CN1370838A - Amidated recombinant polypeptide and its prepn - Google Patents

Abstract

The present invention relates to one kind of nucleic acid constituted matter including at least one nucleic acid sequence encoding alpha-amidated enzyme and one nucleic acid sequence of target polypeptide to be amidated. The present invention further relates to the expression vector and prokaryotic host cell of the said nucleic acid constituted matter. The present invention also relates to the method of utilizing the said nucleic acid constituted matter to express in prokaryotic host to prepare amidated recombinant polypeptide, especially amidated recombinant salmon calcitonin.

Description

A kind of amidated recombinant polypeptide and preparation method thereof

The present invention relates to a kind of nucleic acid construct that comprises the nucleotide sequence of at least a coding for alpha-amidating enzyme and treat the nucleotide sequence of amidated target polypeptides, the invention further relates to the expression vector, the prokaryotic host cell that contain described nucleic acid construct.The invention still further relates to by utilizing described nucleic acid construct in prokaryotic hosts, to express, prepare amidated recombinant polypeptide, especially the method for amidation reorganization thyrocalcitonin.

As everyone knows, in the polypeptide with biologic activity of neural and endocrine system generation, its C-terminal of great majority need be by amidation, and C-terminal α-amide group is most important to the biological activity of polypeptide, avidity (the Fuhlendorff that it may prolong the transformation period of polypeptide or strengthen polypeptide and acceptor, J. etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 87:182-186).α-the amide group of C end is from the translation post-treatment of polypeptide precursor, and by α-amidating enzyme (be designated hereinafter simply as α-AE, be called peptide acyl glycine α-amidation monooxygenase (PAM) again) catalysis, this reaction is the rate-limiting step in the active polypeptide biosynthetic process.The example that belongs to this class active polypeptide has thyrocalcitonin, pitocin, vassopressin, corticotropin releasing homone, thyrotropin releasing hormone, neurokinin, allatostatin, Lem-KI, haematochrome to concentrate hormone, luteinising hormone-releasing hormo, leucopyrokinin, gastrin, pigment dispersion hormone, dermorphin, bombesin, gastrin releasing peptide, neuromedin, pancreastatin, conotoxin M 1, pancreastatin, mellitin, sarcophagid toxin 1A, VIP, α-MSH, MIF-1 etc., and the amidation of its C-end is most important to its biological activity.

Now can utilize recombinant DNA technology in prokaryotic organism, to produce protein and polypeptide with clinical and commercial use value, because the prokaryotic expression system does not have α-AE, therefore production amidation polypeptide can not be used for, but the active polypeptide precursor that the C-end has Gly can be produced.For body before this class reorganization active polypeptide is carried out amidation, the following method of general at present employing: 1) utilize the external catalytic polypeptide precursor of α-AE that obtains to carry out amidation from the animal tissues of natural origin

Engels, (nucleosides and Nucleotide (Nucleosides ﹠amp such as J.W.; Nucleotides) 6:185-195) gene with human growth hormone releasing factor's precursor (hGHCF-Gly) forms inclusion body at expression in escherichia coli, obtain hGHCF-Gly through behind a series of purification steps, separation and purification α-AE from the Niu Chuiti of natural origin utilizes the external amidation of the α-AE catalysis hGHCF-Gly of purifying simultaneously.Because α-AE content is very low in the animal tissues of natural origin, separation and purification difficulty, this method are difficult to enlarge and are used for commercially producing of active polypeptide.2) the external catalytic polypeptide precursor of α-AE that utilizes gene engineering method to obtain carries out amidation

(Bio/Technology such as Ray, 11:64-70) with Chinese hamster ovary celI secreting, expressing rat α-AE, behind hydrophobic chromatography and ion exchange chromatography preliminary purification, utilize the external amidation of salmon calcitonin see calcimar precursor (sCT-Gly) of the α-AE catalysis escherichia coli expression of purifying, the mass ratio (quality of the quality/enzyme of amidated products) of reaction can reach more than 1000, can be used for the scale operation of salmon calcitonin see calcimar.But sCT-Gly expresses respectively in two different expression systems with α-AE, described amidating enzyme and target polypeptides just can need be carried out amidate action behind the purifying respectively, after finishing, amidate action also needs amidated thyrocalcitonin (sCT) is further purified, step is various, complex process, yield is low, the cost height.

People such as Li Yuan disclose the method with recombinant DNA technology precursor (sCT-Gly) of secreting, expressing salmon calcitonin see calcimar in shallow Streptomyces glaucoviolaceus (Streptomyces lividans) in Chinese patent 96105118.3, yet this technology can not directly produce amidated salmon calcitonin see calcimar.

The report that successfully prepares the amidation polypeptide in the eucaryon host expression system is also arranged at present, (peptide (Peptides), 18 (3): 439-44) disclose the method for in COS-7 and Chinese hamster ovary celI system, expressing the sCT-NH2 of biologically active such as Takahashi.Because it is higher that eukaryotic expression system exist to be cultivated cost, strict to working condition, and from nutrient solution, reclaim the problem of aspect such as target polypeptides difficulty, be unsuitable for large-scale commercial production.

Widely used clinically at present biologic activity depends on amidated polypeptide and mainly contains thyrocalcitonin, pitocin, vassopressin etc.The potential amidated polypeptide of other needs clinical and commercial use value is increasing along with having, a kind of more economic, effective polypeptide amidation technology of an urgent demand.In order to simplify processing step, to reduce cost, the present invention has designed a kind of nucleic acid construct, and described nucleic acid construct contains a kind of nucleotide sequence of the amidating enzyme of encoding and the nucleotide sequence that coding is treated amidated target polypeptides at least.The expression vector that will contain this nucleic acid construct by gene engineering method transforms specific prokaryotic hosts, can directly obtain target polypeptides amidated, biologically active through expressing.

One aspect of the present invention relates to the nucleic acid construct that the nucleotide sequence that comprises at least a coding amidating enzyme and coding are treated the nucleotide sequence of amidated target polypeptides.

Another aspect of the present invention relates to the expression vector that contains described nucleic acid construct.Another aspect of the present invention relates to the host cell that contains described expression vector.The invention still further relates to the preparation method of described expression vector and host cell.

Another aspect of the invention relates to by express the target polypeptides of described nucleic acid construct coding in the suitable expressive host of gained, prepares the method for amidated recombinant polypeptide.

Another aspect of the present invention also relates to utilization and has the nucleotide sequence of the amidating enzyme of encoding and coding respectively and treat that the different consistency of amidated target polypeptides expresses carrier, and the same prokaryotic hosts of cotransformation prepares the method for amidation target polypeptides.

Another aspect of the present invention also relate to utilization respectively the expression vector of the nucleotide sequence of encoded amidating enzyme and the prokaryotic hosts that expression vector that coding is treated amidated target polypeptides transforms carry out common cultivation, prepare the method for amidation target polypeptides.

In order to obtain the amidation polypeptide of biologically active, the present invention has prepared a kind of nucleotide sequence of at least a coding for alpha-amidating enzyme and nucleic acid construct that coding is treated the nucleotide sequence of amidated target polypeptides of comprising.1. the nucleotide sequence of coding for alpha-amidating enzyme

Those skilled in the art know, and α-amidating enzyme used among the present invention can include but not limited to the α-amidating enzyme as sources such as pig, ox, mouse, rat, toads from multiple source.The expressive host of amidated target polypeptides and the employing wanted is generally depended in the selection of α-amidating enzyme.In a preferred embodiment of the invention, described α-amidating enzyme comes from the atrial tissue of rat.In a preferred embodiment, encode the nucleotide sequence of described α-amidating enzyme shown in SEQ ID NO:1.

Know as those skilled in the art, because the diversity of α-amidating enzyme mRNA, in the different tissues of the biological or even same biology of different sources, have the α-amidating enzyme of various ways.Therefore, the nucleotide sequence of used coding for alpha-amidating enzyme can have various ways owing to the difference of the different splicing forms of the degeneracy of codon, mRNA among the present invention.Therefore, the present invention comprises that also coding has the nucleotide sequence of polypeptide of the aminoacid sequence of SEQ ID NO:2, and described nucleotide sequence can be different with SEQ ID NO:1 owing to the degeneracy of genetic code.The present invention also comprises the nucleotide sequence of a kind of like this α-amidating enzyme of coding, the nucleotide sequence of itself and SEQ IDNO:1 has homology to a certain degree, and homology is at least about 80%, preferably about 85%, more preferably about 90% even more preferably about 95%, most preferably about 97%.For the present invention, can pass through Wilbur-Lipman method (Wilbur and Lipman, 1983, institute of NAS reports 80:726-730), utilize LASERGENE ^TMMEGALIGN ^TM(Madison WI), uses the degree that identity table and selected multiple reduced parameter are measured identity between two kinds of nucleotide sequences to software for DNASTAR, Inc..The present invention also comprises the nucleotide sequence of a kind of α-amidating enzyme of encoding, its under the low stringent condition, more preferably under the moderate stringent condition even more preferably under the height stringent condition, most preferably under the high stringent condition can with the nucleotide sequence of SEQ ID NO:1 as described here or its complementary strand or its allelic variant and subsequence hybridization thereof (people such as Sambrook, 1989, ibid)." stringent condition " described herein is at least 100 Nucleotide for length, minuent is defined as respectively to high degree stringent condition: according to standard Southern western blot procedure, shear and the salmon sperm DNA of sex change and being respectively for minuent, moderate, height and high degree severity in 25%, 35% or 50% the methane amide 42 ℃ of prehybridizations and hybridization at 5 * SSPE, 0.3%SDS, 200 μ g/ml.Be at least the long probe of 100 Nucleotide for length, use 2 * SSC, 0.2%SDS with this carrier substance washing 3 times at last, each 15 minutes, preferably at least 45 ℃ of wash temperatures (extremely low severity), more preferably at least 50 ℃ (low severity), more preferably at least 55 ℃ (middle severity), more preferably at least 60 ℃ (in-the height severity), even more preferably at least 65 ℃ (high severity), most preferably at least 70 ℃ (high severity).For length is that about 15 Nucleotide are to about 70 Nucleotide, stringent condition is defined as: according to standard Southern western blot procedure, than according to Bolton and McCarthy (1962, institute of NAS reports 48:1390) under the temperature of low 5 ℃-10 ℃ of the Tm that calculates, contain prehybridization, hybridization and post-hybridization washing in 0.9M NaCl, 0.09M Tris-HCl pH7.6,6mM EDTA, 0.5%NP-40,1 * Denhardt solution, 1mM trisodium phosphate, 1mM SODIUM PHOSPHATE, MONOBASIC, 0.1mM ATP and the 0.2mg yeast rna at every ml.For length is about 15 Nucleotide to about 70 Nucleotide, under the temperature than low 5 ℃-10 ℃ of the Tm that calculates, carrier substance is added at 6 * SSC wash one time 15 minutes among the 0.1%SDS, and wash twice with 6 * SSC, each 15 minutes.2. α-amidating enzyme polypeptide

