CN102392041B

CN102392041B - Preparation method of recombinant human corticotropin releasing factor

Info

Publication number: CN102392041B
Application number: CN 201110405033
Authority: CN
Inventors: 张伟; 辛渝; 李树刚; 于廷和; 但国平; 龚会英; 柴新娟; 李晓丽; 曹莉君; 张勇
Original assignee: CHONGQING KERUN BIOMEDICAL R&D Co Ltd
Current assignee: CHONGQING KERUN BIOMEDICAL R&D Co Ltd
Priority date: 2011-12-08
Filing date: 2011-12-08
Publication date: 2013-09-18
Anticipated expiration: 2031-12-08
Also published as: CN102392041A

Abstract

The invention relates to a preparation method of a recombinant human corticotropin releasing factor, characterized in that: by artificially synthesizing a gene sequence of human corticotropin releasing factor mature peptide, the gene sequence is inserted into an escherichia coli expression vector with a thioredoxin label, the target gent fuses with the thioredoxin 3'end, the central part of the vector contains an associated peptide and an enterokinase recognition site, and the expression vector is converted into appropriate escherichia coli to obtain genetic engineering bacteria, the genetic engineering bacteria is subject to optimized high-density fermentation and purification to prepare Trx-CRF fusion protein, then the Trx-CRF fusion protein is subject to enterokinase cutting, and then separation and purification are carried out to obtain the recombinant human corticotropin releasing factor. The prepared recombinant human corticotropin releasing factor has the advantages of high purity, good activity, and low production cost.

Description

A kind of preparation method of CRF

Technical field

The present invention relates to gene engineering technology field, more specifically, relate to a kind of method of utilizing gene recombination technology to prepare the biologically active polypeptides of CRF.

Background technology

Cerebral edema is the severe complication in intracranial tumors and the therapeutic process thereof, and also normal the generation and other kinds cranium disease of brain and damage is the one of the main reasons that causes disordered brain function.Many pathologic processes such as anoxic, wound, infarct, the cerebral edemas that all can occur together such as inflammation poisoning.Can cause a series of physiology and mental symptoms, such as epilepsy, muscle is weak, lacks Harmony and diplopia etc.China's cerebral tumor mortality ratio is arranged the 7th in malignant tumour, and the trend that rises is year by year arranged, and it is 1/10000 that the cerebral tumor sickness rate is sent out in its Central Plains, and the male sex is more than the women.And children's brain tumor sickness rate constantly rises in recent years, has become at present to be only second to the second largest tumour of leukemic children, and sickness rate accounts for 22% of all tumours of children.And finally having 20-30%, other malignant tumours change encephalic over to, the serious harm people's health.

The medicine for the treatment of clinically tumor vessel source property cerebral edema now mainly contains dewatering agent, and diuretic(s) and glucocorticosteroid can alleviate cerebral edema to a certain extent, but in various degree side effect is all arranged.The above two are mainly in kidney function damage and electrolyte disturbance, such as N.F,USP MANNITOL rebound phenomena are arranged, and have Nephrotoxicity, unsuitable life-time service.Heavy dose of cortin, effect is remarkable, but the side effect that produces is very large, can cause patient's hormonal readiness disorderly, aggravation, and diabetes, osteoporosis, passive protective physical fitness descends, and affects development in children etc., therefore is badly in need of seeking the medicine that substitutes hormone.

CRF (corticotropin releasing factor, CRF) is the hormone in a kind of hypothalamus, and it regulates the release of thyroliberin in the pituitary gland in vivo.Research finds that CRF is the inhibitor of mediated brain oedema material, have very strong Ivy extract effect, can alleviate vasogenic brain edema, can substitute adrenocortical hormone, both substantial curative effect can be obtained, the side effect of life-time service cortin can be avoided again.CRF directly acts on the tumor-microvessel structure, the link of reparation hemato encephalic barrier, raise the expression of hemato encephalic barrier differential protein, studies have shown that the substitute that can be used as steroid hormone in the cerebral edema treatment, side reaction (the Tjuvajev et al. that avoids long-term, high-dose to use hormone to cause, Cancer res, 1996,56:1352).CRF both can obtain substantive curative effect, can avoid again the side effect of life-time service cortin.

