only be secreted into its function of extracellular competence exertion, these secretory proteins at cell inner expression exist with the precursor forms that contains presequence (signal peptide), thereby instruct effectively by expressed products by endoplasmic reticulum, cleavedly between the then common product metaphase of presequence (signal peptide) fall.The protein that enters Secretory Pathway is transported to golgi apparatus, and and then is secreted into (Pfeffer, S.R.and Rothman, J.E.Ann.Rev.Biochem.56,829-852,1987) in the substratum of extracellular.

Can use several method in yeast, to express and secrete various heterologous proteins.In general, can make up the DNA that carries coding desired protein and signal peptide according to ordinary method, also prepare by the culture of transformant with this construct transformed yeast host cell then, in suitable nutritional medium, cultivate said yeast cell culture and recovery and the required protein of purifying from the culture supernatant.

As the necessary element of construction of expression vector, be used to promote the signal of desired protein secreting, expressing or signal peptide, allos signal peptide or heterozygote intrinsic and the allos signal peptide that leader sequence can be desired protein self.

A subject matter that is run into when using yeast allos signal peptide is that the allos signal peptide often can not be guaranteed the effective transposition and the cracking of product.As previously mentioned, using cereuisiae fermentum α-mating factor signal peptide to prepare under the recombinant aprotinin situation, may make hardly by correct processing and excretory have a natural N-terminal sequence but enzyme peptide protein matter product.For this reason, the inventor is utilizing the pichia spp Yeast system to express in the practice that presses down the enzyme peptide, substitute the α-mating factor signal sequence of yeast vector itself with the albumin signal sequence, and utilize the suitable restriction enzyme site of introducing in advance this signal sequence to be connected to the upstream that presses down enzyme peptide gene with correct N-terminal sequence.The result is surprisingly found out that, after the recombinant vectors that use so makes up transforms the pichia pastoris phaff cell, has successfully obtained being pressed down the enzyme peptide by correctly cutting, processing and excretory with high yield.

Term used herein " signal peptide " is meant that there is hydrophilic presequence in the N-terminal sequence as the extracellular protein precursor forms of expressing in the yeast cell.Signal peptide is that expressed protein is secreted in the endoplasmic reticulum, and makes that signal peptide is cleaved in this process to be fallen.

People or mammalian plasma albumin gene are positioned on No. 4 karyomit(e), and its primary translation product is that prealbumin is former.Excise signal peptide in secretion process, it is former to generate albumin.Then at one 6 peptide fragment (essence-Gan-figured silk fabrics-phenylpropyl alcohol-essence-essence) of Golgi apparatus, become sophisticated albumin through furin (Furin) excision N-terminal.

Albuminous transport pathway be from rough surfaced endoplasmic reticulum by smooth surfaced endoplasmic reticulum to Golgi complex, enter golgi body by medial Golgi then, secreted to born of the same parents by exocytosis at last.New synthetic albumin at first is cut the formation prealbumin in endoplasmic reticulum, promptly with the active amyloid protein precursor that do not have of N-terminal six amino acid residue sequences (in most of Mammalss, sequence is Arg-Gly-Val-Phe-Arg-Arg) name.

The precursor albumin is transformed into sophisticated protein molecular by enzymolysis and carries out in secretory vesicle.Under the effect of furin (furin), prealbumin is processed to ripe albumin in golgi body or film bubble, be transported to the extracellular at last.

When utilizing this human serum albumin signal peptide to express himself albumin, albumen before cut at P1 place in point of contact at first.After product entered circulation of blood, albumen was cut by protein incision enzyme at point of contact P2 place before the syzygy, thereby produces sophisticated albumin (referring to accompanying drawing 1A).Because first amino acid at P1 place, point of contact is arginine (Arg), and it also is Arg that but the N of enzyme peptide holds first amino acid, can directly be connected the downstream (referring to accompanying drawing 1B) of human serum albumin signal peptide point of contact P1 so press down enzyme peptide gene sequence, thereby be guaranteed the correct cutting of expression product.Because original sequence at cut point P2 place is pressed down the enzyme peptide sequence and is replaced, so after product is secreted into blood, promptly can not carry out the cutting second time again.Guaranteed to have correct cutting and the secretion that correct N-terminal presses down the enzyme peptide thus.

