CN104447982B - A kind of people's relaxain H2 precursors and its preparation method and application - Google Patents

Abstract

The invention provides a kind of artificial C peptide sequences, the sequence has any sequence shown in Seq ID No1 4.Natural or function mutation people's relaxain H2 B chains and A chains can be linked in sequence by the artificial C peptide sequences by B C A, obtain people's relaxain H2 front body structures of restructuring.Present invention also offers the preparation method of recombinant human relaxin element H2 albumen, the B chains and A chains of RLX2 are linked in sequence by B C A by above-mentioned C peptides, after host cell expression people's relaxain H2 precursors are obtained by collecting inclusion body, and the steps such as renaturation being denatured to inclusion body.Precursor obtains the ripe body people relaxain H2 of purifying by protease digestion and through steps such as chromatographies.

Description

A kind of people's relaxain H2 precursors and its preparation method and application

Technical field

The invention belongs to technical field of bioengineering, more particularly to a kind of people's relaxain H2 precursors and preparation method thereof and should With.The invention further relates to the ripe people's relaxain obtained via people relaxain H2 precursors and its application.

Background technology

Relaxain（Relaxin）As a kind of peptide hormone, by Frederick Hisaw in research gestation pelvic girdle Found first during change.Relaxain is considered as initially a kind of pregnancy associated hormone, the March before pregnant women's, in body circulation Relaxain level substantially rise, while changing with haemodynamics and renal hemodynamic；Also there is relaxation pelvis ligament simultaneously, expand Cervix is opened, suppresses uterine smooth muscle Spontaneous Contraction, the effect such as reconstruction of gestational period connective tissue is participated in.Research was found later, Various Tissues have the expression of relaxain in the women of non-pregnancy and males, and with extensive biological action, Including blood vessel dilatation, there is the effect such as anti-inflammatory, extracellular matrix remodeling, angiogenesis and anti-ischemic.Generally believe relaxain at present It is a kind of peptide hormone, such peptide family is by 7 kinds of gene codes, including relaxation gene RLN1, RLN2 and RLN3, and insulin Sample peptide gene INSL3, INSL4, INSL5, INSL6.The relaxain H2 of mankind RLN2 codings is its function product, there is stronger whole body With kidney expansion blood vessel, increase cardiac output, body circulation resistance, pulmonary capillary wedge pressure and NT-proBNP are reduced, improves kidney Function resists body reaction for being harmful to cardiac pumping function etc. to adjust cardiovascular response.Relaxain H2 is with pine in human body The state table former element H2 that relaxes is reached, and relaxation precipitinogen albumen primary structure is arranged by B-C-A order（See Fig. 1）, wherein C chains are by 108 Individual amino acid residue composition, after relaxation precipitinogen generation after the invertase in tissue cuts off C chains, forms ripe relaxain H2.Relaxain H2 is made up of 53 amino acid, and its architectural feature is that have two independent polypeptide chains (A chains and B chains), wherein A Chain is made up of 24 amino acid residues, 29 amino acid residue compositions of B chains；There are two pairs of interchain disulfide bonds to be connected between A, B chain, A There are a pair of intrachain disulfide bonds in chain.Clinical evidence shows that relaxation have vasodilator increase vascular compliance and anti-ischemic effect； In the acute heart failure patient that blood pressure is normal and increases, relaxain can improve expiratory dyspnea and other clinical effectivenesses, and security can Lean on.

Because people's relaxation have good druggability, with high clinical value, demand increasingly increases, therefore The research of people's relaxain H2 preparation methods is promoted.Presently the most common preparation method mainly has following two：

1st, chemical synthesis：E.Bullesbach and C.Schwabe was in 1991 and reported within 2005 RLX chemistry Synthetic method（J.Biol.Chem.1991,266,10754-10761; J.Biol.Chem.2005,280,14586-14590）, Relaxain H2 A chains are synthesized using Fmoc methods, and B chains are synthesized using Boc methods, and building-up process is needed using hydrogen fluoride trealment twice, And need three steps to carry out peptide chain A and peptide chain B combination, three pairs of disulfide bond use different protection groups and different deprotections Agent.This preparation technology is extremely complex, and yield is only 1.4%, the hydrogen fluoride using high toxicity and danger is required in technique, no It is adapted to large-scale production.

By the way that under pH 7 to 12 oxidizing condition free-cysteine form will be reduced in patent US-A-4835251 A chains and the B peptide chains for reducing free-cysteine form mix to produce people's relaxain with bioactivity in an aqueous medium H2.But due to the water-soluble extreme difference of B peptide chains, it is difficult to form correct interchain disulfide bond in A peptide chains and B peptide interchains.

Barlos KK etc.（J Pept Sci. 2010 Apr;16(4):200-11）Reported in 2010 and use Fmoc Method synthesizes peptide chain A and peptide chain B, and the side chain of cysteine uses same protection group, and disulfide bond is formed using random groups are legal.But this Method can not still effectively improve the yield of relaxain 2, and yield is only 48%.And peptide chain B synthesis technique is complicated, is unfavorable for work Industry metaplasia is produced.

