CN1769456A - Recombinant a human peptide production method - Google Patents

Abstract

The invention relates to a method of producing peptides restructured by polypeptide with bioactivity, including following steps: a. Synthesize genes with respect to coding area of peptides amino acids artificially; b Genes gained from step a fuse with 3' side of bacteria thioredoxin genes on the proper expression carriers, among which there are enterokinase or fibrin ferment recognition site dots; c Expression carriers of the coding genes in fusion proteins gained from step b invert proper bacillus colis to get gene engineering bacterias; d said gene engineering bacteria is handled through fermentation, extraction, chromatography, purification steps to prepare peptides- thioredoxin fusion proteins; e said fusion proteins gained from step d is handled by dissection of enterokinase or fibrin ferment, separation, purification to acquire restructed peptides.

Description

The production method of reorganization human peptide

Technical field:

The present invention relates to a kind of preparation method of biologically active polypeptides, particularly a kind of method of utilizing gene recombination technology to prepare the biologically active polypeptides of Na Xili peptide.

Background technology:

Na Xili peptide (Nesiritide) claims that again (Brain Natreuritic Peptide BNP), is to be synthesized and the excretory biologically active substance by cerebral tissue and heart to B-Type natriuretic peptide.Sudoh etc. at first isolate the Na Xili peptide from the pig brain, find also to have in other animal and human's hearts the distribution of Na Xili peptide subsequently, and synthetic Na Xili peptide is brought into play physiological function as neurotransmitter in the brain cell; The Na Xili peptide of the synthetic justacrine of myocardial cell to the blood circulation plays an important role to regulating cardiovascular function and water-electrolyte metabolism then as the increta of heart.Blood plasma BNP content increases the cardiorenal function when the exogenous BNP of intravenous drip can further improve heart failure when in heart failure.Therefore, the reorganization human peptide has important use value clinically.

According to bibliographical information, human peptide (hBNP) is made up of 32 amino-acid residues, and its aminoacid sequence is as follows:

Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His, wherein the 10th and the 26th the intra-residue sulfydryl of Cys form intramolecular disulfide linkage.

Chinese patent 99120613.4 has been announced a kind of method for preparing human peptide, and what this method adopted is the tandem expression technology, both BNP has been connected into ten aggressiveness and directly has been cloned on the expression vector pET22a.Expression product is behind column chromatography purification, placed in-line BNP fusion rotein is cracked into single BNP with the tryptophane lytic reagent, behind column chromatography purification, again disulfide linkage oxidation in the BNP monomer is generated disulfide linkage, unnecessary tryptophane obtains and the on all four BNP of natural human sequence to remove the BNP C-terminal with shuttle peptase Y at last.This processing step is extremely numerous and diverse, and forms intermolecular polymer or isomer easily when the BNP disulfide linkage generates, and yield is extremely low.The present invention has simplified technology greatly, has improved Na Xili peptide output greatly, thus but large-scale industrial production human peptide.

Summary of the invention:

The invention provides a kind of method for preparing human peptide, this method may further comprise the steps:

A. the synthetic people gene DNA fragment of Na Xili peptide ammino acid sequence of encoding;

B. the bacterium thioredoxin gene 3 ' end on step a gene fragment that obtains and the expression vector that suits merges, and the centre contains enteropeptidase or zymoplasm recognition site;

The expression vector recombinant plasmid dna that c. will contain the fusion rotein encoding gene that b. in steps obtains cloning transforms suitable intestinal bacteria and obtains genetic engineering bacterium;

D. this genetic engineering bacterium is after fermentation, through extracting, and chromatography, purification step is prepared human peptide-Trx fusion rotein;

E. the fusion rotein that obtains of steps d separates after enteropeptidase or zymoplasm cutting, and purifying obtains the reorganization human peptide.

Steps d. in fusion rotein in cut recognition site-(Asp) by the enteropeptidase enzyme between Trx and the human peptide _n-Lys-connects, and structure is X-(Asp) _n-Lys-hBNP, wherein X is a Trx, and Asp is aspartic acid (being abbreviated as D), and Lys is Methionin (being abbreviated as K), and hBNP is a human peptide, n is 2,3 or 4, promptly represents n the Asp Asp that is cascaded.

Or

Cutting recognition site X-P2-P3-Pro-P1. by the zymoplasm enzyme between Trx and the human peptide in the fusion rotein connects, structure is X-P2-P3-Pro-P1_hBNP, wherein X is a Trx, P1 is Arg or Lys, P2, P3 are that hydrophobic amino acid is selected from: Leu, Val, ILe, Met, Ala, Cys or Trp, here P2 can be preferably but is not limited to Leu, P3 can be preferably but is not limited to Val, the preferred amino acids sequence of X-P2-P3-Pro-P1_hBNP representative can but be not limited to X-Leu-Val-Pro-Arg-hBNP.

The coding DNA and the aminoacid sequence of described human peptide-Trx fusion rotein are seen sequence table:

Sequence table 1 is the DNA sequences encoding of Na Xili peptide-Trx fusion rotein (the enteropeptidase recognition sequence is DDDDY).

Sequence table 2 is the aminoacid sequence of Na Xili peptide-Trx fusion rotein (the enteropeptidase recognition sequence is DDDDY).