When in Yeast system, express pressing down the enzyme peptide, normally with the signal sequence of yeast secreted protein for example the signal sequence of α-mating factor be connected to the N-terminal that presses down the enzyme peptide, press down the expression of enzyme peptide in order to guiding.Before the processing of former-alpha factor/press down enzyme peptide fusion protein need two kinds of different proteolytic ferments.Be positioned signal peptide on the endoplasmic reticulum and at first between the 19-20 of fusion rotein amino acids, be cut (before consequent-alpha factor/press down the enzyme peptide) in position by glycosylation.And then can be discerned protein incision enzyme (for a kind of Kex2 proteolytic enzyme) cutting of two basic aminoacidss specifically, thereby cause pressing down release and secretion (A.J.Brake, Methods in Enzymology 185,408-421,1990 of enzyme peptide; Das et at.Biotechn.Progress 3,43-48,1987).

Yet, under normal conditions and since Kex2 proteolytic enzyme can not cut have a natural N-terminal sequence " Arg-Pro-Asp " press down the enzyme peptide, therefore be difficult to obtain by equably, processing correctly and excretory press down the enzyme peptide.

In order to solve this difficult problem, we are using when the Yeast system expression presses down the enzyme peptide, utilize the feature of albumin signal sequence guiding cutting basic aminoacids, substitute the signal sequence of α-mating factor with this signal sequence, and the downstream that the enzyme peptide is connected to human serum albumin signal peptide that presses down that will have natural N-terminal sequence, thereby successfully secreting, expressing have a natural N-terminal aminoacid sequence (Arg-Pro-Asp) press down the enzyme peptide.

Under having, the human serum albumin signal peptide that uses among the present invention shows aminoacid sequence: MetLysTrpValThrPheIleSerLeuLeuPheLeuPheSerSerAlaTyrSer (P1) ArgGlyValPheArgArg (P2) AspAlaHisLysSer.Its corresponding nucleotide sequence is: 5 '-ATG AAG TGG GTAACC TTT ATT TCC CTT CTT TTT CTC TTT AGC TCG GCT TAT TCC (P1) AGGGGT GTG TTT CGT CGA (P2) GAT GCA CAC AAG AGT-3 '.Be purpose of the present invention, we have carried out necessary modification to this signal peptide, promptly 5 ' and 3 ' end of human serum albumin signal peptide normal chain and minus strand encoding sequence added respectively help continue after the sequence BstBI and the XhoI restriction enzyme site that are connected, obtain human serum albumin signal peptide encoding sequence as follows: 5 '-- CGAAACGATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTA C-3 ' (BstBI wherein and XhoI restriction enzyme site indicate with underscore) (SEQ ID NO:1); 5 '- TCGAGTAGCCGAGCTAAAGAGAAAAAGAAGGGAAATAAAGGTTACCCACTTCATCGT TT-3 ' (BstBI wherein and XhoI restriction enzyme site indicate with underscore) (SEQ IDNO:2)

First amino acid of prealbumin that is cut in endoplasmic reticulum after (point of contact is P1) is arginine (Arg), and the natural N that presses down the enzyme peptide holds first amino acid also to be arginine, so we can guide the expression that presses down the enzyme peptide with correct N-terminal with human serum albumin signal peptide.To press down the downstream that enzyme peptide gene is connected to the human serum albumin signal peptide sequence, same under the effect of signal peptidase, carry out the first step cutting in endoplasmic reticulum.When secretory protein arrives golgi body or film bubble, owing to do not had recognition sequence Arg-Arg at human serum albumin signal peptide C end, cause furin (Furin) can't carry out the cutting second time again, thereby guaranteed that the enzyme peptide that presses down with correct N-terminal sequence is secreted to the extracellular.In addition, consider to be connected into and press down the enzyme peptide,, design and added an XhoI site (CTCGAG) that helps next step genetic manipulation so we do not change under the situation of signal peptide downstream aminoacid sequence guaranteeing in the human serum albumin signal peptide downstream.