The A of patent CN 102180964 are disclosed prepares the solid phase synthesis process of relaxain -2 by peptide chain A and peptide chain B links. But because of the frequent mispairing of interchain disulfide bond and caused by yield it is relatively low, and need to repeatedly purify, technique is still more complicated, is unfavorable for industry Metaplasia is produced.

2nd, gene engineering research

Utilize the report of recombination expression people's relaxain H2 precursors such as Escherichia coli and yeast both at home and abroad at present.Ding Chuantian Preliminary expression has been carried out in Escherichia coli Deng to people's relaxation precipitinogen（China's experiment and clinical virology magazine, volume 1997,11 the 4 phases, 319-321）, but it does not carry out the purifying of relaxation precipitinogen, and relaxation precipitinogen is not converted into relaxain yet and activity is carried out Determine.Chen Liming etc. realizes people's relaxation precipitinogen H2 expression in Escherichia coli, but relaxation precipitinogen is not carried out into protease Cut and be converted into relaxain H2, protein active is unknown, and the method yield is only 2-3mg/L.Patent US005759807A is disclosed A kind of utilization Escherichia coli Prepare restructuring people's relaxain H2 method, but because it uses MKKNIAFLIK in the method（Or R）R（Or K）, and C peptide chain KRKPTGYGSRKKR, cause K（Or R）- B0 and K（Or R）The generation of-A0 accessory substances, causes pancreas egg White enzyme digestion condition is not easily controlled the complexity with subsequent purification.Han Jia Shans etc.（Products in China magazine 2012, vol.25 No.10）And the A of patent CN 102603888 prepare people relaxain H2 using yeast expression system, its peptide chain C is designed as NGFNG, C peptide chains are cut off using azanol, but this technique causes peptide chain B and A to remain N and G respectively, and relaxain H2 biology is lived Property has a certain degree of influence, as probably due to causing higher structure with the difference of natural acid sequence in primary structure It is incorrect, and then have influence on activity；It may also be caused due to different from the amino acid sequence that human body is natural in animal or human body Drug action and toxic action change.The A of patent CN 102643825 disclose one kind and prepare relaxain using Pichia pastoris The method of wild type and mutant, the method is respectively in 6 histidine peptide chains of N-terminal and B peptide chains junction, and B peptide chains connect with C peptide chains Place is met, and C peptide chains devise three factor protease cutting sites with A peptide chains junction, cause the relaxain B chains of maturation Aminoterminal has an additional glycine, and c-terminus will have additional glycine-arginine, will have one in the aminoterminal of A chains Additional glycine, and relaxain activity is not determined further.And due to the limitation of yeast expression system in itself, Such as slow-growing, the production cycle is longer, and yield is relatively low, causes production cost high, and industrialization effect is unsatisfactory.

The content of the invention

The present invention includes the theme described in the following paragraph arranged in order：

1. a kind of recombinant human relaxin element H2 precursors, B-C-A orders are pressed using artificial C peptides by people's relaxain H2 B chains and A chains Connection, it is characterised in that described artificial C peptides have the amino acid sequence shown in SEQ ID NO any one of 1-4.

2. people's relaxain H2 precursors described in paragraph 1, it is characterised in that people's relaxain H2 B chains and A chains is native sequences Or its function mutation sequence.

3. people's relaxain H2 precursors described in paragraph 1, it is characterised in that connect leader peptide S, leader peptide bag in the N sections of B chains Protein sequence containing purification tag.

4. people's relaxain H2 precursors described in paragraph 3, it is characterised in that the C-terminal of the leader peptide S can also include connects chain.

5. people's relaxain H2 precursors described in paragraph 4, it is characterised in that last amino acid of the connects chain is smart ammonia Sour R or lysine K.

6. people's relaxain H2 precursors described in paragraph 5, it is characterised in that the connects chain is SSGR or SSGK sequences.

7. people's relaxain H2 precursors described in paragraph 6, it is characterised in that the leader peptide S has shown in SEQ ID NO 7 Amino acid sequence.

8. a kind of nucleic acid oligomer sequence, it encodes the recombinant human relaxin element H2 precursors described in paragraph any one of 1-7.

9. the nucleic acid oligomer sequence described in paragraph 8, it is characterised in that with the nucleotide sequence shown in SEQ ID NO 6.

10. a kind of carrier, it includes the nucleic acid oligomer sequence described in paragraph 8 or 9.

11. a kind of host cell, it includes the carrier described in paragraph 10 or the nucleic acid oligomer sequence described in paragraph 8 or 9.

12. the preparation method of the recombinant human relaxin element H2 precursors described in paragraph any one of 1-7, it includes culture paragraph 11 institute The step of host cell and the separation albumen for stating.