Sequence table 3 is the DNA sequences encoding of Na Xili peptide-Trx fusion rotein (the enteropeptidase recognition sequence is DDDY).

Sequence table 4 is the aminoacid sequence of Na Xili peptide-Trx fusion rotein (the enteropeptidase recognition sequence is DDDY).

Sequence table 5 is the DNA sequences encoding of Na Xili peptide-Trx fusion rotein (the enteropeptidase recognition sequence is DDY).

Sequence table 6 is the aminoacid sequence of Na Xili peptide-Trx fusion rotein (the enteropeptidase recognition sequence is DDY).

Sequence table 7 is the DNA sequences encoding of Na Xili peptide-Trx fusion rotein (zymoplasm recognition sequence).

Sequence table 8 is the aminoacid sequence of Na Xili peptide-Trx fusion rotein (zymoplasm recognition sequence).

Preparation method of the present invention is characterized in that, wherein said fermentation is qualified recombinant human Na Xili peptide gene engineering bacteria to be inoculated in the fermentor tank cultivate, and wherein adds IPTG and induces Expression of Fusion Protein.

Preparation method of the present invention, wherein said extraction chromatography purification,

The step that comprises for the fusion rotein of enteropeptidase recognition site is: centrifugal collection thalline, the step of broken bacterium, the broken bacterium liquid supernatant of centrifugal collection is with Zn on the supernatant of collecting ²⁺-Sepharose F.F. metal ion chela and post, with level pad flush away foreign protein, then with the imidazoles wash-out Na Xili peptide-Trx fusion rotein that contains suitable concentration, Na Xili peptide-Trx the fusion rotein of wash-out is removed imidazoles with dialysis or ultrafiltration, do enzyme with highly purified Enterkinase and cut processing, Zn on the fusion rotein that enzyme is cut ²⁺-Sepharose F.F. post removes Trx, and with reversed phase column chromatography on the Na Xili peptide that obtains, the Acetonitrile gradient elution with containing TFA obtains the pure Na Xili peptide of purity more than 95%.

The step that comprises for the fusion rotein of zymoplasm recognition site is: centrifugal collection thalline, broken bacterium, the broken bacterium liquid precipitate (the Thioredoxin-BNP fusion rotein is mainly in centrifugation) of centrifugal collection.The precipitation of collecting is dissolved with the urea of 8M is plain clearly, remove the urea element with the method for dialysis then, directly use zymoplasm (Thrombin) enzyme to cut dialyzate, the solution that enzyme is cut is directly gone up SP-Sepharose F.F. ion exchange column, rhBNP is attracted on the post, and other most of impurity then directly penetrates; With reversed phase column chromatography on the solution that contains rhBNP of wash-out, the Acetonitrile gradient elution with containing TFA can obtain the pure product of purity more than 95%.Remove second cyanogen and trifluoroacetic acid with suitable method, and change suitable damping fluid into, slow or phosphate buffered saline buffer as Citrate trianion.

The present invention also provides the human peptide pharmaceutical composition, and its prescription is composed as follows:

Na Xili peptide 0.5mg

N.F,USP MANNITOL 5～20mg

Citric acid (water) 2.1mg

Lemon sodium (two water) 2.94mg

Or

Na Xili peptide 0.5mg

N.F,USP MANNITOL 5～20mg

SODIUM PHOSPHATE, MONOBASIC 0.93mg

Sodium phosphate dibasic 1.73mg

Sodium-chlor 10mg

The present invention is according to the amino acid coding of the human peptide primary structure of having delivered (this sequence can change the preference principle that genetic code uses according to versatility, degeneracy and the intestinal bacteria of genetic code), this double-stranded DNA complete sequence of synthetic.Principal feature of the present invention is, respectively this encoding gene fragment cloning is gone into suitable expression vector, (preferred as: pET32a or pTDa, be the expression vector of routine, all can on market, have bought, include the thioredoxin gene fragment) the middle formation recombinant plasmid that makes up, then that recombinant plasmid transformed is suitable intestinal bacteria, through screening, preferably: BL21 (λ DE3) or HB101 bacterial strain (be conventional bacterial strain, all can on market, have bought).This genetic engineering bacterium is a fusion partner with Trx (Thioredoxin), amalgamation and expression hBNP, promptly form Na Xili peptide-Trx fusion rotein, after the separation and purification of expressed fusion protein process suitable step, with suitable proteolytic enzyme with distinguished sequence recognition capability as hBNP being cut down from fusion rotein with enteropeptidase (Enterkinase) or zymoplasm (Thrombin), through the high separation purifying, obtain the pure product of hBNP.Through analyses such as-terminal amino acid sequencing, mass spectrum molecular weight determination, isoelectric point determination, peptide figure analysis and biological activity assay, the result shows that human peptide that we are expressed and natural each index of Na Xili peptide are in full accord, and expression efficiency is more than 30% of whole bacterial protein, and the series process that is higher than the prior art report is produced the Na Xili peptide.

The ultimate principle of the molecular cloning method that the present invention uses, and employed raw material in the operation steps, solvent, instrument belongs to the prior art field, as the synthetic method of DNA, and the selection of restriction enzyme site etc., the expression vector that the present invention uses, genetic engineering bacterium etc. can have been bought on market.