At present, XERECEPT chemosynthesis CRF by name by Neurobiological Tech exploitation goes on the market, it is in treatment and reduce oedema, improve nervous symptoms, the aspects such as clinical safety are respond well, mainly are by chemosynthesis, and cost is higher, and affected by technical reason, the scale operation difficulty is large.Therefore, in the urgent need to developing a kind of method of new this medicine of preparation.

Utilize genetic engineering technique, take microorganism as carrier, produce foreign protein and have broad application prospects.Bode Gene Development Co., Ltd., Shanghai (Chinese patent CN01112728.7, CN00136349.2, CN00116452.X, CN00111619.3, CN00111617.7, CN99125755.3) has announced corticotropin releasing factor(CRF) 12.87, corticotropin releasing factor(CRF) 8.8, corticotropin releasing factor(CRF) 10, corticotropin releasing factor(CRF) 23 and corticotropin releasing factor(CRF) 26, with and recombinant expression method.

The recombinant expressed technique of short biologically active peptides is a difficult problem, generally need to by the amalgamation and expression companion, cut fusion tag with special protein incision enzyme.But in actually operating, fusion protein molecule is more much larger than the small peptide that will express, can form sterically hinderedly, often causes the protein incision enzyme cutting efficiency low, perhaps cuts not open at all.

Summary of the invention

The invention provides a kind of preparation method of CRF, the CRF of the method preparation has high expression level amount and yield, and the protein incision enzyme cutting rate is high, and the low advantage of production cost is arranged.

The preparation method of CRF of the present invention has following steps:

1) designs and synthesizes the CRF gene according to the hCRF aminoacid sequence;

2) step 1) gained gene is by the Trx (Thioredoxin on connecting peptide, enteropeptidase recognition site and the suitable carriers; Trx) Trx protein fusion obtains expression vector pET32-CRF;

3) step 2) the expression vector pET32-CRF of gained is transformed into suitable e. coli bl21 (DE3) and obtains CRF engineering bacteria PET32-CRF/BL21 (DE3);

4) the CRF engineering bacteria PET32-CRF/BL21 (DE3) that obtains of step 3) prepares the Trx-CRF fusion rotein through fermentation, purification step;

5) the Trx-CRF fusion rotein that obtains of step 4) is after the enteropeptidase cutting, and separation and purification obtains CRF (rhCRF).

Step 2) building mode of described expression vector is :-Trx-connecting peptide-DDDDK-CRF-, wherein-and DDDDK-is the enteropeptidase recognition site, and D is aspartic acid, and K is Methionin.

The N of enteropeptidase recognition site end introduces structure and is-(GGGGS) flexible peptide linker of n-, and wherein G is glycine, and S is Serine, and n is 0-10.The preferred n of the present invention is 3.

The N terminal sequence that people's thyroliberin discharges gene is SEEPPI-, and wherein S is Serine, and E is L-glutamic acid, and P is proline(Pro), and I is Isoleucine.

The dna sequence dna of the described CRF of step 5) is shown in SEQ ID NO:1.

The corresponding encoding amino acid sequence of the described CRF of step 5) is shown in SEQ ID NO:2.

The described fermentation of step 4) is CRF engineering bacteria PET32-CRF/BL21 (DE3) to be inoculated into carry out the high density fermentation cultivation in the fermentor tank, induces the expression of fusion rotein by adding IPTG.

The step of the described purifying of step 4) comprises centrifugal collection thalline, and broken bacterium, the broken bacterium liquid supernatant of centrifugal collection will be collected Chelating Sepharose Fast Flow chelating chromatography on the supernatant, flush away foreign protein, wash-out purpose fusion rotein.