By the nucleotide sequence of above-mentioned coding α-amidating enzyme of the present invention as can be known, can be used for α-amidating enzyme of the present invention and comprise the α-amidating enzyme that has as the described aminoacid sequence of SEQ ID NO:1.Those skilled in the art are known, can modify the conservative property that described polypeptide carry out one or more amino-acid residues, as insert, deletion or replace, and can not influence the biologic activity of α-amidating enzyme of the present invention basically.Therefore, the invention still further relates to variant, derivative or its fragment of above-mentioned α-amidating enzyme, its aminoacid sequence that has with SEQ ID NO:2 has the to a certain degree aminoacid sequence of identity, at least about 80%, preferably at least about 85%, more preferably at least about 90%, even more preferably at least about 95%, even most preferably at least about 97% identity polypeptide.In a preferred embodiment, described identity polypeptide has aminoacid sequence with SEQ ID NO:2 and differs 5 amino acid, preferably 4 amino acid, more preferably 3 amino acid even more preferably 2 amino acid, 1 amino acid whose aminoacid sequence most preferably.For the present invention, by Clustal method (Higgins, 1989, CABIOS 5:151-153), utilize LASERGENE ^TMMEGALIGN ^TM(Madison WI) measures identity to software for DNASTAR, Inc..Preferably, α of the present invention-amidating enzyme polypeptide can be aminoacid sequence or its allelic variant that comprises SEQ ID NO:2; Or its fragment, as long as this fragment has α-amidating enzyme activity.In a preferred embodiment, polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO:2.In a further preferred embodiment, polypeptide of the present invention comprises aminoacid sequence or its allelic variant of SEQ ID NO:2; Or its fragment, as long as this fragment has α-amidating enzyme activity.In a preferred embodiment, α of the present invention-amidating enzyme polypeptide is made up of the aminoacid sequence of SEQ IDNO:2.The coding target polypeptides nucleotide sequence and amino acid sequence of polypeptide

Of the present inventionly treat that amidated target polypeptides contains multiple polypeptides, this class polypeptide has clinical or medicinal use, and its biologic activity all depends on the amidation of C-end, includes but not limited to thyrocalcitonin, pitocin, vassopressin, corticotropin releasing homone, thyrotropin releasing hormone, neurokinin, allatostatin, Lem-KI, haematochrome concentrates hormone, luteinising hormone-releasing hormo, leucopyrokinin, gastrin, pigment disperses hormone, dermorphin, bombesin, gastrin releasing peptide, neuromedin, pancreastatin, conotoxin M 1, pancreastatin, mellitin, sarcophagid toxin 1A, VIP, α-MSH, MIF-1.It will be understood by those skilled in the art that the nucleotide sequence of the described target polypeptides of coding can be for synthetic or natural origin.As long as the structure that the C-end has with used α-amidating enzyme performance function adapts of selected target polypeptides, thereby can be by α-amidating enzyme amidation.Those skilled in the art also should know, and treat that the expression of amidated target polypeptides should not influence analytic metabolism, the physiological process of selected expressive host.In a preferred embodiment, target polypeptides of the present invention is a thyrocalcitonin, comprises salmon calcitonin see calcimar, chicken calcitonin or pig thyrocalcitonin.In a preferred embodiment, of the present inventionly treat that amidated target polypeptides is a salmon calcitonin see calcimar.In another preferred embodiment, of the present inventionly treat that amidated target polypeptides is that calcitonin-like, derivative or its have the fragment of the biologic activity that is equal to.In a preferred embodiment, coding treats that the nucleotide sequence of amidated salmon calcitonin see calcimar contains the nucleotide sequence of SEQ ID NO:3.In a preferred embodiment, treat that amidated polypeptide is the aminoacid sequence shown in the SEQ ID NO:4.4. nucleic acid construct

As used herein, term " nucleic acid construct " are defined as strand or double-stranded nucleic acid molecule, and be that it can be synthetic or separate from naturally occurring gene, or modifiedly contain that the mode that can not exist with occurring in nature makes up and nucleic acid fragment arranged side by side.Nucleic acid construct of the present invention can be strand or double-stranded DNA or RNA molecule.In a preferred embodiment, nucleic acid construct of the present invention comprises the nucleotide sequence of at least one coding for alpha-amidating enzyme and encodes the described nucleotide sequence for the treatment of amidated target polypeptides.The nucleotide sequence of the coding for alpha-amidating enzyme that is comprised in the nucleic acid construct of the present invention can have multiple situation with the described mutual alignment relation of nucleotide sequence in nucleic acid construct of amidated target polypeptides for the treatment of of coding, two kinds of nucleic acid sequence encodings can be positioned on the same nucleotide chain, also can lay respectively on two nucleotide chains.When two kinds of nucleotide sequences were positioned on the same nucleotide chain, the two can be adjacent or non-conterminous, connects or do not connect, and can have identical or different transcriptional orientation, as long as described α-amidating enzyme can will be treated amidated target polypeptides amidation after expressing.In addition, the two can contain the required control sequence of expression independently of one another two kinds of nucleotide sequences described in the present invention, and described control sequence can be identical or inequality.Selected control sequence depends primarily on the nucleotide sequence of the coding target protein of being controlled and the expressive host of employing.Those of ordinary skills know, can use any can be in selected expressive host the effective control sequence of controlled target expression of polypeptides.In addition, also can comprise two kinds of nucleotide sequences described in the present invention of one or more copies in the construct of the present invention respectively.Be used to regulate 5 ' terminal or 3 ' terminal any side that control sequence that described nucleotide sequence expresses can be positioned at nucleotide sequence, be preferably located in 5 ' side of nucleotide sequence to be expressed, as long as this control sequence can be controlled the expression of target protein matter effectively.In a preferred embodiment of the invention, used control sequence is respectively secretion signal peptide-coding sequence and the upstream sequence thereof from the melC1 gene.

As needs, also can contain in the nucleic acid construct one or more with express other relevant control sequence of contained target polypeptides encoding sequence.In a preferred embodiment of the invention, described nucleic acid construct has the signal peptide sequence that is used for secreting, expressing in 5 ' side of the nucleotide sequence of the nucleotide sequence of coding for alpha-amidating enzyme and coding thyrocalcitonin precursor respectively.In a further preferred embodiment, be positioned at the signal peptide sequence that is used for secreting, expressing of 5 ' side of polypeptide of two kinds of nucleotide sequences in the nucleic acid construct of the present invention all from the melC1 gene.Nucleic acid construct of the present invention, it can comprise that the nucleotide sequence for the treatment of amidated polypeptide with the nucleotide sequence and the coding of coding for alpha-amidating enzyme of the present invention effectively is connected one or more adjusting sequences of ground, is suitably expressing in the host cell in order to guide encoding sequence of the present invention.Should be appreciated that to express to comprise and participate in any step that polypeptide produces, include but not limited to transcribe, post transcriptional modificaiton, translation, posttranslational modification and secretion.

Term " encoding sequence " is defined herein as direct aminoacid sequence nucleotide sequence partly at its protein.Encoding sequence can include but not limited to DNA, cDNA and recombinant nucleic acid sequence.

Can be inserted in the suitable expression with the nucleotide sequence that multiple mode is treated amidated polypeptide with the nucleotide sequence and the coding of described coding for alpha-amidating enzyme, as needs, can process or modify nucleotide sequence before inserting carrier, this depends on expression vector.Utilize the technology of recombinant DNA method modification of nucleic acids sequence to be well known in the art.

At this term " control sequence " is defined as and is included as the necessary or favourable all components of polypeptide expression of the present invention.Every kind of control sequence for the nucleic acid encoding sequence can be self or external source.This class control sequence includes but not limited to: leader sequence, polyadenylation sequence, propeptide sequence, promotor, signal peptide sequence and transcription terminator.At least, control sequence comprises promotor and transcribes and the translation termination signal.Control sequence can have joint, is intended to introduce specific restriction site, so that control sequence is connected with the coding region of the nucleotide sequence of coding target polypeptides.Term " effectively connection " is defined herein as a kind of configuration, and wherein a kind of control sequence is suitably placed the position relevant with the dna sequence encoding sequence, makes this control sequence guide the generation of polypeptide.

Control sequence can be a kind of suitable promoter sequence, i.e. a kind of nucleotide sequence of being discerned by host cell for the expression of nucleotide sequence.Contain the transcriptional control sequence that mediates expression of polypeptides in the promoter sequence.Promotor can be any nucleotide sequence that shows transcriptional activity in selected host cell, comprises mutant, truncation type and heterozygous promotor, and can be outside coding and host cell homology or allogenic born of the same parents or in the born of the same parents obtains the gene of polypeptide.