The concrete operations step of above method can be seen embodiments of the invention.

Advantage of the present invention mainly shows:

(1), there be (connecting with enteropeptidase Enterkinase recognition sequence) in Na Xili peptide-Trx fusion rotein with soluble form among the present invention, perhaps the form with inclusion body exists (connecting with zymoplasm Thrombin recognition sequence) in the plain dissolving of 8M urea back dialysis renaturation, the right-on disulfide linkage of formation of cys on its BNP10 position and 26, avoid fusion protease to cut to carry out renaturation behind the purifying handling, thereby simplify production technique greatly and enhance productivity greatly;

(2), the rhBNP that obtains by above-mentioned technology, the structure of physicochemical properties such as its structure and biologic activity and bibliographical information is in full accord, there is not the existence of any dimer or isomer, also avoid common recombinant protein drug N end front to have more a Met even a plurality of amino-acid residue simultaneously, thereby can reduce its immunogenicity;

(3), expression rate height.

The advantage of the present composition shows:

(1) good stability was placed 18 months under the room temperature, and its every index still can reach every requirement of injection biological products.

Description of drawings:

Fig. 1 pET-32a plasmid map

Fig. 2 pET-32a multiple clone site and sequence

Fig. 3 pET32a-BNP construction of recombinant plasmid synoptic diagram

Fig. 4 Na Xili peptide-Trx fusion rotein (enteropeptidase recognition sequence) expressing fusion protein

Fig. 5 enteropeptidase is handled the Na Xili peptide electrophoretogram of back purifying

Fig. 6 pTDa expression vector

The structure synoptic diagram of Fig. 7 pTDa-BNP recombinant plasmid dna

The expression of Fig. 8 Na Xili peptide-Trx fusion rotein (zymoplasm recognition sequence) (1,2 is that example 1 resulting rhBNP, 3,4 is example 2 resulting rhBNP)

The Na Xili peptide electrophoretogram of Fig. 9 dual mode purifying

Embodiment:

Further specify the present invention by the following examples, but not as limitation of the present invention.

Embodiment 1

1. the selection of expression vector

The pET series expression vector pET32a that selects Novagen company for use is as expression vector, The pET-32a is the coli expression carrier system that is used for efficient amalgamation and expression albumen and polypeptide, and target protein or polypeptide are with the Trx (TrxTag of 109 amino-acid residues ^TM) as fusion partner.(collection of illustrative plates is seen accompanying drawing 1, Fig. 2).

2. the design of gene and synthetic

(sequence can change according to versatility, the degeneracy principle of genetic code according to the human peptide dna encoding sequence of bibliographical information, but not changing that amino acid is formed and arrange) design is encoded into acquaintance BNP sequence, as: 5 ' _ AGC CCT AAA ATG GTA CAG GGT TCT GGT TGC TTC GGT CGT AAA ATG GAC CGTATC AGC TCT TCC AGC GGT CTG GGT TGC AAA GTA CTG CGT CGT CAC TAA_3 '.Simultaneously 5 ' of this sequence-end add Kpn recognition sequence GGTACC and enteropeptidase (Enterokinase) restriction enzyme site encoding sequence GACGACGACGACAAG (promptly encode :-DDDDK-); Add that at 3 ' _ end thereby the recognition sequence CTCGAG of Xho I forms following implementation sequence: 5 ' _ GGT ACC GAC GAC GAC GAC AAG CCT AAAATG GTA CAG GGT TCT GGT TGC TTC GGT CGT AAA ATG GAC CGT ATC AGC TCT TCCAGC GGT CTG GGT TGC AAA GTA CTG CGT CGT CAC TAA CTC GAG_3 '.

Or

Add Kpn recognition sequence GGTACC and enteropeptidase (Enterokinase) restriction enzyme site encoding sequence at 5 ' of this sequence-end GACGACGACAAG (promptly encode :-DDDK-)Add that at 3 ' _ end thereby the recognition sequence CTCGAG of Xho I forms following implementation sequence: 5 ' _ GGT ACC GAC GAC GAC GAC AAG CCT AAAATG GTA CAG GGT TCT GGT TGC TTC GGT CGT AAA ATG GAC CGT ATC AGC TCT TCCAGC GGT CTG GGT TGC AAA GTA CTG CGT CGT CAC TAA CTC GAG_3 '.

Or

Add Kpn recognition sequence GGTACC and enteropeptidase (Enterokinase) restriction enzyme site encoding sequence at 5 ' of this sequence-end GACGACAAG (promptly encode :-DDK-)Add that at 3 ' _ end thereby the recognition sequence CTCGAG of Xho I forms following implementation sequence: 5 ' _ GGT ACC GAC GAC GAC GAC AAG CCT AAA ATG GTACAG GGT TCT GGT TGC TTC GGT CGT AAA ATG GAC CGT ATC AGC TCT TCC AGC GGTCTG GGT TGC AAA GTA CTG CGT CGT CAC TAA CTC GAG_3 '.