The described separation and purification of step 5) is Chelating Sepharose Fast Flow chelating chromatography on the product after enzyme is cut, foreigh protein removing, wash-out target protein.

Above-mentioned steps 1) the synthetic CRF gene in is prior art, can obtain by prior art, such as the method for synthetic.

Step 2) protein gene of Trx described in is that the commercialization carrier carries gene order, wherein is connected by Trx-(GGGGS) n-DDDDK-CRF mode between Trx and the CRF.

The preferred CRF DNA sequences encoding of the present invention is nucleotide sequence shown in the SEQ ID NO:1.

RhCRF aminoacid sequence of the present invention is sequence shown in the SEQ ID NO:2.

Preparation method of the present invention, it is characterized in that, wherein said fermentation is that the CRF engineering bacteria that will meet the demands is inoculated into and carries out high density fermentation in the fermentor tank and cultivate, induce the expression of fusion rotein by adding IPTG, gained rhCRF purity reaches more than 95%.

The present invention is according to the CRF mature peptide amino acid coding of having delivered (this sequence can be carried out same sense mutation to the Preference principle that genetic code uses according to versatility, degeneracy and the intestinal bacteria of genetic code), synthetic gene sequence.Principal feature of the present invention is, this gene clone is entered suitable expression vector (commercialization carrier, preferably as: make up recombinant expression plasmid pET-32a-c (+)), then the intestinal bacteria that recombinant plasmid transformed suited, preferably such as commercialization bacterial strain: BL21 (DE3), the genetic engineering bacterium with high expression level ability that obtains through screening.This project bacterium is take Trx as fusion partner, amalgamation and expression forms the Trx-CRF fusion rotein, after the step separation and purification that expressed albumen process suits, with the suitable proteolytic enzyme with distinguished sequence recognition capability (such as enteropeptidase) CRF is cut down from fusion rotein, through consummateization, obtain the rhCRF sterling.Through analyses such as N-end sequencing, mass spectrum molecular weight determination, isoelectric point determination, peptide figure analysis and biological activity determinations, the result shows that rhCRF that preparation technology of the present invention obtains and natural CRF are in full accord.

Preparation method of the present invention during amalgamation and expression, introduces flexible connecting peptide before the protein endoenzyme enzyme recognition site, solved the low problem of protein incision enzyme cutting efficiency, greatly improves the rate of recovery, and reduces production costs.This preparation method's CRF purity is high, activity is good, production cost is low, but large-scale industrial production CRF.

Advantage of the present invention is mainly manifested in:

(1) the Trx-CRF fusion rotein exists with soluble form among the present invention, by the enteropeptidase cutting, greatly improves expression amount and yield;

(2) introduce the n-of flexible peptide linker-(GGGGS) at the N of enteropeptidase recognition site end, solved the low problem of cutting efficiency, greatly improve the rate of recovery, and reduce production costs;

(3) rhCRF that makes by the inventive method, detect through national biological medicine analytic centre, its physicochemical property and biological activity and CRF are in full accord, the N terminal sequence is consistent with the natural human corticotropin releasing factor(CRF), avoid common escherichia coli expression recombinant protein medicine N end to have more a Met even a plurality of amino-acid residue, thereby reduce its immunogenicity, improved it and become the property of medicine.

The reagent that the present invention uses is the medical agent of market sale; Expression vector, Host Strains are commercialization carrier or the bacterial strain that can buy from the market.

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

Description of drawings

Fig. 1 is pET32-CRF construction of recombinant expression plasmid schematic diagram.

Fig. 2 is the SDS-PAGE electrophorogram behind the rhCRF purifying.Swimming lane 1 is albumen Marker; Swimming lane 2 is sample behind the Trx-CRF fusion protein purification;

Swimming lane

3,4 is Trx-CRF fusion rotein sample after the enteropeptidase enzyme is cut; Swimming lane 5 is sample behind the rhCRF purifying.

Fig. 3 is that the HPLC of sample behind the rhCRF purifying analyzes collection of illustrative plates, and target protein peak retention time is 26.064min; Purity 97%.