13. the preparation method of people's relaxain H2 precursors described in paragraph any one of 1-7, it is characterised in that：

1）Obtain recombination：Obtain the nucleic acid oligomer sequence of expression people's relaxain H2 precursors；

2）Obtain recombinant vector：The nucleic acid oligomer sequence insertion vector plasmid that previous step is obtained, is obtained containing restructuring base The recombinant vector of cause；

3）Obtain genetic engineering bacterium：The recombinant vector conversion host cell that previous step is obtained obtains genetic engineering bacterium；

4）Expression alien gene：Engineering bacteria fermentation expresses recombinant human relaxin element H2 precursor proteins；

5）Collect precursor protein：Thalline is cracked, recombinant human relaxin element H2 precursor proteins are collected；

6）Purify destination protein：Leader peptide S, C peptide is removed by protease digestion, and obtains ripe through chromatography purification step People's relaxain H2 albumen.

14. the preparation method of people's relaxain H2 precursors described in paragraph 13, it is characterised in that the nucleic acid oligomer sequence tool There is the sequence shown in paragraph 8 or 9.

15. ripe people's relaxain H2 albumen made from paragraph 13 or 14.

16. a kind of pharmaceutical composition, ripe people's relaxain H2 albumen described in its paragraph 15 comprising medicine effective quantity.

17. ripe people's relaxation fibroin described in paragraph 15 is preparing treatment angiocardiopathy, lung disease, fibrotic disease Application in the medicine of disease or kidney diaseases.

18. the application described in paragraph 17, the angiocardiopathy includes coronary heart disease, acute coronary artery syndrome, heart failure and the heart Muscle infarction.

To solve in existing Prepare restructuring people relaxain H2 techniques by during relaxation precipitinogen digestion obtains relaxain H2 There is the problem of residual amino is sour in albumen n end or C-terminal, and the problems such as renaturing inclusion bodies, the invention provides a kind of new people The B chains and A chains of people's relaxain H2 precursors are linked in sequence by relaxain H2 precursors, the precursor using artificial C peptides by B-C-A, this hair Bright artificial C peptides have the amino acid sequence shown in SEQ ID NO any one of 1-4.In the present invention, people's relaxain H2 B chains and A chains can be native sequences, or its function mutation sequence.Function mutation sequence refer to native sequences have 90% and Above homology and its ripe body to body circulation and the dilative mutant of renal blood vessels tool.As needed, can be in each chain Protease cleavage site is designed between section, the unnecessary sequence for cutting off.Protease may be selected from trypsase, fibrin ferment, intestines and swash Enzyme and the Xa factors etc..

Under preferable case, people's relaxain H2 precursors of the invention connect leader peptide S in the N-terminal of B chains, and leader peptide includes purifying Label protein sequence.The purification tag can be histidine-tagged, glutathione s-transferase（GST）Or FLAG^®.Histidine Label is conventional purification tag, can be purified by nickel affinity chromatography.It is common it is histidine-tagged be 6 × His（His- Tag）.For enhancing affinitive layer purification effect, it is of the invention use it is histidine-tagged be 10 × His（His-Tag）.More excellent In the case of choosing, leader peptide S of the invention C-terminal can also include connects chain, and last amino acid of connects chain is preferably smart ammonia Sour R.In a more preferred case, connects chain is SSGR sequences.This connection chain-ordering is flexible preferably, can make histidine-tagged Fully exposure, while arginine R accurately can be recognized and cut by trypsase, cuts off leader peptide S sequences completely.Most preferred In the case of, leader peptide S has the amino acid sequence shown in SEQ ID NO 7.

No 7

MGHHHHHHHHHHSSGR

Present invention also offers a kind of nucleic acid oligomer sequence, it encodes recombinant human relaxin described in the case of any of the above-described kind Plain H2 precursors.According to the codon preference of different expression systems, nucleotide sequence can be optimized.Under preferable case, oligomerization Nucleic acid has the nucleotide sequence shown in SEQ ID NO 6.

Present invention also offers a kind of carrier, it includes nucleic acid oligomer sequence described above.

Present invention also offers a kind of host cell, it includes above-mentioned carrier or above-mentioned nucleic acid oligomer sequence.

Present invention also offers a kind of preparation method of recombinant human relaxin element H2 precursors, it includes the above-mentioned host cell of culture The step of with the albumen is separated.The step of protein isolate, includes affinity chromatography, cation exchange, reversed phase chromatography etc..

Specifically, the preparation method of people's relaxain H2 precursors, comprises the following steps：

1）Obtain recombination：Obtain the nucleic acid oligomer sequence of expression people's relaxain H2 precursors；

2）Obtain recombinant vector：The nucleic acid oligomer sequence insertion vector plasmid that previous step is obtained, is obtained containing restructuring base The recombinant vector of cause；

3）Obtain genetic engineering bacterium：The recombinant vector conversion host cell that previous step is obtained obtains genetic engineering bacterium；

4）Expression alien gene：Engineering bacteria fermentation expresses recombinant human relaxin element H2 precursor proteins；

5）Collect precursor protein：Thalline is cracked, inclusion body is collected and washs, inclusion body passes through Ni parents after denaturation, renaturation With chromatography collector's relaxain H2 precursor proteins；

6）Purify destination protein：Leader peptide S, C peptide is removed by protease digestion, and obtains ripe through chromatography purification step People relaxation fibroin.

Under preferable case, it is connected in order that chromatography purification step includes cation chromatography, reversed phase chromatography and cation chromatography etc. The step of.