Employed plasmid pPICZaC is available from Invitrogen company in the inventive method; T carrier and endonuclease are all available from TakaRa company.

Nucleotide sequence involved in the present invention and be used to realize other nucleic acid of the present invention comprises that RNA, cDNA, genomic dna, carrier all can separate to obtain also can standing genetic manipulation, amplification from various sources, and/or by recombinant expressed.

In addition, also can use external synthetic these (strand) nucleic acid of known chemical synthesising technology (for example referring to Adams, J.Am.Chem.Soc.105:661,1983; Belousov, Nucleic Acids Res.25:3440-3444,1997; Blommers, Biochemistry 33:7886-7896,1994; Narang, Meth.Enzymol.68:90,1997).And then synthetic its complementary strand is also annealed them under proper condition together.Perhaps use archaeal dna polymerase, with complementary strand and suitable primer sequence adduction, to obtain required double chain DNA fragment.

The nucleic acid operative technique, for example subclone, probe mark, order-checking, hybridization etc. all have fully at scientific literature and patent documentation and describe (as referring to Sambrook, ed.Molecular Cloning:A LaboratoryManul (2nd.ed.), Vols.1-3, Cold Spring Harbor Laboratory (1998); Current ProtocolsIn Molecular Biology, Ausubel, ed.John wiley ﹠amp; Sons, Inc.New York (1997)).

Can use many known methods to finish the qualitative and quantitative analysis of nucleic acid, carrier, polypeptide and protein etc., for example these methods comprise spectrophotometry, radiography, electrophoresis, high performance liquid chromatography (HPLC), immunoprecipitation, immunodiffusion(ID), immunoelectrophoresis, radioimmunity, enzyme linked immunosorbent assay (ELISA), immunofluorescent test, Southern analysis, Northern analysis, dot blotting analysis, gel electrophoresis (SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or amplification of signal technology etc.

Our experimental result shows, owing to substitute the expression of the signal sequence guiding target protein matter of using α-mating factor usually with the albumin signal sequence, not only make and modify or the enzyme peptide that presses down of sudden change is expressed in the pasteur pichia yeast and become possibility, and can the pichia yeast system with high yield express have a natural N-terminal aminoacid sequence (Arg-Pro-Asp) but the enzyme peptide.Detected result shows that the natural productive rate of enzyme peptide that presses down that uses the inventive method expression is up to about 900mg/L.

Reported and pressed down the expression of enzyme peptide in the intestinal bacteria system, yet, contain three pairs of disulfide linkage owing to press down the enzyme peptide, be easy to take place the mispairing of disulfide linkage during external renaturation, so in intestinal bacteria, but the enzyme peptide is difficult to the activity form of complete two-character surname is expressed, and the output in the secreting, expressing system also generally lower (10mg/L).In addition, press down enzyme peptide secreting, expressing in the yeast saccharomyces cerevisiae system report is also arranged, but as previously mentioned, because α-mating factor signal peptide nickase (Kex2) can not cut N-terminal sequence " Arg-Pro-aspartic acid ", so the output that this expression system secreting, expressing presses down the enzyme peptide is very low equally.Different with above-mentioned prior art, the present invention utilizes the pichia yeast system, utilize the targeting signal peptide of human serum albumin signal peptide to guide expression and the secretion that presses down the enzyme peptide, the result is secretion type expression and obtained having the recombinant aprotinin of correct N-terminal aminoacid sequence successfully, not only other any expression system of the rate ratio of product is all high, and expression product also is easier to purifying.