In the situation that adopts the bacterial expression host cell, guiding the example of the suitable promotor that nucleic acid construct of the present invention transcribes is the promotor that obtains from following gene: the beta galactosidase enzyme gene of shallow Streptomyces glaucoviolaceus, the serpin STI-II gene of long spore streptomycete (Streptomyces longisporus), aminoglycoside phosphotransferase (aph) gene of streptomyces fradiae (Streptomycesfradiae), the promotor of the erythromycin resistance gene (ermE) of thorn sugar chain mould (Streptomyces erythraeus) and alpha-amylase inhibitor (tendamistat) gene of streptomyces tendae (Streptomyces tendae), and in the shallow Streptomyces glaucoviolaceus tipA gene can be by thiostrepton inductive promotor, streptomyces coelicolor (Streptomyces coelicolor) gelase gene (dagA), subtilis type froctosan saccharase gene (sacB), bacillus licheniformis alpha-amylase gene (amyL), bacstearothermophilus produces malt amylase gene (amyM), bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), subtilis xyl A and xyl B gene, intestinal bacteria lac operon, procaryotic β-Nei Xiananmei gene (people such as Villa-Kamaroff, 1978, institute of NAS reports 75:3727-3731) and tac promotor (people such as DeBoer, 1983, institute of NAS reports 80:21-25).At " Scientific Beauty compatriots " (Scientific American), 1980, " the deriving from the useful proteins matter of recombinant bacteria " among the 242:74-94; With people such as Sambrook, 1989, more promotor has been described in the same.Also include but not limited to aphD, tsr, ermE, ermE-up, ssi, vsi, STI-II, β-gal, vph, tipA etc. for available promotor among the host of streptomyces.

Control sequence also can be suitable transcription termination sequence, is promptly stopped a kind of sequence of transcribing by host cell identification.Terminator sequence effectively is connected with 3 of nucleic acid encoding sequence ' end.In selected host cell, there is any terminator of function all to can be used for the present invention.

Control sequence also can be suitable leader sequence, promptly translates the non-translational region of vital mRNA for host cell.Leader sequence effectively is connected with 5 of nucleic acid encoding sequence ' end.In selected host cell, there is any leader sequence of function all to can be used for the present invention.

Control sequence also can be a signal peptide coding region, the aminoacid sequence that its coding is connected with the aminoterminal of polypeptide, and this aminoacid sequence can guide encoded polypeptides to enter the Secretory Pathway of cell.5 of nucleic acid sequence encoding sequence ' end can contain a kind of signal peptide coding region inherently, this coding region with the coding secrete polypeptide the coding region fragment the translation frame in natural the connection.In addition, 5 of encoding sequence ' can to contain a kind of be the signal peptide coding region of external source for encoding sequence to end.When encoding sequence is not when normally containing signal peptide coding region, may need the signal peptide coding region of external source.In addition, the external source signal peptide coding region can be replaced the natural signals peptide-coding region simply to obtain the secretion of enhanced polypeptide.Yet any signal peptide coding region that can guide polypeptide expressed to enter selected secretory host cell approach all can be used for the present invention.

For the effective signal peptide coding region of bacterial host cell is the effective alpha-amylase inhibitor gene of streptomyces tendae in the streptomycete host for example, long spore Streptomyces serine proteases inhibitor STI-II gene, alpha-amylase gene, glucokinase gene, the signal peptide of beta-galactosidase gene and casease melC1 gene, product malt amylase gene from genus bacillus NCIB 11837, the bacstearothermophilus alpha-amylase gene, Bacillus licheniformis subtilisin gene, Bacillus licheniformis β-Nei Xiananmei gene, bacstearothermophilus neutral protease gene (nprT, nprS, nprM) or the signal peptide coding region that obtains in the subtilis prsA gene.Simonen and Palva, 1993, microbiology summary (Microbiological Reviews) 57:109-137 has described more signal peptide.

Control sequence also can be preceding peptide-coding region, its a kind of N-terminal aminoacid sequence of polypeptide that is positioned at of encoding.The polypeptide that obtains is called as proenzyme (proenzyme) or propolypeptide (perhaps being sometimes referred to as proenzyme (zymogen)).The common non-activity of propolypeptide can be converted into sophisticated active polypeptide by the ground of catalysis or autocatalysis from propolypeptide cutting propetide.Preceding peptide-coding region can obtain from bacillus subtilis alkali proteinase gene (aprE), subtilis neutral protease gene (nprT), yeast saccharomyces cerevisiae α-factor gene.

The adjusting sequence that adding can be regulated the expression of polypeptides relevant with the host cell growth also is desirable.The example of regulation system is that those are replied chemistry or physical stimulation (comprising the existence of regulating compound) and cause the regulation system that genetic expression is opened or closed.Regulation system in the prokaryotic system comprises lac, tac and trp operon system etc.

The adjusting sequence of construct can comprise one or more promotors, enhanser, negative regulatory element, transcribe the combination of binding site or these sequences.5. expression vector

Another aspect of the present invention relates to the expression vector that contains described nucleic acid construct, wherein preferably also contains promotor and transcribes and control sequence such as translation termination signal.Above-mentioned multiple nucleic acid and control sequence can effectively link together and produce a kind of recombinant expression vector, and this carrier also preferably comprises one or more suitable restriction sites, so that the purpose nucleotide sequence is in the insertion or the displacement in these sites.In addition, also can express nucleotide sequence of the present invention by the nucleic acid construct that in suitable expression vector, inserts nucleotide sequence or contain this sequence.In construction of expression vector, should make encoding sequence be arranged in carrier, make encoding sequence effectively be connected with the suitable control sequence that is used to express.

Recombinant expression vector can be any carrier (for example plasmid or virus) that can carry out the recombinant DNA operation easily and can cause nucleotide sequence to be expressed.The selection of carrier is generally depended on carrier and is waited to introduce consistency between the host cell of carrier.Carrier can be the plasmid of linearity or closed hoop.

Carrier can be the self-replicating carrier, that is, as the carrier that the outer entity of a kind of karyomit(e) exists, it duplicates and does not rely on THE REPLICATION OF CHROMOSOME, for example, and plasmid, extra-chromosomal element, minichromosome or artificial chromosome.Carrier can contain any element of guaranteed self replication.In addition, carrier also can be a kind of like this carrier, and after introducing host cell, it is incorporated in the genome and with the karyomit(e) of being integrated and duplicates.In addition, also can use a kind of carrier or plasmid or two or more carriers or plasmid, they contain the total DNA that is introduced in the host cell gene group together.

Carrier of the present invention preferably contains one or more makes transformant can obtain the selected marker of convenient screening.Selected marker is a kind of like this gene, and its product provides antibiotics resistance, biocide or virus resistance, heavy metal resistance or the like.The example of bacterium selected marker is the dal gene of subtilis or Bacillus licheniformis, or gives the mark of antibiotics resistance such as penbritin, kantlex, paraxin or tetracyclin resistance or be used for the thiostrepton resistance that streptomycete is screened.

Those skilled in the art know, and target polypeptides to be expressed and selected prokaryotic host cell are depended in the selection of suitable expression.Be applicable to that expression vector of the present invention includes but not limited to the pIJ101 plasmid pIJ680 that derives, pGM16, pSGL1, or their derivative, for example for the expressive host of streptomyces, can be the pIJ680 that has strong promoter aph, other preferred expression vector is referring to Kieser etc., streptomycete genetics is put into practice (PracticalStreptomyces Genetics, The John Innes Foundation, Norwich, 2000), pIJ486/487 for example, pIJ702, pFD666; For escherichia coli host, preferred expression vector is pUR278, pEX2, and pMR100, pKC30, pET-3a etc. are as people such as Sambrook, described in the molecular cloning (second edition, 1989, New York Cold Spring Harbor LabortoryPress); For the genus bacillus expressive host, preferred expression vector can be for example pWVO1, pGA14 etc.

For self-replicating, carrier can further contain make carrier can be in described host cell the replication orgin of self-replicating.The example of bacterium replication orgin is the replication orgin that allows plasmid pBR322, pUC19, pACYC177 and the pACYC184 duplicate in intestinal bacteria, the replication orgin of the pIJ101 that permission is duplicated in streptomycete, pJV1, pSG5, pSGL1, and the replication orgin that allows pUB110, pE194, pTA1060 and the pAM β 1 in genus bacillus, duplicate.Replication orgin can have makes its effect in host cell become temperature sensitive sudden change (for example see, Ehrlich, 1978, institute of NAS reports 75:1433).

Those skilled in the art are known can to insert the generation that strengthens the purpose product in the host cell with the target nucleic acid sequence of the present invention that surpasses a copy.The method that obtaining the nucleotide sequence copy number increases comprises, another copy at least of this sequence is incorporated in the host cell gene group, perhaps make nucleotide sequence contain the selected marker that can increase, by culturing cell in the presence of suitable selective agent, can filter out selected marker that containing the amplification copy, and thereby contain the cell of the nucleotide sequence of other copy.

Be used to connect said elements and be those skilled in the art known (people such as Sambrook, ibid) with the method that makes up recombinant expression vector of the present invention.6. host cell

The present invention also relates to contain the host cell of the recombinant expression vector with nucleic acid construct of the present invention, it is used for the recombinant production of target amidation polypeptide.The expression vector that will contain nucleic acid construct of the present invention is introduced in the host cell, and carrier is remained in wherein as the chromosomal integration body or as the outer carrier of the karyomit(e) of foregoing self replication.Term " host cell " also comprises any filial generation of the parental cell different with parental cell owing to undergo mutation between replicative phase.The gene and the source thereof of coded polypeptide depended in the selection of host cell to a great extent.

Host cell used among the present invention can be a unicellular microorganism, as prokaryotic organism.Can be used for of the present invention useful unicellular be bacterial cell, as gram-positive microorganism, include but not limited to: the kind of streptomyces, for example, the bacterial strain of shallow Streptomyces glaucoviolaceus, comprise for example shallow Streptomyces glaucoviolaceus 1326, TK21, TK24, TK54, TK64, streptomyces coelicolor or mouse ash streptomycete, the bacterial strain of bacillus, for example, Alkaliphilic bacillus, bacillus amyloliquefaciens, bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, bacillus lautus, bacillus lentus, Bacillus licheniformis, bacillus megaterium, bacstearothermophilus, subtilis and bacillus thuringiensis; Or Gram-negative bacteria, as the kind of intestinal bacteria and Rhodopseudomonas.In a preferred embodiment, bacterial host cell is shallow Streptomyces glaucoviolaceus TK24.In a further preferred embodiment, used host cell is shallow Streptomyces glaucoviolaceus TK54.