Present invention also offers the ripe people's relaxain H2 albumen obtained by above-mentioned preparation method.

Present invention also offers a kind of pharmaceutical composition, it includes the maturation obtained by the above method of medicine effective quantity People relaxation fibroin.

The cardiovascular disease for the treatment of is being prepared present invention also offers ripe people's relaxation fibroin that above-mentioned preparation method is obtained Application in disease, lung disease, the medicine of fibrotic conditions or kidney diaseases.The angiocardiopathy includes coronary heart disease, acute coronary Syndrome, heart failure and miocardial infarction.

Beneficial effects of the present invention：Compared with prior art, relaxain H2 precursor fusion proteins and adult form in the present invention Relaxain H2 albumen preparation technology is easy, and yield is high.The C peptides design of innovation and the design of flexible connection peptide chain cause purpose egg High efficient expression can be obtained in vain, while inclusion body is easier to renaturation；The artificial C peptides that the present invention is provided so that relaxain H2 originals are melted Hop protein maturation is more efficient for the digestion process of relaxain H2 albumen, it is to avoid the productions of Arg-A0-relaxain H2 accessory substances It is raw, and the ripe body relaxain H2 albumen obtained has more preferable activity.Design causes relaxain H2 purifying process above More succinct efficiently yield is improved, and is preferably controlled while preparing cost.

Brief description of the drawings

Fig. 1 people's relaxain H2 original albumen primary structure figures；

Fig. 2 people relaxain H2 precursor fusion proteins express PAGE gel electrophoretogram；

Fig. 3 people's relaxain H2 precursor fusion protein amino acid sequence figures；

Fig. 4 trypsase single endonuclease digestion people's relaxain H2 precursor fusion protein RP-HPLC collection of illustrative plates；

Fig. 5 relaxain H2 albumen stoste purity RP-HPLC detects collection of illustrative plates；

Fig. 6 relaxain H2 albumen external activities Elisa is determined.

Embodiment

The present invention is using the method for genetic engineering, the new artificial C peptide sequences of design, and by the C peptides by people's relaxain H2 B chains and A chains being linked in sequence according to B-C-A of precursor, obtain people's relaxain H2 precursor proteins of restructuring.After the protein expression, C peptides are removed by proteolytic cleavage, ripe people's relaxain H2 is obtained.

Specifically, a kind of people's relaxain H2 precursor proteins, are constituted, wherein institute from N-terminal to C-terminal by tetra- sections of peptide chains of S-B-C-A It is nothing or leader peptide to state S peptide chains.The sequence of C peptide chains is selected from SEQ ID NO 1-4 any bar sequence, and particular sequence is as follows：

No 1

RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQSR

No 2

RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQGR

No 3

RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQVR

No 4

RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR

In order to efficiently separate purifying or secretion target protein, leader peptide sequences usually include the label for being easy to isolate and purify Albumen or tag polypeptide（Tag）.Conventional has glutathione-S-transferase（glutathione S-transferase, GST), hexahistine peptide（His-Tag), a-protein (protein A) and cellulose binding site (cellulose binding domain) etc..The shape of fusion protein is made up of with target protein particularity albumen or polypeptide Formula, can be separated and be purified to target protein using the special nature of described label protein or tag polypeptide after expression.Such as His-Tag is specifically bound with Ni-Chelating Sepharose posts.Described label protein or tag polypeptide can be pure Fusion sequence is removed with site-specific protease digestion after change, fibrin ferment, enterokinase and the Xa factors is such as can use, to obtain Target protein.And recognize specific and more preferable exposed purification tag, leader peptide sequences to strengthen the cleavage site of protease It may also comprise connects chain.In the present invention, the end amino acid of connects chain is preferably arginine R, and connection chain-ordering is preferably SSG.B peptide chains and A peptide chains can be people's relaxain H2 native sequences, or its function mutation sequence.Function mutation sequence Refer to that there is 90% and above homology and its ripe body to body circulation and renal blood vessels have dilative with native sequences Mutant.Under preferable case, the amino acid sequence of A peptide chains and B peptide chains is as shown in SEQ ID No8 and 9, and particular sequence is as follows：

The A chains of No 8

DSWMEEVIKLCGRELVRAQIAICGMSTWS

The B chains of No 9

QLYSALANKCCHVGCTKRSL ARFC

In a specific embodiment, its amino acid sequence of S-B-C-A peptide chains is as shown in SEQ ID NO. 5.Meanwhile, Those skilled in the art it can be appreciated that with the amino acid sequence shown in SEQ ID NO. 5 have 90% or more homology and its into Ripe body to body circulation and renal blood vessels have dilative mutant, also within protection scope of the present invention.