As a kind of serpin and active pharmaceutical ingredient, the basic role that presses down the enzyme peptide is the hydrolase of proteolysis that pathologic raises is reduced to normal level, for example be used for the treatment of acute pancreatitis, wound hemorrhage shock, suppress plasmin activity, protect thrombocyte, reduce (J.McMichanet al.Circulatory Shock 9 such as hemorrhage and oozing of blood, 107,1982; L.M.Auer et al.Acta Neurochir.49,207,1979).In addition, press down the enzyme peptide and also can be used for treating the pathologic liver tissue injury (referring to No. 03135997.3 Chinese patent application) that a variety of causes causes.

What the present invention relates to presses down the enzyme peptide and can use separately, and the form that also can be used as the pharmaceutical composition of various different dosage forms is used.(referring to Remington ' sPharmaceutical Sciences, Mack Pub.Co.Eaton Pa.1980), prepares pharmaceutical composition of the present invention with the unit metering form that is suitable for oral or non-oral administration can to use the known method in pharmaceutical industry field.For example, the enzyme peptide that presses down as the significant quantity of activeconstituents can be mixed equably with pharmaceutically acceptable carrier or vehicle, make solution or form of suspension be suitable in intravenously, intramuscular, the tissue lumen, intracutaneous and gi tract such as the subcutaneous pharmaceutical composition of administration outward; Perhaps also can make the pharmaceutical composition of being suitable for of forms such as tablet, capsule, powder agent, emulsion, granule through the gi tract administration.

According to pharmaceutical composition of the present invention, wherein employed pharmaceutically acceptable carrier or vehicle will have different selections according to the formulation of prepared pharmaceutical composition of the present invention.

The preparation that is suitable for the gi tract external administration can contain sterilized water or vehicle commonly used such as salt solution, polyoxyethylene glycol, oil and hydrogenated naphthalene.Particularly, can use biocompatible, biodegradable lactide polymer, poly (lactide-co-glycolide), polyoxyethylene/polyoxypropylene multipolymer, with the slow release of control activeconstituents as vehicle.Be suitable in the preparation of gi tract external administration at other, also comprise contain salicylic rectal administration preparation and contain glycocholate contain the clothes drug-delivery preparation.

Specifically, can use vehicle such as lactose, glucose, sucrose, N.F,USP MANNITOL and methylcellulose gum, disintegrating agents such as starch, sodium alginate, calcium carboxymethylcellulose and crystalline cellulose, lubricant such as Magnesium Stearate and talcum, cakingagents such as gelatin, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose gum and hydroxypropylcellulose, with tensio-active agents such as sucrose fatty ester, Spans, and ancillary component such as tinting material, sweeting agent, spices, dispersion agent, prepare tablet with ordinary method.

Can use vehicle such as lactose and N.F,USP MANNITOL, disintegrating agents such as starch, cakingagents such as gelatin prepare granule with ordinary method.

Perhaps, can use vehicle such as lactose and sucrose, prepare powder agent with ordinary method.Use gelatin, water, sucrose, gum arabic, sorbyl alcohol, glycerine, crystalline cellulose, Magnesium Stearate and talcum etc., prepare capsule with ordinary method.

Can make water, physiological saline, vegetables oil (as sweet oil and peanut oil), ethyl oleate and propylene glycol equal solvent, Sodium Benzoate, solubilizing agent such as sodium salicylate and urethanum, isotonic agent such as sodium-chlor and glucose, microbiotic such as penicillin and Streptomycin sulphate and other anti-mycotic agents, sanitass such as phenol, cresols, contraposition hydroxybenzoate, trichloro-butyl alcohol, degree rice phenol and Sorbic Acid, antioxidants such as xitix and trisodium phosphate prepare the injectable agent with ordinary method.

In addition, also can use known method and auxiliary composition in the pharmaceutical industry, pharmaceutical composition of the present invention is made microcapsule or liposome agent.