The method of introducing recombinant expression vector in bacterial host cell can be that for example protoplast transformation (is seen, for example, Chang and Cohen, 1979, molecule General Genetics (MolecularGeneral Genetics) 168:111-115), use experience attitude cell (is seen, for example, Young and Spizizin, 1961, bacteriology magazine (Journal of Bacteriology) 81:823-829, or Dubnau and Davidoff-Abelson, 1971, molecular biology magazine 56:209-221), electroporation (is seen, for example, Shigekawa and Dower, 1988, biotechnology (Biotechniques) 6:742-751) or bonding method (see, for example, Koehler and Thorne, 1987, bacteriology magazine 169:5771-5278).Can be for the method for utilizing the same prokaryotic expression host of consistency plasmid cotransformation referring to people such as Sambrook, ibid and Hopwood, D.A. wait the people, streptomycete genetic manipulation (Genetic Manipulationof Streptomyces), The John Innes Foundation, Norwich, the method described in 1985.7. the preparation method of reorganization amidation polypeptide

The present invention also relates to prepare the method for amidation polypeptide, comprise that (a) cultivates the host cell that has recombinant expression vector that obtains as mentioned above under suitable condition, produces the culture supernatant that contains target amidation polypeptide with biologic activity; (b) from culture supernatant, reclaim this polypeptide.

In a preferred embodiment of the invention, the amidation reorganization thyrocalcitonin method that acquisition has biologic activity comprises: (a) cultivate the streptomycete host cell under the condition that helps the generation of α-amidating enzyme and thyrocalcitonin, wherein this described host cell transforms with the expression vector that contains just like the nucleotide sequence shown in SEQ ID NO:1 and 3 or its variant; And (b) directly from culture supernatant, reclaim the amidation thyrocalcitonin.

Another aspect of the present invention also relates to the same prokaryotic hosts of expression vector cotransformation that coding that utilization contains the expression vector of nucleotide sequence of the amidating enzyme of encoding and consistency is respectively treated the nucleotide sequence of amidation target polypeptides, for example streptomyces, bacillus or colibacillary bacterial strain prepare the method for amidation target polypeptides.Wherein containing the nucleotide sequence of described coding amidating enzyme and the expression vector of the nucleotide sequence that coding is treated amidated target polypeptides respectively is consistency.Consistency described herein is meant that different plasmids can coexist in same host cell.Judge whether to belong in those skilled in the art's ken for the experiment of consistency plasmid.Optionally be used to express the consistency plasmid of target amidation polypeptide among the present invention, for the bacterial strain of streptomyces, include but not limited to pIJ101 and derive plasmid and pSGL1 and the plasmid of deriving thereof, pIJ101 and derive plasmid and pJV1 and the plasmid of deriving thereof, pIJ101 and derive plasmid and pSG5 and the plasmid of deriving thereof; For the bacterial strain of Colibacter, include but not limited to pUC18 and derive plasmid and p15A and the plasmid of deriving thereof, pUC18 and derive plasmid and pSC101 and the plasmid of deriving thereof.Different carriers can carry out simultaneously to host's conversion, or carries out successively.Those skilled in the art know, and selected expression vector should adapt with selected expressive host.In another preferred embodiment of the present invention, acquisition has the amidation thyrocalcitonin method of biologic activity, comprises that (a) is with the encode expression plasmid cotransformation prokaryotic host cell of the nucleotide sequence for the treatment of amidated target polypeptides of containing of expression plasmid of nucleotide sequence that contains the amidating enzyme of encode and consistency; (b) under the condition that helps the generation of α-amidating enzyme and target polypeptides, cultivate the gained prokaryotic host cell; And (c) from culture, reclaim the amidation target polypeptides.

In another preferred embodiment of the present invention, the amidation thyrocalcitonin method that acquisition has biologic activity comprises: (a) under the condition that helps the generation of α-amidating enzyme and thyrocalcitonin, mixed culture is treated the independently streptomycete host cell that the different expression vectors of the nucleotide sequence of amidation target polypeptides transform by nucleotide sequence that contains the amidating enzyme of encoding respectively and coding; And (b) from coculture, reclaim the amidation thyrocalcitonin.The streptomycete host cell of cultivating altogether among the present invention through transforming respectively can be same type of one of any type in the multiple streptomycete expressive host cited as the front or dissimilar bacterial strains, as long as selected expression host cell can effectively produce the amidation thyrocalcitonin in the culturing process altogether.

In amidation polypeptide production method of the present invention, with method well known in the art culturing cell in being suitable for the nutritional medium that polypeptide produces and under the aerobic culture condition.For example; can be in laboratory or industrial fermentation jar by shake-flask culture, small-scale or large scale fermentation (comprise continuously ferment, batch fermentation, fed-batch fermentation or solid state fermentation) culturing cell, the suitable medium neutralization make polypeptide can express and/or isolating condition under carry out.Cultivate in the proper nutrition substratum of the carbon source that contains appropriate amount and nitrogenous source and inorganic salt etc. with method well known in the art.Suitable medium can obtain or according to disclosed composition (for example from commercial suppliers, disclosed in the catalogue of American type culture collection) preparation, also can be referring to Hopwood, people such as D.A., ibid) described in those be suitable for the substratum of the strain growth of streptomyces.Be secreted into situation in the fermention medium for polypeptide, then can directly from fermention medium, reclaim this polypeptide.In the present invention, describedly be suitable in the streptomycete expressive host, carrying out condition that α-amidating enzyme and thyrocalcitonin express basically as Hopwood, D.A. wait the people, ibid) described in method described in, adopt as YEME substratum, TSB substratum, LB substratum, at 20-37 degree centigrade, preferably at 22-30 degree centigrade, more preferably under 25-28 degree centigrade in containing shaking in the bottle and/or cultivating in the laboratory scale automatic fermentor tank of this substratum by above-mentioned prepared streptomycete host.After fermentation is carried out about 30-200 hour under the described conditions, preferably carry out 60-180 hour after, more preferably carry out after 80-140 hour finishing fermentation.Preferably determine at fermentation termination by amount of monitoring cumulative amidation target polypeptides in the fermentation culture and the upgrowth situation of monitoring expressive host.

In one embodiment of the invention, use multiple method well known in the art to detect amidation thyrocalcitonin and biological activity thereof directly or indirectly.These detection methods can comprise: HPLC, flight mass spectrum method, n terminal amino acid sequencing, radioimmunity (RIA) or enzyme immunity (EIA) method, the interior serum calcium level mensuration of body etc.

The amidation target polypeptides that obtains can reclaim by means commonly known in the art.For example, available ordinary method reclaims polypeptide from nutritional medium, and these methods are including, but not limited to centrifugal, filtration, extraction, spraying drying, evaporation or precipitation.In one embodiment of the invention, adopt the centrifugal mycelium of removing expressive host streptomycete in the nutrient solution, reclaim supernatant liquor.For guaranteeing the activity of gained target polypeptides, the pH that regulates the gained supernatant liquor is to suitable scope.Obtain the amidation polypeptide through ammonium sulfate precipitation.

Polypeptide of the present invention can be with several different methods purifying well known in the art, these methods including, but not limited to: chromatography is (for example, ion-exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method (for example, the isoelectrofocusing of preparation type), differential solubility method (for example, ammonium sulfate precipitation), SDS-PAGE or extraction (are seen, for example, " protein purification " be J.-C.Janson and Lars Ryden volume (ProteinPurification), VCH Publishers, New York, 1989).In one embodiment of the invention, adopt multistep volume-exclusion chromatography method to carry out purifying.8. the beneficial effect of the invention

The present invention is by gene engineering method, made up a kind of nucleic acid construct that at least a coding for alpha-AE and coding are treated amidated target polypeptides nucleotide sequence that contains.In one embodiment of the invention, by having made up the nucleic acid construct that contains coding rat α-AE (SEQ ID NO:1) and salmon calcitonin see calcimar precursor (SEQ ID NO:3), the gained nucleic acid construct is imported the expression vector pIJ-mel-AE/mel-sCT that makes up through gene engineering method, transform shallow Streptomyces glaucoviolaceus TK54 with this expression vector, obtain the shallow Streptomyces glaucoviolaceus of recombinant bacterial strain [pIJ-mel-AE/mel-sCT], acquisition can direct secretion produces the genetic engineering bacterium of sCT-NH2.In addition, the present invention is also by the cotransformation method, the same streptomycete host cell of different expression vector cotransformations that has α-amidating enzyme and salmon calcitonin see calcimar precursor encoding sequence respectively with consistency obtains the shallow Streptomyces glaucoviolaceus of recombinant bacterial strain [pMS680+pSGLN-mel-AE].By cultivating recombinant bacterial strain as above-mentioned gained, perhaps by cultivating altogether respectively by containing the amidating enzyme expression vector and containing the shallow Streptomyces glaucoviolaceus of streptomycete recombinant bacterial strain [pMS680] and the shallow Streptomyces glaucoviolaceus [pSGLN-mel-AE] of reorganization thyrocalcitonin expression vector, directly from fermented liquid, reclaim the gained sCT-NH2, simplified the processing step that obtains the amidation target polypeptides, improve yield, thereby greatly reduced production cost.

Now the present invention is described in detail in conjunction with following the drawings and specific embodiments.

Fig. 1 is the synoptic diagram of the nucleic acid construct of the α-AE gene that has control sequence respectively and salmon calcitonin see calcimar precursor-gene.

Fig. 2 is the synoptic diagram that contains the recombinant expression vector pIJ-mel-AE/mel-sCT of nucleic acid construct.

Fig. 3 is for containing the synoptic diagram of the recombinant expression vector pMS680 of salmon calcitonin see calcimar precursor encoding gene and control sequence separately.

Fig. 4 is for containing the synoptic diagram of the recombinant expression vector pSGLN-mel-AE of α-AE encoding gene and control sequence separately.

Fig. 5 is the high pressure liquid chromatographic analysis result of amidation thyrocalcitonin.The retention time of gained sCT-NH2 of the present invention is 16.61 minutes among the figure, and the retention time of commercially available sCT-NH2 (Sigma company, standard substance) is 16.79 minutes.Both retention time difference is within the system errors for measurement scope.

Fig. 6 is the flight mass spectrum analytical results of sCT-NH2.