Synthetic method is referring to " molecular cloning handbook cold spring port second edition

3. the Kpn and the Xho I site that above-mentioned synthetic nucleic acid fragment are inserted into PET32a form recombinant plasmid, and transformed into escherichia coli BL21 efficiently expresses genetic engineering bacterium through screening promptly to get.Relevant working method is referring to " molecular cloning handbook cold spring port second edition

Make up collection of illustrative plates and see accompanying drawing 3

With microbionation in LB or other suitable culture base, long during to suitable concentration such as mid-log phase when engineering bacteria, (as 0.1～0.5mM), visible significantly expressing fusion protein band is seen to add the IPTG of suitable concentration

Accompanying drawing 4:

5. with the seed liquor of overnight incubation, be inoculated in the fermentor tank suitable culture base (in LB, TB or other appropriate media) and cultivate, microbial culture adds IPTG inducing culture certain hour (as: 3-4 hour) during to suitable degree.Centrifugal receipts bacterium is suspended in (as phosphate buffered saline buffer, Tris-HCL damping fluid etc.) in the suitable damping fluid with thalline, the broken bacterium of excusing from death ripple or high-pressure homogenization.The bacterium liquid of fragmentation is centrifugal, collect supernatant liquor, abandon precipitation.

The supernatant of collecting (or handling after filtration again) is gone up Zn ²⁺(chelating-Sepharose F.F. is through the Zn of suitable concentration such as 100mM for-Sepharose F.F. metal ion chela and post ²⁺SO4 or Zn ²⁺Cl ₂Handle), with level pad (as 20-50mM phosphate buffered saline buffer, Tris-HCL damping fluid etc.) flush away foreign protein, then with the 100mM imidazoles wash-out Na Xili peptide-Trx fusion rotein that contains suitable concentration.

Na Xili peptide-Trx the fusion rotein of wash-out is removed imidazoles with dialysis or ultrafiltration, with highly purified Enterkinase (as the EK of Invitrogen company ^Max) handle, but the Enterkinase enzyme of condition reference literature or Invitrogen company uses the recommendation condition.

Zn on the fusion rotein that enzyme is cut ²⁺-Sepharose F.F. post removes Trx (condition is with the first step purifying), the rhBNP that obtains is gone up reversed phase column chromatography (filler can be chosen reverse phase filler such as C4, c8, Resource), with the Acetonitrile that contains finite concentration water (containing finite concentration) gradient elution, can obtain the pure product of purity more than 95% as 0.1% trifluoroacetic acid.With suitable method (as vacuum pump transfer or with the method for ion-exchange) remove second cyanogen and trifluoroacetic acid, and change suitable damping fluid into, as phosphate buffered saline buffer or citrate buffer.

The rhBNP electrophoresis accompanying drawing 5 that obtains behind the purifying:

Embodiment 2

1, the selection of expression vector

Select for use this breadboard pTDa as expression vector (seeing accompanying drawing 6)

2, the design of gene and synthetic

According to intestinal bacteria the preference principle that genetic code uses has been designed encoding mature human peptide sequence: 5 ' _ AGC CCT AAA ATG GTA CAG GGT TCT GGT TGC TTC GGT CGT AAA ATG GACCGT ATC AGC TCT TCC AGC GGT CTG GGT TGC AAA GTA CTG CGT CGT CAC TAA (sequence can change according to versatility, the degeneracy principle of genetic code, but does not change amino acid composition and arrangement).Simultaneously add EcoRI recognition sequence GAA TTC and zymoplasm (Thrombin) restriction enzyme site (LVPR-X) encoding sequence CTG GTG CCT CGT at 5 ' of this sequence-end; Add that at 3 ' _ end thereby the recognition sequence GTC GAC of Sal I forms following final implementation sequence: 5 ' _ GAA TTC CTG GTG CCT CGT AGC CCTAAA ATG GTA CAG GGT TCT GGT TGC TTC GGT CGT AAA ATG GAC CGT ATC AGC TCTTCC AGC GGT CTG GGT TGC AAA GTA CTG CGT CGT CAC TAA GTC GAC_3 '.Synthetic method is referring to " molecular cloning handbook cold spring port second edition

2, above-mentioned synthetic nucleic acid fragment is inserted into EcoRI and the Sal I site formation recombinant plasmid of pTDa, and transformed into escherichia coli HB101 promptly obtains efficiently expressing genetic engineering bacterium through screening.Relevant working method is referring to " molecular cloning handbook cold spring port second edition (the structure synoptic diagram of recombinant plasmid dna is seen accompanying drawing 7)

3, with microbionation in LB or other suitable culture base, long during to suitable concentration such as mid-log phase when engineering bacteria, add suitable concentration IPTG (as 0.1～0.5mM), visible significantly expressing fusion protein band, see accompanying drawing 8:

The seed liquor of 4, spending the night is inoculated in the fermentor tank suitable culture base (in LB, TB or other appropriate media) and cultivates, and microbial culture adds IPTG inducing culture certain hour (as: 3-4 hour) during to suitable degree.Centrifugal receipts bacterium is suspended in (as phosphate buffered saline buffer, Tris-HCL damping fluid etc.) in the suitable damping fluid with thalline, the broken bacterium of excusing from death ripple or high-pressure homogenization.The bacterium liquid of fragmentation is centrifugal, and collecting precipitation is abandoned supernatant liquor.