Pharmaceutical composition of the present invention also can contain one or more synthetic, other activeconstituentss with collaborative or booster action natural or recombinant sources except that pressing down enzyme peptide or its analogue or the mutant of containing as the natural of primary activity composition or recombinate and produce.Under the situation that is used for the treatment of liver tissue injury (for example acute or subacute severe hepatitis, acute viral hepatitis, active period chronic viral hepatitis, toxic hepatitis and the Secondary cases liver injury that causes of other reasons and liver failure etc.), these activeconstituentss comprise but are not only limited to have immunostimulation or regulate other compositions active, that virus replication suppresses activity, functional liver cell repairing activity, for example can be Interferon, rabbit, interleukin, thymosin, pHGF and fibroblast growth factor etc.

In general, the oral administration dosage of pharmaceutical composition of the present invention is 0.1-100 unit/kg/ days, is preferably 1-80 unit/kg/ days, is preferably 5-60 unit/kg/ days; Intraperitoneal or intramuscular injection dosage are 0.05-100 unit/kg/ days, are preferably 0.1-80 unit/kg/ days, are preferably 0.5-50 unit/kg/ days; The intravenous injection dosage is 0.1-100 unit/kg/ days, is preferably 0.5-80 unit/kg/ days, is preferably 1-50 unit/kg/ days.Certainly, it will be appreciated by those skilled in the art that, should be for treating the required definite dosage of patient effectively according to the character of illness to be treated or pathological state, severity, patient's age, body weight and general health state, the formulation of used medicine, patient are to the susceptibility and the tolerance of used medicine, and factors such as the route of administration of using, determine by the clinician according to the principle of individuation.

Description of drawings

Figure 1A shows albuminous signal peptide sequence and albuminous cutting mode; Figure 1B shows albuminous signal peptide sequence and the mode of connection that presses down the enzyme peptide.

Fig. 2 shows the structure that is used to express the recombinant expression plasmid pPICZC-HSAsp-BPTI that presses down enzyme peptide secretory protein.

Fig. 3 shows 1% agarose gel electrophoretogram of the recombinant plasmid that has connected the human serum albumin signal peptide encoding sequence.Wherein swimming lane 1 is with BstBI and the plasmid (pPICZC-HSAsp) that is connected with the human serum albumin signal peptide encoding sequence again after the XhoI enzyme is cut; Swimming lane 2 is dna molecular amount marks.Swimming lane 3 is to cut with the XhoI enzyme with BstBI but be not connected the original plasmid of human serum albumin signal peptide encoding sequence.

Fig. 4 shows 1% agarose gel electrophoretogram that presses down enzyme peptide gene sequence.Wherein swimming lane 1 is the dna sequence dna that coding presses down the enzyme peptide; Swimming lane 2 is dna molecular amount marks.

Fig. 5 shows the additional 1% agarose electrophoresis collection of illustrative plates that presses down enzyme peptide gene sequence that restriction enzyme site is arranged.Wherein swimming lane 1 is the dna sequence dna that coding presses down the enzyme peptide; Swimming lane 2 is dna molecular amount marks.

Fig. 6 shows that the SDS-PAGE that presses down the enzyme peptide prod of secreting, expressing analyzes.Wherein swimming lane 1 is spissated culture supernatant part; Swimming lane 2 is the purified products through cation-exchange chromatography; Swimming lane 3 is to press down the enzyme poly saccharide peptide standard product.

Embodiment

The following example is intended to further illustrate rather than limit the present invention.It will be appreciated by those skilled in the art that, under the prerequisite that does not deviate from the spirit and principles in the present invention, all will fall in the claim scope that awaits the reply of the present invention any parallel change of the present invention and change.

Embodiment 1: the structure of recombinant plasmid pPICZ C-HSAsp-BPTI

Present embodiment is described for example and is used to express construction strategy and the basic skills (referring to accompanying drawing 2) that the present invention presses down the recombinant expression plasmid pPICZC-HSAsp-BPTI of enzyme peptide.