Fig. 4 is rhCRF sample N end sequencing result.4-1:N end order-checking examining report; 4-2:N end 1-7 amino-acid residue collection of illustrative plates; 4-3:N end 8-15 amino-acid residue collection of illustrative plates.

Fig. 5 is the mass spectroscopy collection of illustrative plates.

Embodiment

Embodiment 1 expresses the structure of rhCRF engineering bacteria

1. the CRF gene design is with synthetic

According to the hCRF aminoacid sequence of logining among the GenBnak (accession number: CAA23834.1), choose 41 amino acid gene coded sequences of mature peptide, and take into account that codon preference and gene order optimization carry out same sense mutation in intestinal bacteria.And connect successively nucleic acid coding sequence corresponding to enteropeptidase recognition sequence (DDDDK), connecting peptide (GGGGSGGGGSGGGGS) at the gene 5 ' end, introduce KpnI recognition sequence GATTCC at 5 ' end of full sequence simultaneously, 3 ' end is introduced NotI recognition sequence GCGGCCGC.The gene order that designs is carried out full gene synthetic (Shanghai is given birth to the worker and finished).

2. pET32-CRF/BL21 (DE3) engineering bacteria makes up

2.1 expression vector pET32-CRF makes up

With synthetic gene sequence, use the KpnI/NotI double digestion, simultaneously with PET-32a-c (+) carrier KpnI/NotI double digestion.Electrophoresis on 1.2% sepharose of 1 * TAE preparation, object tape separate when better, downcut with the blob of viscose of blade with the object tape place, cut unnecessary glue as far as possible, are placed in the 1.5 clean ml eppendorf pipes.Reclaim test kit with gel and reclaim purified genes and carrier.

With cutting gene and the carrier through the KpnI/NotI double digestion of glue recovery, with T4 dna ligase 16 ^oThe C connection is spent the night.Connect product and transform DH5 α competence, screening transforms bacterial strain, and carries out sequence verification, makes up and obtains expression vector pET32-CRF, and concrete structure is seen Fig. 1, is stored in the DH5 α bacterial strain called after: PET32-CRF/DH5 α.

2.2 the structure of engineering bacteria pET32-CRF/BL21 (DE3)

(1) BL21 (DE3) competent cell preparation

Adopt Calcium Chloride Method to prepare e. coli bl21 (DE3) competent cell, its method is as follows:

The single bacterium colony of picking BL21 (DE3) in 25ml LB liquid nutrient medium, 37 ^OC, 250 r/min incubated overnight; Second day is got 1ml incubated overnight liquid and is inoculated among the 100ml LB, and 37 ^oIt is about 0.375 that C, 250 r/min are cultured to OD600, approximately 2.5 hours; Nutrient solution divided install in the aseptic polypropylene tube of precooling, in placing 5-10 minute on ice, then in 4 ^OC, centrifugal 7 minutes of 2000 r/min; Abandon supernatant, with the ice-cold CaCl of 10ml ₂The resuspended bacterial sediment of solution, 4 ^OC, centrifugal 5 minutes of 1600 r/min; Abandon supernatant, with the ice-cold CaCl of 10ml ₂The resuspended bacterial sediment of solution is placed on ice and is spent the night, and makes the thalline natural subsidence for subsequent use.

(2) expression plasmid preparation

Inoculation pET32-CRF/DH5 α bacterial strain is to 20mlLB liquid (containing penbritin 100ug/mL), 37 ^oC, the 250rpm overnight incubation.Collect the 3mL thalline, prepare plasmid with the plasmid purification test kit, and send part plasmid sequence verification consistent with implementation sequence.