No 5

MGHHHHHHHHHHSSGRDSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQVGQVELGGGPGAGSLQ PLALEGSLQSRQLYSALANKCCHVGCTKRSLARFC

During protein renaturation, people's relaxain H2 precursors of the denaturation of acquisition are 8.5-11 in pH and there is low concentration Denaturant and redox couple existence condition under, low temperature renaturation.During renaturation, fusion protein concentration is controlled in 0.5-2mg/ml； Renaturation temperature is 4 DEG C -15 DEG C；It is preferred that denaturant be urea, concentration is 0.5-2M.Redox couple is usually mercaptan and oxidation Type mercaptan dimer；Dithiothreitol (DTT), dithioerythritol, cysteine, reduced form with strong reducing property may be selected in mercaptan Glutathione, β mercaptoethanols；The optional oxidized form of glutathione of oxidized form mercaptan dimer, cystine.In the present invention, mercaptan With oxidized form mercaptan dimer ratio（Mol ratio）For 1:5-1:10.

It will be further described below by specific embodiment.It should be appreciated, however, that specific embodiment is only used for solution The present invention is released, the protection domain being not intended to limit the present invention.The instrument use herein arrived, equipment, reagent, method etc. as Do not specialize, be instrument commonly used in the art, equipment, reagent and method.

The people's relaxain H2 precursor fusion proteins expression system construction of embodiment 1 and expression identification

Artificial synthesized people's relaxain H2 precursor fusion protein gene orders RLXH2（Invitrogen is synthesized）, from 5 ' end to 3 ' ends include the leading peptide gene sequence with 10*His label proteins, relaxain H2 peptide chain 1 B genes sequence, peptide chain C bases successively Because of sequence and peptide chain A gene orders；Wherein 5 ' ends introduce NcoI restriction enzyme sites, and 3 ' ends introduce the strong terminators of TAATAA and XhoI enzymes Enzyme site；People's relaxain H2 precursor protein genes sequence of this synthesis is optimized according to e. coli codon preferences, excellent Sequence after change（SEQ ID No6）It is as follows：

The gene order of artificial synthesized people's relaxain H2 precursor fusion proteins is inserted into pUCK carriers, obtained PUCK-RLXH2 plasmids.

By RLXH2 gene clonings into pET28a (+) carrier, pET-RLXH2 prokaryotic expression carriers, RLXH2 genes are built Under regulation and control in T7 strong promoters and RBS sequences, high efficient expression can be obtained, structure is comprised the following steps that：By pUCK- RLXH2 plasmids carry out NcoI and XhoI double digestions respectively with pET-28a (+) plasmid；Agarose gel electrophoresis is separated, and glue reclaim RLXH2 genetic fragments and pET28a carrier segments；By T4 DNA ligases, after genetic fragment is connected with carrier segments, Routine transformation DH5 α competent cells, obtain people's relaxain H2 precursor fusion protein expression plasmids pET-RLXH2.Extract after plasmid Gene sequencing is carried out, correct strain and plasmid is retained.

By plasmid pET-RLXH2 routine transformation host e. colis BL21（DE3）, pass through card that resistance LB flat boards, screening Obtain positive clone molecule pET-RLXH2/BL21 (DE3).

Picking pET-RLXH2/BL21 (DE3) positive clone molecule is inoculated in LB fluid nutrient mediums（Containing 50 μ g/ml Kan⁺）, 150rpm, 37 DEG C of culture 12h；Subsequent 1:50（V/V）Switching LB fluid nutrient mediums（Kan⁺）, 200rpm, 37 DEG C of cultures；Treat bacterium solution When OD600 reaches 0.8, IPTG to final concentration 0.5mM is added, in 200rpm, 37 DEG C of cultures, induced fusion protein expression.Induction 4 After hour, thalline is collected；Ultrasonication thalline, 12000rpm centrifugations, collects bacteria break supernatant and precipitation respectively.Pass through 15% SDS- PAGE electrophoresis carries out expressing quantity analysis, and target person relaxain H2 precursor fusion proteins are inclusion body, and expression quantity accounts for thalline The 15% of total protein concentration.（As shown in Figure 2）

Expressed carries histidine-tagged people's relaxain H2 precursor proteins amino acid sequence such as SEQ ID NO.5 institutes Show (as shown in Figure 3).

The people's relaxain H2 of embodiment 2 original fusion protein engineering bacterium fermentations

1）PET-RLXH2/BL21 (DE3) bacterium solution is taken, by 1:1000 are inoculated in LB fluid nutrient mediums（Containing 50 μ g/ml Kan⁺）In, 37 DEG C, 150rpm cultures.

2）When bacterium solution OD600 reaches 4, according to 1:20（V/V）Ratio is seeded to the fermentation tank containing 30L TB culture mediums In；Dissolved oxygen controls 30%, pH controls 7.0,37 DEG C of temperature control, fermentation tank speed of agitator and dissolved oxygen auto-associating.Every 30min Sampling, determines zymotic fluid OD600 values.

3）When fermentation tank bacterium solution OD600 reaches 10, final concentration of 0.5mM IPTG, induced fusion protein expression are added. Now dissolved oxygen control is in 30%, pH controls 7.0, and temperature control is at 37 DEG C, fermentation tank speed of agitator and dissolved oxygen auto-associating.Often Hour sampling, determines zymotic fluid OD600 values.

4）After induction 4 hours, stop fermenting and determining now zymotic fluid OD600 up to 25.10000rpm centrifuges zymotic fluid, Thalline is collected, thalline weight in wet base 1090g is obtained.