At first, synthetic two (normal chain and minus strands) are as follows, and 5 ' and 3 ' end all comprises the human serum albumin signal peptide encoding sequence of BstBI and XhoI restriction enzyme site:

5 '-- CGAAACGATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTA C-3 ' (BstBI wherein and XhoI restriction enzyme site indicate with underscore) (SEQ IDNO:1) 5 '- TCGAGTAAGCCGAGCTAAAGAGAAAAAGAAGGGAAATAAAGGTTACCCACTTCATCGT TT-3 ' (BstBI wherein and XhoI restriction enzyme site indicate with underscore) (SEQ ID NO:2).

Article two, after the external annealing of mononucleotide chain warp, obtain the bifilar nucleotide coding sequence of human serum albumin signal peptide that two ends have BstBI and XhoI restriction enzyme site respectively.After 1% agarose gel electrophoresis reclaims, in the presence of the T4DNA ligase enzyme, this fragment is connected among the plasmid pPICZaC (Invitrogen) that uses the cutting of BstBI and XhoI restriction endonuclease in advance, obtain containing the carrier pPICZC-HSAsp of human serum albumin signal peptide (HSAsp), promptly added the recombinant plasmid of human serum albumin signal peptide.BstBI and XhoI enzyme are cut qualification result and are shown, remove original α-mating factor and connected the endonuclease bamhi length behind the signal peptide sequence and be about 60bp, do not carry out signal peptide sequence metathetical fragment length and then be about 300bp (referring to accompanying drawing 3) in corresponding site.

Secondly, use suitable primer to obtain containing the dna fragmentation that presses down enzyme peptide complete genome sequence from the total DNA cloning of ox lung with polymerase chain reaction (PCR) method.Two primers of PCR reaction are respectively P1:5 '--CTGTGATGACCTTCCCTCTTTAACCG-3 ' (SEQ ID NO:3) P2:5 '-TTCTGAGCGCCACCACTTTCCCTA-3 ' (SEQ ID NO:4).

(1) 1:94 ℃ of sex change of PCR reaction is 4 minutes.(2) PCR reaction 2:, 94 ℃ of sex change 30 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ were extended 28 circulations 1 minute.(3) 3:94 ℃ of sex change of PCR reaction is 45 seconds, and 72 ℃ were extended 2 minutes.The length of pcr amplification product is 300bp (referring to accompanying drawing 4).Then, be connected in the T carrier (TakaRa), obtain plasmid T-BPTI.

Then, use synthetic Oligonucleolide primers P1:5 '-CAAAGCCTCGAGACCTGACTTCTGCCTA-3 ' (containing the XhoI restriction enzyme site) (SEQ ID NO:5); P2:5 '-GGTTCTAGAGCCCTTAAGCACC-3 ' (containing the XbaI enzyme cutting site) (SEQ ID NO:6) take T-BPTI as template pcr amplification obtain the encoding dna fragmentation of BPTI, the about 203bp of length.

(1) 1:94 ℃ of sex change of PCR reaction is 4 minutes.(2) PCR reaction 2:, 94 ℃ of sex change 30 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ were extended 28 circulations 1 minute.(3) 3:94 ℃ of sex change of PCR reaction is 45 seconds, and 72 ℃ were extended 2 minutes.Obtain amplified production BPTI encoding sequence through 29 circulations.(referring to accompanying drawing 4)

The PCR product is after 1% agarose gel electrophoresis reclaims, with endonuclease XhoI and XbaI double digestion, and in the presence of the T4DNA ligase enzyme, this fragment is connected among the plasmid pPICZC-HSAsp with same restriction endonuclease digestion, thereby obtain recombinant expression plasmid pPICZC-HSAsp-BPTI.With the sequenator CEQ2000 of U.S. Beckman company order-checking, the result shows that human serum albumin signal peptide all has and known identical nucleotide coding sequence with BPTI.

Embodiment 2: the expression of recombinant aprotinin and fermentative production

This enforcement is enumerated and is described the abduction delivering of enzyme peptide protein matter in the pichia yeast host cell and the high density fermentation of thalline thereof of pressing down of the present invention.