5, precipitation is dissolved with the 8M urea, and dilution refolding carries out enzyme with Thrombin and cuts (operation instruction of reference enzyme).

6, carry out purifying with the SP-FF column chromatography after enzyme is cut.

The rhBNP that obtains is gone up reversed phase column chromatography, and (filler can be chosen C ₄, C ₈, reverse phase filler such as Resource), with the second cyanogen that contains finite concentration water (containing finite concentration) gradient elution, can obtain the pure product of purity more than 95% as 0.1% trifluoroacetic acid.With suitable method (as vacuum pump transfer or with the method for ion-exchange) remove second cyanogen and trifluoroacetic acid, and change suitable damping fluid into, as phosphate buffered saline buffer or citrate buffer.

6, the rhBNP electrophoresis accompanying drawing 9 that obtains behind the purifying:

Embodiment 3

Physical and chemical property determining according to the rhBNP of embodiment 1 or 2 gained

1, terminal 15 determined amino acid sequences of N-

With the reorganization human peptide that above-mentioned two kinds of methods obtain, carry out the terminal mensuration of N-, Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met

2, molecular weight mass spectroscopy

Adopt the FAB method to measure, the molecular weight of the reorganization human peptide that two kinds of methods obtain is 3462, and is consistent with theoretical prediction.

3, Determination of biological activity

Utilize Phenylephrine and its receptors bind on blood vessel, the generation vascular effect that contracts, the resting tension of blood vessel is improved, and stable maintenance is at certain level, utilize rhBNP to combine then with its natriuretic peptide receptor on blood vessel, generation by the variation of antiotasis, is measured the biological activity of rhBNP to the expansion vascular effect of rabbit thoracic aorta bar.Resulting rhBNP shows tangible expansion vascular effect.

Sequence table

＜110〉Chengdu Xima Biotechnology Co., Ltd.