5）A small amount of thalline is taken, thalline is resuspended with buffer solution 20mM Tris pH8.0,0.5M NaCl, albumen loading is added and delays Fliud flushing boiling water bath 10min, 15% SDS-PAGE electrophoresis detection people's relaxain H2 precursor fusion protein expression quantity accounts for whole bacterial protein 16%。

The people's relaxain H2 precursor fusion protein renaturation of embodiment 3

1）Use buffer solution（20mM Tris pH8.0,150mM NaCl）According to 1:10（W/v, W/V）Bacterium is resuspended Body, 10000rpm centrifugation 15min, washing thalline.By thalline again with 1:10（W/V）It is resuspended, is existed using high pressure homogenizer Bacterium is broken under the conditions of 800bar twice, break bacterium rate up to more than 95%.Broken bacterium solution is centrifuged 20 minutes through 12000rpm, is obtained inclusion body and is sunk Form sediment.It is about 106g that 30L zymotic fluids, which obtain inclusion body weight in wet base,.

2）Step 1）Middle inclusion body also includes many thalline foreign proteins in addition to destination protein, can be by using low concentration denaturant Most of foreign protein is removed.It is guanidine hydrochloride or urea that denaturant, which is typically selected,.By inclusion body according to 1:20（W/V）It is resuspended in and forgives Body cleaning solution（20mM Tris pH9.0、2M Urea、1% Triton X-100、 2mM EDTA 1% Tween-20）, utilize High pressure homogenizer twice of homogeneous under the conditions of 800bar, is subsequently agitated for washing 15min, inclusion body is collected by centrifugation in 12000rpm；Weight After backwashing is washed 1 time；Inclusion body is washed with deionized water in third time, and inclusion body, gained is collected by centrifugation in agitator treating 15min, 12000rpm Inclusion body purity is up to 75%.

3）By step 2）Gained inclusion body is resuspended in inclusion body and redissolves liquid（20mM DTT, 20mM Tris, 150mM NaCl, 8M urea, pH9.0）In, adjustment people's relaxain precursor fusion protein concentration is stirred at room temperature 2h, can make to forgive in 20-40mg/ml Body protein fully dissolves.

4）By step 3）Redissolution liquid press 1:20（V/V）Ratio be slowly added into batches containing 3mM cystines and 0.5mM In 20mM Tris pH10, the 150mM NaCl solutions of cysteine, 4 DEG C are stirred renaturation 24 hours, and renaturation yield is 65%.

It is prepared by the purifying of the people relaxain H2 protein chromatographics of embodiment 4

1）The renaturation solution that embodiment 3 is obtained is purified by nickel affinity chromatography.Concretely comprise the following steps Chelating- Sepharose F.F. posts（GE, BPG100/500 chromatographic column, gel volume 1L）By level pad（20mM Tris PH8.0,50mM imidazoles, 0.5M NaCl）100 ml/min are balanced after three column volumes, and by renaturation solution loading, loading flow velocity is 100 ml/min；Use affine lavation buffer solution（20mM Tris pH8.0,100mM imidazoles, 0.5M NaCl）100 ml/min are rushed It is washed till baseline；Then use elution buffer（20mM Tris-HCl pH8.0,450 mM imidazoles, 0.5M NaCl）Elution.People pine Element H2 precursor fusion protein purity of relaxing reaches more than 80%, and protein content is 43 mg/ml, and sample volume is about 1000 after elution ml。

2）Protease double digestion removes leader peptide and C peptides：Because between leader peptide S and peptide chain B, between peptide chain B and peptide chain C, Three tryptic digestion sites are designed between peptide chain C and peptide chain A, therefore leader peptide S can remove by tryptic digestion With C peptides.Comprise the following steps that：Utilize cross-flow ultrafiltration system（Millipore MINI PELLICON）, by 1）Eluent lead to Cross 3kD film bags（Millipore 3K PLBC-C 0.1 SQM）Ultrafiltration, is replaced as tryptic digestion buffer solution（20mM Tris PH9,0.1M NaCl）, adjustment people relaxain H2 precursor fusion proteins are 10mg/ml；According to 1:1000（Weight ratio, W/W）Add Trypsase, 25 DEG C of enzymolysis 30min；Then 1U is added in digestion system according to every 2mg people's relaxain H2 precursor fusion proteins The ratio of protaminase, adds protaminase, 25 DEG C of digestion 4h, and sampling per hour carries out C18 RP-HPLC monitoring digestion processes, split Solution adds phosphoric acid to pH3.5 termination albumen enzyme effects at once when completing.

3）Cation chromatographic purifying relaxain H2：Level pad（50mM Tris, 50mM NaCl, pH=7.0）Balance SP-Sepharose F.F chromatographic columns（GE companies XK50/60 chromatographic columns, the ml of gel volume 400）, by step 2）In collection liquid Sample, flow velocity is 50 ml/min；Three cylinders of chromatographic column are cleaned with the mM Tris-HCl pH7.0 of buffer solution 20,0.1 M NaCl Product, then elutes destination protein with the mM Tris-HCl pH7.0 of buffer solution 20,0.6 M NaCl and collects eluting peak, obtains thick Pure adult form relaxain H2.