Behind the recombinant plasmid pPICZ C-HSAsp-BPTI that makes among the restriction enzyme PmeI digestion embodiment 1, grow in BMGY substratum (1% yeast extract with resulting digestion product conversion, 2% peptone, 1% glycerine, 100mM potassium phosphate buffer PH6.0,1.34%YNB, 4 * 10-5% vitamin H) the pichia yeast strain X-33 (Invitrogen) in, and 30 ℃ of yeast host cells that cultivation is transformed according to a conventional method.Then, picking and cultivate the positive monoclonal bacterium colony.Work as OD ₆₀₀Nm reaches at 2～6 o'clock, and frozen part bacterial classification is with BMGY substratum (1% yeast extract, 2% peptone, 1% glycerine, 100mM potassium phosphate buffer PH6.0,1.34%YNB, 4 * 10 ^-5The % vitamin H) is replaced with BMMY substratum (1% yeast extract, 2% peptone, 0.5% methyl alcohol, 100mM potassium phosphate buffer PH6.0,1.34%YNB, 4 * 10 ^-5The % vitamin H) abduction delivering.Induce in the process every sampling in 24 hours, with SDS-PAGE electrophoretic method monitoring protein expression product.SDS-PAGE analyzes demonstration, has the band (referring to accompanying drawing 5) of an about 6KDa of molecular weight in the expression product.During expressing, utilize known trypsinase suppress experiment quantitative analysis expression product biologic activity (referring to Cara B, Marks V, Peter N, et al, [J] .JBC, 1986,261 (16): 7115-7118).

Then, in 28 ℃, with 30L scale fermentation culture positive strain.For this reason, the basic salt culture medium (H of preparation 30L FM21 ₃PO ₄3.5ml/L, CaSO ₄2H ₂O 0.15g/L, K ₂SO ₄2.4g/L, MgSO ₄7H ₂O1.95g/L, KOH 0.65g/L), 121 ℃ of autoclavings 30 minutes treat to add when temperature drops to 30 ℃ of left and right sides blended trace element (PTM1) and vitamin H, and thalline is inoculated in 80 liters of fermentor tanks (New Brunswick scientific), carry out conventional high density fermentation.Fermentation condition is: temperature: 28-30 ℃; Agitator speed of rotation: 600 rev/mins (rpm); PH:3.30 (uses NH ₄OH and/or H ₃PO ₄Proofread and correct).

By control, make that dissolved oxygen amount remains on more than 20% in the culture, and make other parameters maintain steady state conditions such as stir speed (S.S.), jar internal pressure, air flows.

After inducing beginning,, take by weighing the weight in wet base or the dry weight of thalline, and leave and take supernatant and carry out its lytic activity and SDS-PAGE electrophoretic analysis, thereby determine the accumulative total effect that protein prolongs in time in the different time sampling.Fermentation continues 30 hours approximately.The thalline weight in wet base is about 300g/L during fermentation ends, and the concentration of recombinant aprotinin is about 900mg/L.

Embodiment 3: the purifying of recombinant aprotinin

After cultivation is finished, utilize the centrifugation technique of standard that yeast cell is separated with nutrient solution, discard the yeast thalline after, collect supernatant.Add the 200mM NaAc-HAc PH4.0 damping fluid of about 1/10 volume in the supernatant, make its final concentration reach about 20mM.Use 20mM NaAc-HAc (pH4.0) equilibrated Sepharose SP FF cation-exchange chromatography column chromatography for separation through in advance, and use solution A (20mMNaAc-HAc PH4.0) and solution B (2M Nacl, 20mM NaAc-HAc PH4.0) to carry out wash-out successively.Behind the wash-out, collect each component peaks and carry out SDS-PAGE and press down the activation analysis of enzyme peptide.