＜120〉production method of reorganization human peptide

<160>8

<210>1

<211>579

<212>DNA

＜213〉artificial sequence

<220>

<400>1

atgagcgata?aaattattca?cctgactgac?gacagttttg?acacggatgt?actcaaagcg 60

gacggggcga?tcctcgtcga?tttctgggca?gagtggtgcg?gtccgtgcaa?aatgatcgcc 120

ccgattctgg?atgaaatcgc?tgacgaatat?cagggcaaac?tgaccgttgc?aaaactgaac 180

atcgatcaaa?accctggcac?tgcgccgaaa?tatggcatcc?gtggtatccc?gactctgctg 240

ctgttcaaaa?acggtgaagt?ggcggcaacc?aaagtgggtg?cactgtctaa?aggtcagttg 300

aaagagttcc?tcgacgctaa?cctggccggt?tctggttctg?gccatatgca?ccatcatcat 360

catcattctt?ctggtctggt?gccacgcggt?tctggtatga?aagaaaccgc?tgctgctaaa 420

ttcgaacgcc?agcacatgga?cagcccagat?ctgggtaccg?acgacgacga?caagagccct 480

aaaatggtac?agggttctgg?ttgcttcggt?cgtaaaatgg?accgtatcag?ctcttccagc 540

ggtctgggtt?gcaaagtact?gcgtcgtcac?taactcgag 579

<210>2

<211>190

<212>PRT

＜213〉artificial sequence

<220>

<400>2

Met?Ser?Asp?Lys?Ile?Ile?His?Leu?Thr?Asp?Asp?Ser?Phe?Asp?Thr

1 5 10 15

Asp?Val?Leu?Lys?Ala?Asp?Gly?Ala?Ile?Leu?Val?Asp?Phe?Trp?Ala

20 25 30

Glu?Trp?Cys?Gly?Pro?Cys?Lys?Met?Ile?Ala?Pro?Ile?Leu?Asp?Glu

35 40 45

Ile?Ala?Asp?Glu?Tyr?Gln?Gly?Lys?Leu?Thr?Val?Ala?Lys?Leu?Asn

50 55 60

Ile?Asp?Gln?Asn?Pro?Gly?Thr?Ala?Pro?Lys?Tyr?Gly?Ile?Arg?Gly

65 70 75

Ile?Pro?Thr?Leu?Leu?Leu?Phe?Lys?Asn?Gly?Glu?Val?Ala?Ala?Thr

80 85 90

Lys?Val?Gly?Ala?Leu?Ser?Lys?Gly?Gln?Leu?Lys?Glu?Phe?Leu?Asp

95 100 105

Ala?Asn?Leu?Ala?Gly?Ser?Gly?Ser?Gly?His?Met?His?His?His?His

110 115 120

His?His?Ser?Ser?Gly?Leu?Val?Pro?Arg?Gly?Ser?Gly?Met?Lys?Glu

125 130 135

Thr?Ala?Ala?Ala?Lys?Phe?Glu?Arg?Gln?His?Met?Asp?Ser?Pro?Asp

140 145 150

Leu?Gly?Thr?Asp?Asp?Asp?Asp?Lys?Set?Pro?Lys?Met?Val?Gln?Gly

155 160 165

Ser?Gly?Cys?Phe?Gly?Arg?Lys?Met?Asp?Arg?Ile?Ser?Ser?Ser?Ser

170 175 180

Gly?Leu?Gly?Cys?Lys?Val?Leu?Arg?Arg?His

185 190

<210>3

<211>576

<212>DNA

＜213〉artificial sequence

<220>

<400>3

atgagcgata?aaattattca?cctgactgac?gacagttttg?acacggatgt?actcaaagcg 60

gacggggcga?tcctcgtcga?tttctgggca?gagtggtgcg?gtccgtgcaa?aatgatcgcc 120

ccgattctgg?atgaaatcgc?tgacgaatat?cagggcaaac?tgaccgttgc?aaaactgaac 180

atcgatcaaa?accctggcac?tgcgccgaaa?tatggcatcc?gtggtatccc?gactctgctg 240

ctgttcaaaa?acggtgaagt?ggcggcaacc?aaagtgggtg?cactgtctaa?aggtcagttg 300

aaagagttcc?tcgacgctaa?cctggccggt?tctggttctg?gccatatgca?ccatcatcat 360

catcattctt?ctggtctggt?gccacgcggt?tctggtatga?aagaaaccgc?tgctgctaaa 420

ttcgaacgcc?agcacatgga?cagcccagat?ctgggtaccg?acgacgacaa?gagccctaaa 480

atggtacagg?gttctggttg?cttcggtcgt?aaaatggacc?gtatcagctc?ttccagcggt 540

ctgggttgca?aagtactgcg?tcgtcactaa?ctcgag 576

<210>4

<211>189

<212>PRT

＜213〉artificial sequence

<220>

<400>4

Met?Ser?Asp?Lys?Ile?Ile?His?Leu?Thr?Asp?Asp?Ser?Phe?Asp?Thr

l 5 10 15

Asp?Val?Leu?Lys?Ala?Asp?Gly?Ala?Ile?Leu?Val?Asp?Phe?Trp?Ala

20 25 30

Glu?Trp?Cys?Gly?Pro?Cys?Lys?Met?Ile?Ala?Pro?Ile?Leu?Asp?Glu

35 40 45

Ile?Ala?Asp?Glu?Tyr?Gln?Gly?Lys?Leu?Thr?Val?Ala?Lys?Leu?Asn

50 55 60

Ile?Asp?Gln?Asn?Pro?Gly?Thr?Ala?Pro?Lys?Tyr?Gly?Ile?Arg?Gly

65 70 75

Ile?Pro?Thr?Leu?Leu?Leu?Phe?Lys?Asn?Gly?Glu?Val?Ala?Ala?Thr

80 85 90

Lys?Val?Gly?Ala?Leu?Ser?Lys?Gly?Gln?Leu?Lys?Glu?Phe?Leu?Asp

95 100 105

Ala?Asn?Leu?Ala?Gly?Ser?Gly?Ser?Gly?His?Met?His?His?His?His

110 115 120

His?His?Ser?Ser?Gly?Leu?Val?Pro?Arg?Gly?Ser?Gly?Met?Lys?Glu

125 130 135

Thr?Ala?Ala?Ala?Lys?Phe?Glu?Arg?Gln?His?Met?Asp?Ser?Pro?Asp

140 145 150

Leu?Gly?Thr?Asp?Asp?Asp?Lys?Ser?Pro?Lys?Met?Val?Gln?Gly?Ser

155 160 165

Gly?Cys?Phe?Gly?Arg?Lys?Met?Asp?Arg?Ile?Ser?Ser?Ser?Ser?Gly

170 175 180

Leu?Gly?Cys?Lys?Val?Leu?Arg?Arg?His

185 189

<210>5

<211>573

<212>DNA

＜213〉artificial sequence

<220>

<400>5

atgagcgata?aaattattca?cctgactgac?gacagttttg?acacggatgt?actcaaagcg 60

gacggggcga?tcctcgtcga?tttctgggca?gagtggtgcg?gtccgtgcaa?aatgatcgcc 120

ccgattctgg?atgaaatcgc?tgacgaatat?cagggcaaac?tgaccgttgc?aaaactgaac 180