Embodiment

The operation of used molecular biology experiment is unless stated otherwise all according to people such as Sambrook in the following example of the present invention, and the source is the same, and adopts Hopwood, people such as D.A., the source is the same, described in method carry out.Unless stated otherwise, used restriction enzyme and modifying enzyme are all available from GIBCO/BRL company, and used reaction conditions all carries out according to the condition that manufacturer is recommended.The preparation of embodiment 1 melC1 secretion signal peptide-coding sequence and upstream sequence thereof, the preparation of salmon calcitonin see calcimar precursor sCT-Gly encoding sequence, and have the preparation 1.melC1 secretion signal peptide-coding sequence of streptomycete expression plasmid pMS680 of salmon calcitonin see calcimar precursor sCT-Gly encoding sequence (mel+sCT-Gly) of melC1 secretion signal peptide-coding sequence and upstream sequence thereof and the preparation of upstream sequence thereof

The signal peptide of known melC1 from antibiosis streptomycete (S.antibioticus) ATCC 10382 is formed (Katz etc. by 30 amino acid, general microbiology magazine (J GenMicrobiol), 129:2703,1983), its signal peptidase identification cleavage site be AlaArgAla ↓.According to known melC1 sequence (people such as Bernan V, gene, 1985,37:101-110) PCR primer P1 and P2 have been synthesized in design, wherein P2 changes the codon of last amino acid Ala into GCC by GCG in order to introduce Pst I site.P1：5’GC TGATCACGTCAGTTTTCG?3’(SEQ?ID?NO：5)

BcllP2：5’TGCA CTGCAGGCGCGGGCGGCGGGAAG?3’(SEQ?IDNO：6) PstI

Utilize above-mentioned primer to P1 and P2, total DNA with antibiosis streptomycete ATCC 10382 is a template, obtain the dna fragmentation of a 370bp by following PCR reaction amplification: carry out the PCR reaction with cumulative volume 50 μ l, 95 ℃ of sex change add the Taq archaeal dna polymerase after 10 minutes, by 94 ℃ of sex change 1 minute, annealed 1.5 minutes for 36 ℃, 72 ℃ are extended 1.5 minutes circulating reactions 5 times, and then by 94 ℃ of sex change 1 minute, annealed 1 minute and 10 seconds for 42 ℃, 72 ℃ were extended 1.5 minutes, and circulating reaction 25 times extended 10 minutes at 72 ℃ at last.Reclaim gained PCR fragment reaction from polyacrylamide gel electrophoresis, cut through restriction enzyme PstI and BclI enzyme, connect with the T4 ligase enzyme, be cloned into Pst I and the Bam HI site of the pUC19 that cuts through Pst I and Bam HI enzyme, wherein the enzyme of BamHI and BclI is cut the sticky end complementation, acquisition has the recombinant plasmid pUC19-mel of melC1 secreting signal peptide and upstream sequence thereof, (instrument model 373A) measures this fragment sequence through ABI company automatic sequencer, shows that extension increasing sequence conforms to fully with the sequence of bibliographical information.2. have the preparation of nucleic acid construct of the salmon calcitonin see calcimar precursor sCT-Gly encoding sequence (mel+sCT-Gly) of melC1 secreting signal peptide and upstream sequence thereof

Utilize the method described in Chinese patent 96105118.3 basically, by pcr amplification, use following primer: P3:5 ' TGCACTGCAGCAACCTCAGCACCTGT 3 ' (SEQ ID NO:7) P4:5 ' GCGGATCCTACTAGCCCGGCGTACCACTTCCCG 3 ' (SEQID NO:8)

Amplification obtains the dna fragmentation of 117 base pairs from the salmon dna profiling.Gained PCR product is cut the corresponding restriction enzyme site of rear clone in escherichia coli plasmid pUC19 through Pst I and Bam HI enzyme, obtain recombinant plasmid pUC19-sCT, (instrument model 373A) measures this fragment sequence through ABI company automatic sequencer, shows that extension increasing sequence conforms to fully with known salmon calcitonin see calcimar encoding sequence.

With reference to the method described in Chinese patent 96105118.3, as above gained pUC19-sCT cuts with PstI and BamHI enzyme, the pUC19-mel of aforementioned preparation cuts with EcoRI and PstI enzyme, with be connected with the escherichia coli plasmid pUC19 that the BamHI enzyme is cut by EcoRI, obtain having the recombinant plasmid pUC19-mel-sCT of the salmon calcitonin see calcimar precursor sCT-Gly encoding sequence (mel+sCT-Gly) of melC1 secreting signal peptide and upstream sequence thereof.3. have the preparation of streptomycete expression plasmid pMS680 of the salmon calcitonin see calcimar precursor sCT-Gly encoding sequence (mel+sCT-Gly) of melC1 secreting signal peptide and upstream sequence thereof

Utilize Hopwood, D.A. wait the people, ibid, described in the protoplast transformation method of PEG mediation, the protoplastis for preparing shallow Streptomyces glaucoviolaceus TK54, with reference to method used in the Chinese patent 96105118.3, the fragment of the 4.6kb that streptomycete plasmid vector pIJ680 is obtained with Sac I and Xba I double digestion, simultaneously will be as above the fusion gene of the 0.5kb that obtains with Sac I and Xba I double digestion of gained pUC19-mel-sCT, connect the back and transform the treated shallow Streptomyces glaucoviolaceus TK54 that becomes protoplastis, obtained to have the thiostrepton resistance transformant of (50 μ g/ml).Plasmid in the extraction transformant carries out enzyme and cuts evaluation, and the result shows the streptomycete expression plasmid pMS680 that contains the salmon calcitonin see calcimar precursor sCT-Gly encoding sequence (mel+sCT-Gly) that has melC1 secreting signal peptide and upstream sequence thereof in the transformant.In expression plasmid pMS680, fusion gene is in the strong promoter downstream of neomycin phosphotransferase gene (aph), except the promotor of utilizing melC1 own, also can utilize the strong promoter of aph to express salmon calcitonin see calcimar.The shallow Streptomyces glaucoviolaceus bacterial strain of the reorganization MS680 that has the recombinant expression plasmid pMS680 that makes up as mentioned above has been preserved in Chinese microbial preservation management committee common micro-organisms preservation center (CGMCC), preserving number CGMCC0262 on April 24th, 1996.Embodiment 2: have α-amidating enzyme encoding sequence (preparation of the preparation 1. rats α-AE encoding sequence of the nucleic acid construct of mel+ α-AE) of melC1 secretion signal peptide-coding sequence and upstream sequence thereof

Utilize the known acid guanidinium isothiocyanate-phenol-chloroform single stage method in this area from the rat atrial tissue, extract total RNA (with reference to Chomczynski P etc., analytical biochemistry (AnalBiochem.), 162:156-159).Rat atrial tissue (about 1 gram) is placed solution D (the GIT 4mol/L of precooling, Trisodium Citrate 25mmol/L, pH7.0, sarcosyl 0.5%, 2 mercapto ethanol 0.1mol/L) in, use glass homogenizer homogenate under the condition of ice bath, transfer in the centrifuge tube then, add 1ml 2mol/L NaAc (pH4.0) successively, all abundant mixing after 10ml water-saturated phenol and the 2ml chloroform-primary isoamyl alcohol (49: 1), a kind of composition of every adding, last violent jolting 10 seconds, ice bath cooling 15 minutes, 4 ℃ down 12,000 rev/mins centrifugal 20 minutes, draw water and add the 10ml Virahol,-20 ℃ of precipitations are after 1 hour, 4 ℃ down 12,000 rev/mins centrifugal 20 minutes.Gained precipitation with the dissolving of 3ml solution D, is added 2 times of volume of ethanol ,-20 ℃ of precipitations 1 hour, under 4 ℃ 12,000 rev/mins centrifugal 20 minutes, with 80% washing with alcohol twice, vacuum is drained, and is dissolved in the distilled water that DEPC handled ,-70 ℃ of preservations are standby.

GIBCO/BRL Corporation's Super script is adopted in reverse transcription ^TMPreamplificationSystem for First Strand cDNA Synthesis test kit.Will be as the total RNA12 μ of above-mentioned gained l (1-5 μ g), 20U RNasin and 2 μ g random hexamer primers, behind the mixing 70-80 ℃ sex change 10-20 minute, ice bath 1 minute, add 30U RNasin more successively, 2 μ l, 10 * damping fluid, 1 μ l 10mmol/L dNTPs, 2 μ l 0.1mmol/L DTT, 1 μ l reversed transcriptive enzyme (200U), 20 ℃ of temperature were bathed after 10 minutes, and 42 ℃ were extended 50 minutes, and 99 ℃ were heated 6 minutes, 5 minutes termination reactions of last 95 ℃ of heating, the ice bath cooling adds 1 μ l RNase H (2U), and 37 ℃ are incubated 20 minutes, obtain the reverse transcription product ,-20 ℃ of preservations are standby.

(amino terminal sequence of ripe α-AE designs forward primer P3 and reverse primer P4 267:4008-4015) and in the rat atrial tissue for people such as Eipper BA, journal of biological chemistry according to the cDNA sequence of known rat α-AE.

P5：5’AGCCCACTTTCTGTCTTTAAG3’(SEQ?ID?NO：9)

P6：5’TCAAACTGACCGATGCTCCATT3’(SEQ?ID?NO：10)

With as above gained reverse transcription product is that template is carried out pcr amplification.Contain in the 50 μ l reaction systems: reverse transcription product 2-3 μ l, primer P3 and P4 are respectively 10pmol, each 10nmol of dNTP, 1 * PCR damping fluid (Mg ⁺⁺Concentration is 1mmol/L), Taq polysaccharase 2U.Place the pcr amplification instrument (MJ Research, Inc.) increase by following condition: 94 ℃ of sex change entered circulating reaction after 5 minutes, 93 ℃ of sex change 40 seconds, 59 ℃ of annealing 35 seconds, 70 ℃ were extended 15 circulations 2.5 minutes; 93 ℃ of sex change 40 seconds, 59 ℃ of annealing 35 seconds, 70 ℃ are extended 2 minutes (each circulation back increases by 10 seconds), 20 circulations; Last 70 ℃ were extended 8.5 minutes.Obtain the cDNA fragment of coding for alpha-AE of 2.1kb by pcr amplification.With the T4 archaeal dna polymerase equating of gained pcr amplified fragment, be cloned into that the SmaI enzyme is cut and in the pUC18 of CIAP dephosphorylation, obtain having the recombinant vectors pUC18-AE of the nucleotide sequence of coding for alpha-amidating enzyme, measure target fragment through ABI company automatic sequencer (instrument model 373A), prove that the sequence of this dna fragmentation conforms to fully with the α of expection-AE encoding gene.