Through the isolating enzyme peptide crude product that presses down of Sepharose SP FF cation-exchange chromatography again through Sourece ^TMThe anti-phase hydrophobic chromatography of 30RPC is further purified.Contain to press down and add with initial buffer A (0.1%TFA) balance Sourece after the active sample of enzyme peptide adds 1/10 volume 1%TFA ^TMThe anti-phase hydrophobic chromatography post of 30RPC carries out gradient elution with solution A (0.1%TFA) and solution B (100% acetonitrile), collects each component peaks and carries out SDS-PAGE and press down the activation analysis of enzyme peptide, and measure protein concentration according to the OD280 absorption value.

Mass spectrometric detection result shows that the molecular weight that presses down the enzyme peptide of the present invention's preparation is 6508.7Da.Measure the protein N-terminal sequence with the Edman hydrolysis method, the result shows, terminal 15 aminoacid sequences of protein N are with natural but the enzyme peptide sequence is in full accord.

Embodiment 4: the biologic activity analysis of expression product

Utilize trypsinase to suppress the biologic activity that experiment (with reference to Cara B, Marks V, PeterN, et al) comes the quantitative analysis expression product.Briefly, this method comprises that at first (50mMTris-HCl, PH 8.0,10mM MgCl to 600 μ l buffer systems ₂, 10mM CaCl ₂) in add 200 μ l trypsin solutions (100U/mlBuffer), and then add the representation supernatant of 200 μ l different concns.Place under the room temperature reaction after about 20～30 minutes in this mixture, add 50 μ l substrate N-benzoyl-L arginine-β naphthalene amino acid solution (BAPNA) (20mM is dissolved in DMSO).React after 5 minutes, measure the variation (with respect to control tube) of absorbancy in the 405nm place.As a result, the enzyme peptide performance that presses down with natural N-terminal aminoacid sequence (Arg-Pro-Asp) that visible the inventive method makes has very significant trypsin inhibition activity (inhibiting rate is about 80%).In addition, use this method to record the productive rate with recombinant aprotinin of natural N-terminal aminoacid sequence of the present invention and be about 900mg/L.

Sequence table

＜110〉Jinlin Shengyuan Science ﹠. Technology Co., Ltd.

＜120〉prepare the method for recombinant aprotinin

<140>

<141>

<160>6

<210>1

<211>58

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic human serum albumin signal peptide dna encoding sequence normal chain.

<400>1

CGAAACGATG?AAGTGGGTAA?CCTTTATTTC?CCTTCTTTTT?CTCTTTAGCT?CGGCTTAC

<210>2

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic human serum albumin signal peptide dna encoding sequence minus strand.

<400>2

TCGAGTAAGC?CGAGCTAAAG?AGAAAAAGAA?GGGAAATAAA?GGTTACCCAC

TTCATCGTTT

<210>3

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.

<400>3

CTGTGATGAC?CTTCCCTCTT?TAACCG

<210>4

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<400>4

TTCTGAGCGC?CACCACTTTC?CCTA

<210>5

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<400>4

CAAAGCCTCG?AGACCTGACT?TCTGCCT?A

<210>6

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<400>4

GGTTCTAGAG?CCCTTAAGCA?CC

Claims

1. method of using pichia pastoris phaff system secreting, expressing recombinant aprotinin, this method comprises cultivates the pichia pastoris phaff bacterial strain that comprises reproducible expression vector, said carrier carries the gene and the signal sequence that directly is connected at its upstream that coding presses down the enzyme peptide, and allow secreting, expressing in suitable nutritional medium but the dna sequence dna of enzyme peptide, from the culture supernatant, reclaim then and purifying is required presses down the enzyme peptide, be characterised in that directly being connected the signal sequence that presses down enzyme peptide gene upstream in the said carrier is people or Mammals albumin signal sequence.

2. according to the method for claim 1, be characterised in that said press down the enzyme peptide be have a correct natural N-terminal aminoacid sequence press down the enzyme peptide.

3. according to the method for claim 1, be characterised in that said albumin signal sequence has the nucleotide sequence shown in the SEQ ID NO:1 and SEQ ID NO:2 in the sequence table.