atcgatcaaa?accctggcac?tgcgccgaaa?tatggcatcc?gtggtatccc?gactctgctg 240

ctgttcaaaa?acggtgaagt?ggcggcaacc?aaagtgggtg?cactgtctaa?aggtcagttg 300

aaagagttcc?tcgacgctaa?cctggccggt?tctggttctg?gccatatgca?ccatcatcat 360

catcattctt?ctggtctggt?gccacgcggt?tctggtatga?aagaaaccgc?tgctgctaaa 420

ttcgaacgcc?agcacatgga?cagcccagat?ctgggtaccg?acgacaagag?ccctaaaatg 480

gtacagggtt?ctggttgctt?cggtcgtaaa?atggaccgta?tcagctcttc?cagcggtctg 540

ggttgcaaag?tactgcgtcg?tcactaactc?gag 573

<210>6

<211>188

<212>PRT

＜213〉artificial sequence

<220>

<400>6

Met?Ser?Asp?Lys?Ile?Ile?His?Leu?Thr?Asp?Asp?Ser?Phe?Asp?Thr

1 5 10 15

Asp?Val?Leu?Lys?Ala?Asp?Gly?Ala?Ile?Leu?Val?Asp?Phe?Trp?Ala

20 25 30

Glu?Trp?Cys?Gly?Pro?Cys?Lys?Met?Ile?Ala?Pro?Ile?Leu?Asp?Glu

35 40 45

Ile?Ala?Asp?Glu?Tyr?Gln?Gly?Lys?Leu?Thr?Val?Ala?Lys?Leu?Asn

50 55 60

Ile?Asp?Gln?Asn?Pro?Gly?Thr?Ala?Pro?Lys?Tyr?Gly?Ile?Arg?Gly

65 70 75

Ile?Pro?Thr?Leu?Leu?Leu?Phe?Lys?Asn?Gly?Glu?Val?Ala?Ala?Thr

80 85 90

Lys?Val?Gly?Ala?Leu?Ser?Lys?Gly?Gln?Leu?Lys?Glu?Phe?Leu?Asp

95 100 105

Ala?Asn?Leu?Ala?Gly?Ser?Gly?Ser?Gly?His?Met?His?His?His?His

110 115 120

His?His?Ser?Ser?Gly?Leu?Val?Pro?Arg?Gly?Ser?Gly?Met?Lys?Glu

125 130 135

Thr?Ala?Ala?Ala?Lys?Phe?Glu?Arg?Gln?His?Met?Asp?Ser?Pro?Asp

140 145 150

Leu?Gly?Thr?Asp?Asp?Lys?Ser?Pro?Lys?Met?Val?Gln?Gly?Ser?Gly

155 160 165

Cys?Phe?Gly?Arg?Lys?Met?Asp?Arg?Ile?Ser?Ser?Ser?Ser?Gly?Leu

170 175 180

Gly?Cys?Lys?Val?Leu?Arg?Arg?His

185 188

<210>7

<211>507

<212>DNA

＜213〉artificial sequence

<220>

<400>7

atgtctgata?aaattattca?tctgactgat?gattcttttg?atactgatgt?acttaaggca 60

gatggtgcaa?tcctggttga?tttctgggca?cactggtgcg?gtccgtgcaa?aatgatcgct 120

ccgattctgg?atgaaatcgc?tgacgaatat?cagggcaaac?tgaccgttgc?aaaactgaac 180

atcgatcaca?acccgggcac?tgcgccgaaa?tatggcatcc?gtggtatccc?gactctgctg 240

ctgttcaaaa?acggtgaagt?ggcggcaacc?aaagtgggtg?cactgtctaa?aggtcagttg 300

aaagagttcc?tcgacgctaa?cctggccggc?tctggatccg?gtgatgacga?tgacaaggta 360

cctatgcatg?agctcgagat?cttcgaattc?ctggtgcctc?gtagccctaa?aatggtacag 420

ggttctggtt?gcttcggtcg?taaaatggac?cgtatcagct?cttccagcgg?tctgggttgc 480

aaagtactgc?gtcgtcacta?agtcgac?507

<210>8

<211>166

<212>PRT

＜213〉artificial sequence

<220>

<400>8

Met?Ser?Asp?Lys?Ile?Ile?His?Leu?Thr?Asp?Asp?Ser?Phe?Asp?Thr

1 5 10 15

Asp?Val?Leu?Lys?Ala?Asp?Gly?Ala?Ile?Leu?Val?Asp?Phe?Trp?Ala

20 25 30

Glu?Trp?Cys?Gly?Pro?Cys?Lys?Met?Ile?Ala?Pro?Ile?Leu?Asp?Glu

35 40 45

Ile?Ala?Asp?Glu?Tyr?Gln?Gly?Lys?Leu?Thr?Val?Ala?Lys?Leu?Asn

50 55 60

Ile?Asp?Gln?Asn?Pro?Gly?Thr?Ala?Pro?Lys?Tyr?Gly?Ile?Arg?Gly

65 70 75

Ile?Pro?Thr?Leu?Leu?Leu?Phe?Lys?Asn?Gly?Glu?Val?Ala?Ala?Thr

80 85 90

Lys?Val?Gly?Ala?Leu?Ser?Lys?Gly?Gln?Leu?Lys?Glu?Phe?Leu?Asp

95 100 105

Ala?Asn?Leu?Ala?Gly?Ser?Gly?Ser?Gly?Asp?Asp?Asp?Asp?Lys?Val

110 115 120

Pro?Met?His?Glu?Leu?Glu?Ile?Phe?Glu?Phe?Leu?Val?Pro?Arg?Ser

125 130 135

Pro?Lys?Met?Val?Gln?Gly?Ser?Gly?Cys?Phe?Gly?Arg?Lys?Met?Asp

140 145 150

Arg?Ile?Ser?Ser?Ser?Ser?Gly?Leu?Gly?Cys?Lys?Val?Leu?Arg?Arg

155 160 165

His

166

Claims (10)

1, a kind of preparation method of human peptide may further comprise the steps:

A. the corresponding gene in synthetic human peptide amino acid coding region;

B. the gene that obtains of step a and the bacterium thioredoxin gene 3 on the expression vector ' end merges, and the centre contains enteropeptidase or zymoplasm recognition site;

C. will containing in steps, the clone of b has the expression vector recombinant plasmid dna transformed into escherichia coli of fusion rotein encoding gene to obtain genetic engineering bacterium;

D. after this genetic engineering bacterium ferments, through extraction, chromatography, purification step is prepared Na Xili peptide-Trx fusion rotein;

E. the fusion rotein of steps d, after enteropeptidase or zymoplasm cutting, through separating, purifying, human peptide.