With the pUC18-AE of Restriction Enzyme DpnI cutting gained, the dna fragmentation of separation and purification 2.02kb from the low melting-point agarose gel; Similarly, the pUC19-mel with Restriction Enzyme PstI cutting obtains as mentioned above with the equating of T4 archaeal dna polymerase, sloughs 5 ' phosphate group with CIAP again.With above-mentioned two fragments with 3-5: 1 concentration ratio mixes, and adds the T4 dna ligase and connects by the condition of manufacturer's recommended.The preparation of used escherichia coli jm109 competent cell, conversion and plasmid extraction step are all by people such as Sambrook, and ibid, described in method carry out.Above-mentioned connection product is converted into the competent cell of e. coli jm109, coat the LB agar plate that contains penbritin 100 μ g/ml, 37 ℃ of overnight incubation, picking colony, extraction plasmid wherein carries out enzyme and cuts evaluation, cuts the result according to enzyme and identifies the segmental size and Orientation of insertion.Selection has the insertion fragment of expectation size and correct direction, thus the segmental recombinant plasmid pUC19-mel-AE of α-AE that obtains encoding and have the melC1 secreting signal peptide.Because the fusion gene both sides can be few for the restriction enzyme site that utilizes among the plasmid pUC19-mel-AE, structure for the ease of expression vector, with SmaI/HindIII above-mentioned coding melC1 secreting signal peptide and the segmental fusion gene of α-AE are scaled off from pUC19-mel-AE, and mend flat with Klenow, then according to being similar to top condition of contact, be connected with the SmaI cutting and through the pUC18 of CIAP dephosphorylation processing, obtain having the segmental recombinant plasmid pUC18-mel-AE of α-amidating enzyme of coding melC1 secreting signal peptide.Embodiment 3: contain the preparation of streptomyces expression vector of the nucleic acid construct of mel+ α-AE fragment and mel+sCT-Gly fragment

With gained expression plasmid pMS680 among the XbaI enzyme cutting embodiment 1, the pUC18-mel-AE that handles with same endonuclease digestion and dephosphorylation is connected, screen recon behind the transformed into escherichia coli, enzyme is cut and is identified the segmental size and Orientation of insertion in the recombinant plasmid, obtain pUC18-mel-AE/MS680, the fusion gene of melC1 secretion signal peptide-coding sequence and upstream sequence thereof and α-AE inserts the downstream of sCT-Gly gene in this plasmid, and its transcriptional orientation is consistent with the sCT-Gly gene.Cut pUC18-mel-AE/MS680 with the EcoRI/HindIII enzyme, the big fragment of separation and purification, the Klenow enzyme is mended flat back and is added the connection of T4 dna ligase, is used to transform shallow Streptomyces glaucoviolaceus TK54, the transformant that has obtained to have the thiostrepton resistance.Plasmid in the extraction transformant carries out enzyme and cuts evaluation, and the result shows that transformant contains expression plasmid pIJ-mel-AE/mel-sCT, with the shallow Streptomyces glaucoviolaceus of gained transformant called after genetic engineering bacterium [pIJ-mel-AE/mel-sCT].Embodiment 4: the fermentation culture of the fermentation culture of the shallow Streptomyces glaucoviolaceus of genetic engineering bacterium [pIJ-mel-AE/mel-sCT] of generation amidation thyrocalcitonin and the shallow Streptomyces glaucoviolaceus of separation and purification 1. genetic engineering bacteriums [pIJ-mel-AE/mel-sCT] of amidation thyrocalcitonin

To be inoculated in the following fermention medium as the shallow Streptomyces glaucoviolaceus of genetic engineering bacterium [pIJ-mel-AE/mel-sCT] of preparation as described in the embodiment 3, its component is: glucose 5.5%, casein hydrolysate 3%, yeast extract 3%, trace element 3% (ZnCl ₂.7H ₂O 0.02%, FeSO ₄.H ₂O 0.1%, CuCl ₂.H ₂O 0.0025%, H ₃BO ₃.10H ₂O 0.00056%, MnSO ₄.7H ₂O 0.1%, CaCl ₂.H ₂O 0.01%, (NH ₄) ₆Mo ₇O ₂₄.4H ₂O0.001%).28 ℃ of shaking culture 35 hours are inoculated in the fresh substratum of the same race with 10% inoculum size again, continue to cultivate about 65 hours in 28 ℃.2. the separation of amidation thyrocalcitonin, purifying:

Press above-mentioned condition culturing engineering bacterium after 65 hours, 3000 rev/mins centrifugal 12 minutes, get supernatant liquor and abandon mycelia, carry out macroporous adsorbent resin X5 column chromatography decolouring (fermented liquid: resin (volume)=1: 12.5), collect effluent liquid, further carry out hydrophobic chromatography (Phenyl SepharoseHigh Performance post, effluent liquid: medium (volume)=1: 25), (contain 1.5M (NH with the 50mM phosphoric acid buffer ₄) ₂SO ₄) carry out wash-out.With the amidation thyrocalcitonin in the detection of the EIA described in the embodiment 5 monitor flows fluid, merge the fraction that contains the amidation thyrocalcitonin, through the molecular weight that dams is that 1000 ultra-filtration membrane utilizes Amicon (U.S.) ultrafiltration instrument (operational condition is with reference to the instrument specification sheets) to carry out ultrafiltration, and the gained fraction is concentrated and desalination.The sample that ultrafiltration obtains is through lyophilize, and is standby in-20 ℃ of preservations.And then adopt preparation type high pressure liquid chromatography to be further purified.Preparative column is Supelcosil ^TMLC-308,25cm * 10.0mm.Mobile phase A: acetonitrile, B:0.05% trifluoroacetic acid.Flow velocity 2ml/ branch, gradient A:10%-80%, B:90%-20%, 0 minute-60 minutes time, ultraviolet detection wavelength: 220nm.With the amidation thyrocalcitonin in the detection of the EIA described in the embodiment 5 monitor flows fluid, collect the fraction that contains the amidation thyrocalcitonin.Embodiment 5: the detection of sCT-NH2 and identify the high pressure liquid chromatographic analysis of 1. sCT-NH2s:

Adopt the analysis mode high pressure liquid chromatographic analysis to detect the amidation thyrocalcitonin, used analytical column is Supelcosil ^TMLC-308,5cm * 2.6mm.Mobile phase A: acetonitrile, B:0.05% trifluoroacetic acid; Flow velocity: 1.15ml/ branch, gradient A:5%-80%, B:95%-20%; Time: 0 minute-50 minutes, detect wavelength: 220nm.The sCT-NH2 standard substance are available from U.S. Sigma company, Fig. 5 is the high pressure liquid chromatographic analysis figure as the sCT-NH2 of preparation as described in the embodiment 4, the result shows, the gained sCT-NH2 is consistent with the standard substance retention time, shows the shallow Streptomyces glaucoviolaceus of engineering strain of the present invention [pIJ-mel-AE/mel-sCT] but the amidated salmon calcitonin see calcimar of secreting, expressing.2. the flight mass spectrum analysis of sCT-NH2:

Adopt MALDI-TOF mass spectrometric determination salmon calcitonin see calcimar molecular weight.Show consistently with Sigma company standard product after measured as embodiment 4 gained sCT-NH2 molecular weight, molecular weight is 3430.7.Known non-amidated salmon calcitonin see calcimar molecular weight is 3492, and this shows that the shallow Streptomyces glaucoviolaceus of engineering strain of the present invention [pIJ-mel-AE/mel-sCT] secreting, expressing salmon calcitonin see calcimar is that the C end is amidated.Fig. 6 is the flight mass spectrum analytical results of gained sCT-NH2 of the present invention.3. the N terminal amino acid sequence of sCT-NH2 is analyzed:

Adopt the ABI Procise of company 491 type automatic analyzer for amino acidss to carry out the N terminal amino acid sequence analysis of sCT-NH2.Thus 15 amino acid of gained genetically engineered salmon calcitonin see calcimar N end of the present invention are carried out sequential analysis, its N end 1-15 amino acids is followed successively by: Cys, Ser, Asn, Leu, Ser, Thr, Cys, Val, Leu, Gly, Lys, Leu, Ser, Gln, Glu.The result shows that gained sCT-NH2 of the present invention and natural salmon calcitonin see calcimar are in full accord.4. the biological activity determination A. of sCT-NH2 adopts enzyme immunoassay (EIA) to measure the external activity of thyrocalcitonin

Enzyme immunoassay provides operation steps to carry out with EIA detection kit (available from U.S. PENISULALABORATORIES company) according to company.Get 50 μ l testing sample solutions and salmon calcitonin see calcimar (sCT) standard solution that contains different concns respectively, in the different holes of application of sample to 96 hole enzyme plate (flat board is in advance with albumin A bag quilt), in every hole, add antibody and the biotin labeled sCT of 25 μ l of 25 μ l sCT subsequently successively, spend the night in 4 ℃ of placements.Dull and stereotyped 5 times of the damping fluid 300 μ l/ holes washing that provides with detection kit under the room temperature, add streptavidin-horseradish peroxidase reagent 100 μ l in every sample well then, room temperature was placed 1 hour, and with dull and stereotyped 5 times of damping fluid 300 μ l/ holes washing, every sample well adds 100 μ l substrate solution (H again ₂O ₂, TMB), placed 1 hour under the room temperature, add 100 μ l 2N hydrochloric acid respectively with termination reaction to each hole, in half an hour, mark colorimeter 450nm colorimetric estimation result with enzyme.Adopt as above method to detect the sCT-NH2 of gained among the embodiment 4, experimental result shows, the shallow Streptomyces glaucoviolaceus of engineering strain [pIJ-mel-AE/mel-sCT] but the secreting, expressing sCT-NH2, secretory volume reaches peak value when being cultured to 70-90 hour.The average secretion expression level of amidation thyrocalcitonin in the fermentation sample of 6 different batches is 10.3mg/L.B. adopt and reduce the activity in vivo that rat blood serum calcium level method is measured thyrocalcitonin