(3) engineering bacteria transforms and screening

BL21 (DE3) competent cell with preparation, inferior daily pipettor removes most of supernatant, remaining approximately 1 ml solution, mixing gently, get the competent cell of the fresh preparation of 100 μ l in 1.5 ml centrifuge tubes, the expression plasmid that adds the above-mentioned preparation of 1 μ l, light mixing was placed 30 minutes on ice.Then heat-shocked 90 seconds in 42 ℃ of water-baths is put in 2 minutes immediately on ice, adds 800 μ l room temperature liquid LB and cultivates based on 37 ℃ of 150r/min and cultivated 1.5 hours.Get 50-200 μ l bacterium liquid coating LB dull and stereotyped (containing penbritin 100ug/mL), cultivate 16-20 hour h for 37 ℃.Picking list bacterium colony PCR checking makes up and obtains escherichia coli expression engineering bacteria PET32-CRF/BL21 (DE3).The most finally ferment by shaking flask screening high expression level amount bacterial strain and to use engineering bacteria.

The fermentation of embodiment 2 engineering bacteria pET32-CRF/BL21 (DE3)

With the seed liquor of overnight incubation, be inoculated in the suitable substratum (such as LB, TB or other appropriate media) of fermentor tank and cultivate, microbial culture is to OD ₆₀₀Be about 25, add such as IPTG and induced 3-4 hour.Centrifugal collection thalline is suspended in (25mm Tris-HCl, 150 mmNaCl, PH8.0) in the suitable damping fluid with thalline, and high-pressure homogenization breaks bacterium, and broken liquid is centrifugal, collects supernatant liquor, abandons precipitation.

Embodiment 3 rhCRF purifying

This broken bacterium liquid supernatant is splined on through 0.2M NiSO4 processes and with Ni2+-Chelating Sepharose Fast Flow (GE Healthcare) the chelating chromatography media of 20mM Tris-HCl (pH8.0) balance, with 100mM imidazoles (containing 20mM Tris-HCl, pH8.0) wash-out; The purpose fusion rotein of collecting adds 0.5U enteropeptidase (50mM Tris-HCl, 2mM CaCl2,0.1% Tween-20, pH8.0) enzyme under 4 ℃ of conditions by every 1mg fusion rotein and cuts approximately 16h of fusion rotein through desalting treatment.

Enzyme is cut target protein and is splined on Ni2+-Chelating Sepharose Fast Flow, and target protein and fusion tag be hanging column simultaneously.But target protein hanging column bonding force is low, with lower concentration imidazoles (50mM) wash-out target protein, collects the eluted protein peak.SDS-PAGE detection molecules amount is 4700Da approximately, and purity greater than 97%, is seen Fig. 2, Fig. 3 greater than 95%, HPLC purity assay.

Embodiment 4 rhCRF structural identifications

(1) 15 determined amino acid sequences of N-terminal

The rhCRF that aforesaid method is prepared, carry out the N-terminal determined amino acid sequence, measurement result is as follows: N-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-C, in full accord through national biological medicine analytic centre's detection and expected results, see that Fig. 4-1 is to Fig. 4-3.

(2) mass spectroscopy

The rhCRF determining molecular weight that aforesaid method prepares is 4757.7, detects unanimously with theoretical prediction through national biological medicine analytic centre, sees Fig. 5.

Embodiment 5 rhCRF biological activity determinations

CRF (corticotropin releasing factor(CRF)) has the function of pituicyte secretion ACTH (thyroliberin) in the control agent, the quantitative response of ACTH the active height of CRF.

(1) separate tissue: mouse pituicyte excision.

(2) tissue digestion: the tissue block cellular segregation adopts the method that combines with digestion of shearing.The mouse hypophysis that scales off is cut into less fragment, pituicyte is dissolved in the D-Hanks damping fluid of proper volume, wherein contain 0.5% (w/v) collagenase (Type II) and 0.5 (w/v) BSA, process 45min in 45 ℃ of lower placements.

(3) collect Digestive system: filter with filter screen (100 order filtering net) and remove the large fully fragment of tissue of digestion that reaches not, the cell suspension of collecting fully washs three times through the Hanks damping fluid.