2, preparation method according to claim 1 is characterized in that,

Steps d. in fusion rotein in cut recognition site-(Asp) by the enteropeptidase enzyme between Trx and the human peptide _n-Lys-connects, and structure is X-(Asp) _n-Lys-hBNP, wherein X is a Trx, and Asp is an aspartic acid, and Lys is a Methionin, and hBNP is a human peptide, n is 2,3 or 4; Or steps d. in fusion rotein in cut recognition site X-P2-P3-Pro-P1. by the zymoplasm enzyme between Trx and the human peptide and connect, structure is X-P2-P3-Pro-P1-hBNP, wherein X is a Trx, P1 is Arg or Lys, and P2, P3 are that hydrophobic amino acid is selected from: Leu, Val, ILe, Met, Ala, Cys or Trp.

3, preparation method according to claim 2 is characterized in that, wherein said structure is: the fusion rotein of X-P2-P3-Pro-P1-hBNP is X-Leu-Val-Pro-Arg-hBNP, and wherein X is a Trx, and its aminoacid sequence is seen sequence table 8.

4, preparation method according to claim 2 is characterized in that, wherein said structure is X-(Asp) _nThe fusion rotein of-Lys-hBNP is:

X-Asp-Asp-Asp-Asp-Lys-hBNP, aminoacid sequence are seen sequence table 2

X-Asp-Asp-Asp-Lys-hBNP, aminoacid sequence are seen sequence table 4

Or X-Asp-Asp-Lys-hBNP, aminoacid sequence is seen sequence table 6

Wherein X is a Trx.

5. preparation method according to claim 3 is characterized in that, described structure is:

The fusion rotein of X-Leu-Val-Pro-Arg-hBNP, its DNA sequences encoding is seen sequence table 7.

6. preparation method according to claim 4 is characterized in that, described structure is:

The aminoacid sequence of X-Asp-Asp-Asp-Asp-Lys-hBNP, its DNA sequences encoding is seen sequence table 1,

The amino acid of X-Asp-Asp-Asp-Lys-hBNP, its DNA sequences encoding is seen sequence table 3,

Or the aminoacid sequence of X-Asp-Asp-Lys-hBNP, its DNA sequences encoding is seen sequence table 5,

Wherein X is a Trx.

7. preparation method according to claim 1 is characterized in that, wherein said expression vector is pET32a or pTDa.

8. preparation method according to claim 1 is characterized in that, wherein said intestinal bacteria are BL21 or HB101 bacterial strain.

9. preparation method according to claim 1 is characterized in that,

Wherein said fermentation is qualified recombinant human Na Xili peptide gene engineering bacteria to be inoculated in the fermentor tank cultivate, and adopts IPTG to induce Expression of Fusion Protein;

Wherein said extraction, chromatography, purification step, the step that comprises for the fusion rotein of enteropeptidase recognition site is: centrifugal collection thalline, the step of broken bacterium, the broken bacterium liquid supernatant of centrifugal collection is with Zn on the supernatant of collecting ²⁺-Sepharose F.F. metal ion chela and post, with level pad flush away foreign protein, then with the imidazole buffer wash-out Na Xili peptide-Trx fusion rotein that contains suitable concentration, Na Xili peptide-Trx the fusion rotein of wash-out is removed imidazoles with dialysis or ultrafiltration, do enzyme with highly purified Enterkinase and cut processing, enzyme cuts finishes Zn on the solution of back ²⁺-Sepharose F.F. post removes Trx, with the reversed phase column chromatography on the Na Xili peptide solution that contains that obtains, with the Acetonitrile gradient elution that contains TFA, De Naxili peptide;

The step that comprises for the fusion rotein of zymoplasm recognition site is:

Centrifugal collection thalline, broken bacterium, the broken bacterium liquid precipitate of centrifugal collection, the precipitation of collecting is dissolved with the urea of 8M is plain clearly, remove the urea element fully with the method for dialysis then, dialyzate is directly cut with the zymoplasm enzyme, solution after enzyme cut is directly gone up SP-Sepharose F.F. ion exchange column, and rhBNP is attracted on the post, and other most of impurity then directly penetrates; With reversed phase column chromatography on the solution that contains rhBNP of wash-out, with the Acetonitrile gradient elution that contains TFA, De Naxili peptide.

10, a kind of Na Xili peptide medicine composite, its prescription is composed as follows:

Na Xili peptide 0.5mg

N.F,USP MANNITOL 5～20mg

Citric acid (water) 2.1mg

Lemon sodium (two water) 2.94mg

Or

Na Xili peptide 0.5mg

N.F,USP MANNITOL 5～20mg

SODIUM PHOSPHATE, MONOBASIC 0.93mg

Sodium phosphate dibasic 1.73mg

Sodium-chlor 10mg

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