The physiological function of known salmon calcitonin see calcimar is for reducing free calcium level in the serum.The thyrocalcitonin intracorporeal active experiment is carried out according to the method described in 98 years versions of British Pharmacopoeia among the present invention.Laboratory animal is the SD rat, and is male, body weight 158.9 ± 9.2 grams, and 25 animals are divided into 5 groups at random and carry out.Calcium liquid reagent box is to give birth to biotechnology high-tech company product in Beijing, adopts OCPC method (Ray Sarkar, BC and Chauhan, UPS, analytical biochemistry 20:155) to measure calcium ion content in the serum.Testing sample and standard substance are respectively with physiological saline solution.Drinking-water is not limit in experimental rat administration fasting in eve, and the subcutaneous injection medicine with the anesthesia of 3% vetanarcol, was got aortic blood after 1 hour, and not anti-freezing is got serum after room temperature leaves standstill, and adopts the calcium level in the OCPC method mensuration serum.Adopt the thyrocalcitonin of method mensuration embodiment 4 described acquisitions as mentioned above, the result shows the genetically engineered salmon calcitonin see calcimar of the shallow Streptomyces glaucoviolaceus of engineering strain of the present invention [pIJ-mel-AE/mel-sCT] secreting, expressing than work＞4000IU/mg, with the active basically identical of natural salmon calcitonin see calcimar.In view of sCT-NH2 activity not is lower than 200IU/mg, above-mentioned experimental result shows that the secreted thyrocalcitonin of the shallow Streptomyces glaucoviolaceus of engineering strain of the present invention [pIJ-mel-AE/mel-sCT] is amidated salmon calcitonin see calcimar.Embodiment 6: the α that have melC1 secretion signal peptide-coding sequence and the upstream sequence thereof-amidating enzyme encoding sequence compatible with the expression plasmid pMS680 (structure of the expression plasmid pSGLN-mel-AE of mel+ α-AE)

Utilize people such as Hopwood, ibid, described in method from styreptomyces globispotus strain (Streptomyces globisporus) C-1027 (FERM BP-1299), extract plasmid, cut through agarose gel electrophoresis and enzyme and to identify and confirm that the gained plasmid is pSGL1 (the gentle Li Yuan of Li Xiao, China's microbiotic magazine, 1992,17 (5): 326-332).

With Xba I enzyme cut and dephosphorylation Processing Example 2 in gained pUC18-mel-AE, with as above-mentioned gained plasmid pSGL1 through the big fragment (about 4.4kb) of Sal I/Xba I double digestion and plasmid pIJ680 small segment (neomycin resistance gene through Xho I/Xba I double digestion, about 1.2kb) connects, wherein the enzyme of XhoI and SalI is cut the sticky end complementation, connect product transformed into escherichia coli JM109, through recombinant screen, enzyme is cut and is identified the segmental size and Orientation of insertion in the recombinant plasmid, obtain plasmid pUC18-mel-AE/SGLN, cut pUC18-mel-AE/SGLN with the EcoRI/HindIII enzyme, the big fragment of separation and purification, the Klenow enzyme is mended flat back and is added the connection of T4DNA ligase enzyme, be used to transform shallow Streptomyces glaucoviolaceus TK54, the substratum screening that has 10 μ g/ml Xin Meisus according to the method utilization of similar embodiment 1 has the transformant of neomycin resistance, obtains expressing the shallow Streptomyces glaucoviolaceus of reorganization [pSGLN-mel-AE] of rat α-amidating enzyme.Plasmid in the extraction transformant carries out enzyme and cuts evaluation, and the result shows the expression plasmid pSGLN-mel-AE that contains α-AE in the transformant really.Embodiment 7: prepare the protokaryon recombinant strains with the consistency plasmid pMS680 and the pSGLN-mel-AE cotransformation streptomycete of expressing thyrocalcitonin and amidating enzyme respectively

According to bibliographical information, pIJ101 and derive plasmid and pSGL1 and the plasmid of deriving thereof are consistency plasmid (Hong Bin and Li Yuan, microorganism journal, 38 (4): 256-260).According to being similar to the method described in the embodiment 1, will be as the recombinant expression plasmid pMS680 and the shallow Streptomyces glaucoviolaceus TK54 of pSGLN-mel-AE cotransformation of gained in above-mentioned embodiment 1 and 6, utilization has the culture plate of 50 μ g/ml thiostreptons and 10 μ g/ml Xin Meisus, and screening has the transformant of thiostrepton and neomycin resistance simultaneously.The gained transformant is the shallow Streptomyces glaucoviolaceus of recombinant strains [pMS680+pSGLN-mel-AE] that has α-AE gene and salmon calcitonin see calcimar precursor-gene on different consistency plasmids respectively.Embodiment 8: the fermentation culture of the shallow Streptomyces glaucoviolaceus of separation and purification 1. recombinant strains [pMS680+pSGLN-mel-AE] of fermentation culture of the shallow Streptomyces glaucoviolaceus of recombinant strains [pMS680+pSGLN-mel-AE] and amidation thyrocalcitonin

To be inoculated in as the shallow Streptomyces glaucoviolaceus of genetic engineering bacterium [pMS680+pSGLN-mel-AE] of preparation as described in the embodiment 7 as described in example 4 above in the fermention medium.28 ℃ of shaking culture 35 hours are inoculated in the fresh substratum of the same race with 10% inoculum size again, continue to cultivate about 65 hours in 28 ℃.2. the separation of amidation thyrocalcitonin, purifying:

Cultivate this genetic engineering bacterium after 65 hours by above-mentioned condition, 3000 rev/mins centrifugal 12 minutes, get supernatant liquor and abandon mycelia, carry out macroporous adsorbent resin X5 column chromatography decolouring (fermented liquid: resin (volume)=1: 12.5), collect effluent liquid, further carry out hydrophobic chromatography (Phenyl SepharoseHigh Performance post, effluent liquid: medium (volume)=1: 25), (contain 1.5M (NH with the 50mM phosphoric acid buffer ₄) ₂SO ₄) carry out wash-out.With the amidation thyrocalcitonin in the detection of the EIA described in the embodiment 5 monitor flows fluid, merge the fraction that contains the amidation thyrocalcitonin, through the molecular weight that dams is that 1000 ultra-filtration membrane utilizes Amicon (U.S.) ultrafiltration instrument (operational condition is with reference to the instrument specification sheets) to carry out ultrafiltration, and the gained fraction is concentrated and desalination.The sample that ultrafiltration obtains is through lyophilize, and is standby in-20 ℃ of preservations.And then adopt preparation type high pressure liquid chromatography to be further purified.Preparative column is Supelcosil ^TMLC-308,25cm * 10.0mm.Mobile phase A: acetonitrile, B:0.05% trifluoroacetic acid.Flow velocity 2ml/ branch, gradient A:10%-80%, B:90%-20%, 0 minute-60 minutes time, ultraviolet detection wavelength: 220nm.With the amidation thyrocalcitonin in the detection of the EIA described in the embodiment 5 monitor flows fluid, collect the fraction that contains the amidation thyrocalcitonin.The average secretion expression level of amidation thyrocalcitonin in the fermentation sample of 6 different batches is 9.8mg/L.Embodiment 9 utilizes the common cultivation of reorganization shallow Streptomyces glaucoviolaceus [pSGLN-mel-AE] and the shallow Streptomyces glaucoviolaceus of reorganization [pMS680] to prepare the amidation thyrocalcitonin

According to the method described in the embodiment 1, transform shallow Streptomyces glaucoviolaceus TK24 and TK54 with pSGLN-mel-AE and pMS680 respectively, utilization has the culture plate of 50 μ g/ml thiostreptons and 10 μ g/ml Xin Meisus, screening has the transformant of thiostrepton and neomycin resistance respectively, obtains the shallow Streptomyces glaucoviolaceus TK24[pSGLN-mel-AE of recombinant bacterial strain] and the shallow Streptomyces glaucoviolaceus TK54[pMS680 of recombinant bacterial strain].With reference to the fermentation culture conditions among the embodiment 4, cultivate shallow Streptomyces glaucoviolaceus TK24[pSGLN-mel-AE respectively at 28 ℃] and shallow Streptomyces glaucoviolaceus TK54[pMS680] seed 35 hours, two kinds of inoculation culture things of gained are with 1 then: the volume ratio of 1-5 is inoculated in as described in example 4 above the fermention medium, cultivates 65 hours in 28 ℃.Measure the expression of amidation thyrocalcitonin in the coculture according to the EIA detection method described in the embodiment 5.When treating that expression amount reaches maximum, stop fermentation, according to the separation purification method described in the embodiment 4, separation and purification gained sCT-NH2 from coculture, the average secretion expression level in the fermentation sample of 6 different batches is 9.2mg/L.