(4) cell cultures: in the cell suspension of collecting, add the DMEM nutrient solution and (contain 10% foetal calf serum, 20nM HEPES, 50 μ g/ml penicillin, 50 μ g/ml Streptomycin sulphates, 6 μ g/ml bacitracins), placing 95% air to add 5% carbon dioxide gas mixture cultivated 4-5 days.Then the cell of collecting adds specimen or standard substance and cultivates 2h with the DMEM nutrient solution washing that does not contain serum, and the ACTH assay is treated in-20 ℃ of preservations.

(5) mensuration of ACTH content: adopt the ELISA test kit of ACTH concentration determination in the rat cell culture supernatant to carry out detection by quantitative, detailed process reference reagent box described method carries out.

The result: as reference, rhCRF biological activity and the reference substance of preparation are suitable with natural CRF reference substance (Bachem).

Sequence table

＜110〉Chongqing Kerun Biomedical R﹠D Co., Ltd.

＜120〉a kind of preparation method of CRF

<130>

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 123

<212> DNA

＜213〉dna sequence dna of CRF

<400> 1

agcgaagaac cgccgattag cctggatctg acctttcatc tgctgcgcga agtgctggaa 60

atggcgcgcg cggaacagct ggcgcagcag gcgcatagca accgcaaact gatggaaatt 120

att 123

<210> 2

<211> 41

<212> PRT

＜213〉aminoacid sequence of CRF

<400> 2

Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg

1 5 10 15

Glu Val Leu Glu Met Ala Arg Ala Glu Gln Leu Ala Gln Gln Ala His

20 25 30

Ser Asn Arg Lys Leu Met Glu Ile Ile

35 40

Claims

1. the preparation method of a CRF, the dna sequence dna of described CRF is shown in SEQ ID NO:1, and the preparation method of this releasing hormone has following steps:

1) design and synthesize the CRF gene according to the hCRF aminoacid sequence, the dna sequence dna of this gene is shown in SEQ ID NO:1;

2) step 1) gained gene obtains expression vector pET32-CRF by the Trx protein fusion in connecting peptide, enteropeptidase recognition site and pET-32a-c (+) carrier;

3) step 2) the expression vector pET32-CRF of gained is transformed into e. coli bl21 (DE3) and obtains CRF engineering bacteria pET32-CRF/BL21 (DE3);

5) the Trx-CRF fusion rotein that obtains of step 4) is after the enteropeptidase cutting, and separation and purification obtains CRF.

2. preparation method according to claim 1 is characterized in that: step 2) building mode of described expression vector is :-Trx-connecting peptide-DDDDK-CRF-, wherein-and DDDDK-is the enteropeptidase recognition site, and D is aspartic acid, and K is Methionin.

3. preparation method according to claim 2 is characterized in that: the connecting peptide structure of the N end of enteropeptidase recognition site is-(GGGGS) n-, and wherein G is glycine, and S is Serine, and n is 0-10.

4. preparation method according to claim 3, it is characterized in that: described n is 3.

5. preparation method according to claim 1, it is characterized in that: the corresponding encoding amino acid sequence of the described CRF of step 5) is shown in SEQ ID NO:2.

6. described preparation method according to claim 1, it is characterized in that, the described fermentation of step 4) is CRF engineering bacteria pET32-CRF/BL21 (DE3) to be inoculated into carry out the high density fermentation cultivation in the fermentor tank, induces the expression of fusion rotein by adding IPTG.

7. described preparation method according to claim 1, it is characterized in that, the step of the described purifying of step 4) comprises centrifugal collection thalline, broken bacterium, the broken bacterium liquid supernatant of centrifugal collection, to collect Chelating Sepharose Fast Flow chelating chromatography on the supernatant, the flush away foreign protein, wash-out purpose fusion rotein.

8. described preparation method according to claim 1 is characterized in that, the described separation and purification of step 5) is Chelating Sepharose Fast Flow chelating chromatography on the product after enzyme is cut, foreigh protein removing, wash-out target protein.