4）Reverse phase chromatography relaxain H2：Adult form people's relaxain is separated using the Source 15RPC gels of GE companies H2 albumen, chromatographic column is the FINE LINE100LP chromatographic columns of GE companies, and gel volume is 1.2 L.With buffer solution (5% acetonitrile+ 0.1%TFA) balance, by step 3）Thick pure sample liquid is diluted to 1 ~ 1.5 mg/ml loadings, then uses buffer solution（5% acetonitrile, 0.1%TFA）Balance, flow velocity is 100 ml/min；With the 20%-60% acetonihile gradient elutions containing 0.1%TFA.Analyzed through HPLC, The relaxain H2 destination proteins peak purity of collection is more than 98%.

5）Cation chromatography removes acetonitrile：Level pad（50mM Tris, 50mM NaCl, pH=7.0）Balance SP- Sepharose F.F chromatographic columns（GE companies XK50/60 chromatographic columns, the ml of gel volume 400）, by step 4）Collection liquid loading, stream Speed is 50 ml/min；Chromatographic column is cleaned with the mM Tris-HCl pH7.0 of buffer solution 20,0.1 M NaCl, acetonitrile and three is removed Fluoroacetic acid, then with the mM Tris-HCl pH7.0 of buffer solution 20,0.6 M NaCl）Elution destination protein simultaneously collects eluting peak. Analyzed through HPLC, the relaxain H2 purity of protein of collection is up to more than 95%（Fig. 5）, it is pine after 0.22 μm of filter aseptic filtration Relaxation element H2 stostes, are computed relaxain H2 concentration more than 2 mg/ml, yield is about 2.65 g, and final products, which meet medicine, to be declared Quality standard.

Other the artificial C peptides of embodiment 5

According to above-described embodiment, the people's relaxain H2 precursors connected by different C peptides are prepared respectively, and sequence is as follows（Underscore Part is C peptide sequences）：

79S people's relaxain H2 is former：

MGHHHHHHHHHHSSGRDSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQVGQVELGGGPGAGSLQ PLALEGSLQSRQLYSALANKCCHVGCTKRSLARFC

79G people's relaxain H2 is former：

MGHHHHHHHHHHSSGRDSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQVGQVELGGGPGAGSLQPLALEGSL QGRQLYSALANKCCHVGCTKRSL ARFC

79V people's relaxain H2 is former：

MGHHHHHHHHHHSSGRDSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQVGQVELGGGPGAGSLQ PLALEGSLQVRQLYSALANKCCHVGCTKRSLARFC

79A people's relaxain H2 is former：

MGHHHHHHHHHHSSGRDSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQVGQVELGGGPGAGSLQ PLALEGSLQARQLYSALANKCCHVGCTKRSLARFC

Proteolytic cleavage result shows that four kinds of C peptides can eliminate wild type C peptides makes one relaxation precipitinogen H2 in protease cracking The Arg-A0- people's relaxain accessory substance produced in maturation.Trypsase single endonuclease digestion actrapid monotard H2 original fusion proteins, product Relaxain H2 corresponding position HPLC are unimodal（Fig. 4).In these four novel C peptides, preferably 79S-C peptides, its expression quantity compared with It is other three kinds high.Annealing efficiency and digesting efficiency are excellent compared with other three kinds（Table 1).

The recombinant mature body people's relaxain H2 determinations of activity of embodiment 6

Use RPMI1640 complete mediums（Containing 10% FBS, 0.05mM 2-Mercaptoethand, 0.11g/L acetone Sour sodium）THP-1 cells are cultivated, the cell in exponential phase is inoculated in 96 porocyte culture plates, per the μ l of hole 180（1× 10⁵Cells/well）, cultivate after 12h, add through sample diluting liquid（PRIM1640 basal mediums, 1% BSA, 100mM IBMX, 10μM Forskolin）The restructuring relaxain H2 obtained by 2 doubling dilution embodiments 4, per the μ l of hole 20, in CO₂37 DEG C of incubator 20 min are cultivated, culture medium supernatant is removed after 600g centrifugations.100 μ l cell pyrolysis liquids are added per hole（0.1M HCl, 0.5% Triton-X100）37 DEG C of cell lysis 30min, 600g centrifugation 5min.The μ l of lysate supernatant 50 are taken, by competing Elisa methods, Detect that each hole cAMP contains using Monoclonal Anti-cAMP Antibody Based Direct cAMP detection kits Amount.Elisa detections data analyzes through four parametric regressions, and calculatings ED50 is 0.3ng/ml, recombinate that relaxain H2 is active and existing system Preparation Method is compared to more excellent（Such as Fig. 6）

Sequence table

<110>Shenzhen occasion riel bio tech ltd

<120>A kind of people's relaxain H2 precursors and its preparation method and application