SEQ ID NO：1AGCCCACTTTCTGTCTTTAAGAGGTTTAAAGAAACTACCAGATCATTTTCCAATGAATGCCTTGGTACCATTGGACCAGTCACCCCTCTTGATGCATCAGATTTTGCGCTGGATATTCGCATGCCTGGGGTTACACCTAAAGAGTCTGACACATACTTCTGCATGTCCATGCGTCTGCCTGTGGATGAGGAAGCCTTCGTGATTGACTTCAAGCCTCGTGCCAGCATGGATACTGTCCACCATATGCTGCTGTTTGGATGCAATATGCCCTCGTCCACTGGAAGTTACTGGTTTTGTGATGAAGGAACCTGTACAGATAAAGCCAATATTCTATATGCCTGGGCAAGGAATGCTCCCCCCACCCGGCTCCCGAAAGGTGTTGGATTCAGAGTTGGAGGAGAAACTGGAAGCAAATACTTCGTCCTTCAAGTTCACTATGGCGATATCAGTGCTTTTCGAGATAATCACAAAGACTGCTCTGGCGTGTCCGTACATCTCACACGTGTGCCCCAGCCTTTAATTGCGGGCATGTACCTTATGATGTCTGTTGACACTGTCATACCACCAGGAGAGAAAGTAGTGAATGCTGACATTTCGTGCCAATACAAAATGTATCCAATGCATGTGTTTGCCTACAGAGTCCACACTCACCATTTAGGTAAGGTGGTGAGCGGATACAGAGTAAGAAACGGACAGTGGACACTGATTGGACGCCAGAACCCCCAGCTGCCACAGGCTTTCTACCCTGTGGAACACCCCGTTGATGTTACTTTTGGTGATATACTGGCAGCCAGATGTGTGTTCACTGGTGAAGGGAGGACAGAGGCCACCCACATCGGCGGCACTTCTAGTGACGAAATGTGTAACCTGTACATCATGTATTACATGGAAGCCAAATATGCACTTTCCTTCATGACCTGTACAAAGAACGTGGCTCCAGATATGTTCAGAACTATCCCAGCAGAGGCCAATATCCCAATTCCTGTCAAACCGGACATGGTTATGATGCACGGGCATCACAAAGAAGCAGAAAACAAAGAAAAGAGTGCTTTAATGCAGCAGCCAAAACAGGGAGAGGAAGAAGTATTAGAGCAGGATTTCCATGTGGAAGAAGAACTGGACTGGCCTGGAGTGTACTTGTTACCAGGCCAGGTTTCTGGGGTGGCCCTGGATTCTAAGAATAACCTAGTGATTTTCCACAGAGGTGACCATGTTTGGGATGGAAACTCTTTTGACAGCAAGTTTGTTTACCAGCAAAGAGGTCTTGGGCCAATTGAAGAAGACACCATCCTGGTCATTGACCCAAATAATGCTGAAATCCTCCAGTCCAGTGGCAAGAACCTGTTTTATTTACCACACGGCTTGAGCATAGATACAGATGGAAATTATTGGGTCACAGATGTGGCTCTCCACCAGGTGTTCAAATTGGACCCGCATAGCAAAGAAGGCCCGCTCTTAATTCTGGGAAGGAGCATGCAACCTGGGAGTGACCAAAATCATTTCTGCCAGCCCACCGATGTGGCTGTGGAGCCCAGTACTGGAGCTGTCTTCGTGTCAGACGGTTACTGTAACAGTCGGATTGTGCAGTTTTCACCAAGCGGAAAGTTCGTCACCCAGTGGGGAGAAGAGTCCTCTGGAAGCAGTCCTAGGCCAGGCCATTTCAGTGTTCCTCACAGTTTGGCCCTTGTGCCTCATTTGGACCAGTTGTGTGTGGCAGACAGGGAAAATGGCCGAATCCAATGCTTCAAAACTGACACCAAAGAATTTGTGAGAGAGATTAAGCACGCATCATTTGGAAGGAATGTCTTTGCCATTTCATATATACCAGGTTTCCTCTTTGCCGTAAACGGGAAGCCTTACTTTGGAGACCAAGAGCCCGTGCAAGGATTTGTGATGAACTTTTCCAGTGGGGAAATTATAGACGTCTTCAAGCCAGTACGCAAGCACTTCGACATGCCTCATGATATTGTGGCTTCTGAAGATGGGACTGTGTACATTGGAGACGCACACACAAACACCGTGTGGAAGTTCACCCTGACTGAAAAAATGGAGCATCGGTCAGTCTGASEQ ID NO：2SPLSVFKRFKETTRSFSNECLGTIGPVTPLDASDFALDIRMPGVTPKESDTYFCMSMRLPVDEEAFVIDFKPRASMDTVHHMLLFGCNMPSSTGSYWFCDEGTCTDKANILYAWARNAPPTRLPKGVGFRVGGETGSKYFVLQVHYGDISAFRDNHKDCSGVSVHLTRVPQPLIAGMYLMMSVDTVIPPGEKVVNADISCQYKMYPMHVFAYRVHTHHLGKVVSGYRVRNGQWTLIGRQNPQLPQAFYPVEHPVDVTFGDILAARCVFTGEGRTEATHIGGTSSDEMCNLYIMYYMEAKYALSFMTCTKNVAPDMFRTIPAEANIPIPVKPDMVMMHGHHKEAENKEKSALMQQPKQGEEEVLEQDFHVEEELDWPGVYLLPGQVSGVALDSKNNLVIFHRGDHVWDGNSFDSKFVYQQRGLGPIEEDTILVIDPNNAEILQSSGKNLFYLPHGLSIDTDGNYWVTDVALHQVFKLDPHSKEGPLLILGRSMQPGSDQNHFCQPTDVAVEPSTGAVFVSDGYCNSRIVQFSPSGKFVTQWGEESSGSSPRPGQFSVPHSLALVPHLDQLCVADRENGRIQCFKTDTKEFVREIKHASFGRNVFAISYIPGFLFAVNGKPYFGDQEPVQGFVMNFSSGEIIDVFKPVRKHFDMPHDIVASEDGTVYIGDAHTNTVWKFTLTEKMEHRSVSEQ ID NO：35’TGC TCC AAC CTC AGC ACC TGT GTG CTG GGC AAA CTG TCC CAA GAG CTG CATAAA TTG CAG ACG TAC CCC CGC ACC AAC ACG GGA AGT GGC ACG CCT GGC 3’SEQ ID NO：4Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu His Lys Leu Gln Thr Tyr Pro ArgThr Asn Thr Gly Ser Gly Thr Pro GlySEQ ID NO：51：5’GCTGATCACGTCAGTTTTCG3’SEQ ID NO：62：5’TGCACTGCAGGCGCGGGCGGCGGGAAG3’SEQ ID NO：73：5’TGCACTGCAGCAACCTCAGCACCTGT3’SEQ ID NO：84：5’GCGGATCCTACTAGCCCGGCGTACCACTTCCCG3’SEQ ID NO：95：5’AGCCCACTTTCTGTCTTTAAG3’SEQ ID NO：106：5’TCAAACTGACCGATGCTCCATT3’

Claims (21)

1. a nucleic acid construct is treated the nucleotide sequence of amidated target polypeptides comprising the nucleotide sequence of at least a coding amidating enzyme with encoding.

2. the described nucleic acid construct of claim 1 wherein saidly treats that amidated target polypeptides is that thyrocalcitonin, pitocin, vassopressin, corticotropin releasing homone, thyrotropin releasing hormone, neurokinin, allatostatin, Lem-KI, haematochrome concentrate hormone, luteinising hormone-releasing hormo, leucopyrokinin, gastrin, pigment and disperse hormone, dermorphin, bombesin, gastrin releasing peptide, neuromedin, pancreastatin, conotoxin M 1, pancreastatin, mellitin, sarcophagid toxin 1A, VIP, α-MSH, MIF-1.

3. the described nucleic acid construct of claim 1, wherein said target polypeptides is a salmon calcitonin see calcimar.

4. the described nucleic acid construct of claim 1, wherein said amidating enzyme is rat α-amidating enzyme.

5. recombinant expression vector that contains the described nucleic acid construct of claim 1, wherein said expression vector also contains expression control sequenc, comprises promotor, enhanser, terminator sequence.

6. prokaryotic expression host of containing the described expression vector of claim 5, it is selected from bacterial strain and the intestinal bacteria of streptomyces, bacillus.

7. the described prokaryotic expression host of claim 6, it is shallow Streptomyces glaucoviolaceus TK54, TK24, TK21, TK64 or 1326.

8. the described prokaryotic expression host of claim 7, wherein said expression vector is the plasmid that derives from the plasmid of pIJ101 or derive from pSGL1.

9. the described prokaryotic expression host of claim 8, wherein said recombinant expression plasmid is pIJ-mel-AE/mel-sCT.

10. method for preparing the amidation polypeptide, it comprises that (1) transforms suitable prokaryotic hosts with the recombinant expression vector that has the nucleic acid construct described in the claim 1; (2) the prokaryotic expression host cell of cultivation gained under the condition that helps the generation of α-amidating enzyme and target polypeptides, (3) reclaim the amidation target polypeptides from culture.

11. the method for claim 10 wherein saidly treats that amidated target polypeptides is a salmon calcitonin see calcimar.

12. the method for claim 10, wherein said prokaryotic hosts are shallow Streptomyces glaucoviolaceus TK54, TK24, TK21, TK64 or 1326.

13. the method for claim 10 contains pIJ-mel-AE/mel-sCT among the wherein said recombinant expressed host.

14. a method for preparing the amidation polypeptide, it comprises that (a) is with the encode expression plasmid cotransformation prokaryotic host cell of the nucleotide sequence for the treatment of amidated target polypeptides of containing of expression plasmid of nucleotide sequence that contains the amidating enzyme of encode and consistency; (b) under the condition that helps the generation of α-amidating enzyme and target polypeptides, cultivate gained prokaryotic expression host cell; And (c) from culture, reclaim the amidation target polypeptides.

15. the method for claim 14 wherein saidly treats that amidated target polypeptides is a salmon calcitonin see calcimar.

16. the method for claim 14, wherein said prokaryotic hosts are shallow Streptomyces glaucoviolaceus TK54, TK24, TK21, TK64 or 1326.

17. the method for claim 14, wherein said expressive host contains pMS680 and pSGLN-mel-AE.

18. a method for preparing the amidation polypeptide, it comprises that (a) is respectively with the expression plasmid of the nucleotide sequence that contains the amidating enzyme of encode and contain two of the expression plasmid conversions prokaryotic host cells independently that coding is treated the nucleotide sequence of amidated target polypeptides; (b) under the condition that helps the generation of α-amidating enzyme and target polypeptides, cultivate gained prokaryotic expression host cell altogether; And (c) from culture, reclaim the amidation target polypeptides.

19. the method for claim 18 wherein saidly treats that amidated target polypeptides is a salmon calcitonin see calcimar.

20. the method for claim 18, wherein said prokaryotic hosts are selected from shallow Streptomyces glaucoviolaceus TK54, TK24, TK21, TK64 or 1326 independently of one another.

21. the method for claim 18 contains pMS680 or pSGLN-mel-AE respectively in wherein said two kinds of expressive hosts.

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