<130> 2014HBWT003

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 1

Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly

1 5 10 15

Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

20 25 30

Gln Ser Arg

35

<210> 2

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 2

Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly

1 5 10 15

Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

20 25 30

Gln Gly Arg

35

<210> 3

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 3

Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly

1 5 10 15

Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

20 25 30

Gln Val Arg

35

<210> 4

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 4

Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly

1 5 10 15

Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

20 25 30

Gln Ala Arg

35

<210> 5

<211> 101

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 5

Met Gly His His His His His His His His His His Ser Ser Gly Arg

1 5 10 15

Asp Ser Trp Met Glu Glu Val Ile Lys Leu Cys Gly Arg Glu Leu Val

20 25 30

Arg Ala Gln Ile Ala Ile Cys Gly Met Ser Thr Trp Ser Arg Arg Glu

35 40 45

Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly

50 55 60

Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Ser Arg

65 70 75 80

Gln Leu Tyr Ser Ala Leu Ala Asn Lys Cys Cys His Val Gly Cys Thr

85 90 95

Lys Arg Ser Leu Ala Arg Phe Cys

100

<210> 6

<211> 326

<212> DNA

<213> Artificial Sequence

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ccatgggcca tcatcatcat catcatcatc atcatcacag cagcggccgt gactcttgga 60

tggaagaagt tatcaaactg tgcggtcgtg aactggttcg tgctcagatc gctatctgcg 120

gtatgtctac ctggtctcgt cgtgaagctg aagacctgca ggttggtcag gttgaactgg 180

gtggtggtcc gggtgctggt tctctgcagc cgctggctct ggaaggttct ctgcagtctc 240

gtcagctgta ctctgctctg gctaacaaat gctgccacgt tggttgcacc aaacgttctc 300

tggctcgttt ctgctaataa ctcgag 326

<210> 7

<211> 16

<212> PRT

<213> Artificial Sequence

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Met Gly His His His His His His His His His His Ser Ser Gly Arg

1 5 10 15

<210> 8

<211> 29

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 8

Asp Ser Trp Met Glu Glu Val Ile Lys Leu Cys Gly Arg Glu Leu Val

1 5 10 15

Arg Ala Gln Ile Ala Ile Cys Gly Met Ser Thr Trp Ser

20 25

<210> 9

<211> 24

<212> PRT

<213> Artificial Sequence

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Gln Leu Tyr Ser Ala Leu Ala Asn Lys Cys Cys His Val Gly Cys Thr

1 5 10 15

Lys Arg Ser Leu Ala Arg Phe Cys

20

Claims (7)

1. a kind of recombinant human relaxin element H2 precursors, are connected people's relaxain H2 B chains and A chains by B-C-A orders using artificial C peptides Connect, it is characterised in that described artificial C peptides have the amino acid sequence shown in SEQ ID NO any one of 1-4, people's relaxain H2 Precursor is the sequence shown in following any bar：

MGHHHHHHHHHHSSGRDSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQVGQVELGGGPGAGSLQPLAL EGSLQSRQLYSALANKCCHVGCTKRSLARFC；

MGHHHHHHHHHHSSGRDSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQVGQVELGGGPGAGSLQPLAL EGSLQGRQLYSALANKCCHVGCTKRSL ARFC；

MGHHHHHHHHHHSSGRDSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQVGQVELGGGPGAGSLQPLAL EGSLQVRQLYSALANKCCHVGCTKRSLARFC；

MGHHHHHHHHHHSSGRDSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQVGQVELGGGPGAGSLQPLAL EGSLQARQLYSALANKCCHVGCTKRSLARFC；

Wherein MGHHHHHHHHHHSSGRD is leader peptide S sequences.

2. a kind of nucleic acid oligomer sequence, it encodes any bar people's relaxain H2 precursors described in claim 1.

3. the nucleic acid oligomer sequence described in claim 2, it is characterised in that with the nucleotide sequence shown in SEQ ID NO 6.

4. a kind of carrier, it includes the nucleic acid oligomer sequence described in Claims 2 or 3.

5. a kind of host cell, it includes the carrier described in claim 4 or the nucleic acid oligomer sequence described in Claims 2 or 3.

6. the preparation method of the recombinant human relaxin element H2 precursors described in claim 1, it includes cultivating the place described in claim 5 The step of chief cell is with the recombinant human relaxin element H2 precursors described in claim 1 are separated.

7. the preparation method of people's relaxain H2 precursors described in claim 1, it is characterised in that：

1）Obtain recombination：The nucleic acid oligomer sequence of expression people's relaxain H2 precursors is obtained, the nucleic acid oligomer sequence has Sequence shown in Claims 2 or 3；

2）Obtain recombinant vector：By step 1）The nucleic acid oligomer sequence insertion vector plasmid of acquisition, acquisition contains recombination Recombinant vector；

3）Obtain genetic engineering bacterium：By step 2）The recombinant vector conversion host cell of acquisition obtains genetic engineering bacterium；

4）Expression alien gene：Engineering bacteria fermentation expresses recombinant human relaxin element H2 precursor proteins；

5）Collect precursor protein：Thalline is cracked, recombinant human relaxin element H2 precursor proteins are collected；

6）Purify destination protein：Leader peptide S, C peptide is removed by protease digestion, and through the ripe people of chromatography purification step acquisition Relaxain H2 albumen.