CN102060921B - High-stability VPAC2 type receptor specific activator MHDBAY, preparation method and application thereof - Google Patents

Abstract

The invention discloses a high-stability VPAC2 type receptor specific activator MHDBAY, a preparation method and application thereof. Compared with the traditional VPAC2 type recombinant receptor specific activator, the recombinant activator MHDBAY has higher stability and physiological activity, and through key amino acid mutation, the specific activation activity of the recombinant activator MHDBAY to the VPAC2 type receptor is obviously improved. The in vitro stability of the recombinant activator MHDBAY is about 25 times of that of BAY55-9837 and about 8 times of that of chemical synthesis MHDBAY polypeptide. Proved through experiments, the selective activation activity of the recombinant activator MHDBAY to the VPAC2 type receptor is obviously higher than that of BAY55-9837. The recombinant activator MHDBAY can be applied to preparing medicaments having the functions of treating diabetes mellitus and complications thereof, treating hyperglycemia, treating hypertension, treating thrombosis, treating adiposes, treating cardiovascular diseases, and the like.

Description

High stability VPAC2 receptor specific agonist MHDBAY and preparation method thereof and application

Technical field

The present invention relates to gene engineering technology field; be particularly related to a kind of high stability VPAC2 receptor specific agonist MHDBAY and preparation method thereof; with its medicine for the preparation for the treatment of VPAC2 receptor relative disease; as: the insulin secretion that promotes dependence on the glucose; improve body to the emergency capability of high density blood sugar; effective blood sugar concentration that reduces, the effects such as protection Pancreatic beta cells function and increase β cell quantity and adjusting function of immune system.

Background technology

Pituitary adenylate cyclase activating peptide (PACAP) is the nerve polypeptide that important biomolecule is learned function that has that is found nineteen ninety by pituitary secretion, is the newcomer in secretin/glucagon/VIP family.PACAP has two kinds of existence form: PACAP38, is comprised of 38 amino acid; PACAP27 is comprised of 27 amino acid of PACAP38 nitrogen end.PACAP improves second couriers' such as target cell cAMP concentration, thereby brings into play extensive and important biological function by activating its special g protein coupled receptor.PACAP has three class g protein coupled receptor: PAC1, VPAC1 and VPAC2; Three receptoroids distribute different in organism, and role is not identical yet.Wherein the VPAC2 receptor is distributed in the tissues such as pancreas islet; closely related with energy metabolism; from clinical point; specific PACAP derivative as VPAC2 receptor specific agonist can reduce blood glucose levels effectively; the potentiality of protection Instreptozotocin Induced and prevention disease progression; and without the hypoglycemia risk; the any stadium that can be used for diabetes; as the nerve polypeptide with wide application; having the complex therapy effect that is better than other drug for second phase and three sugar futures urine patient, is the newtype drug of generally acknowledged treatment type ii diabetes.

PACAP and VPAC2 receptor acting are main to be joined with adenylate cyclase (AC) lotus root: by the cAMP signal transduction pathway, activate AC, catalysis ATP is converted into cAMP, cAMP activates its deopendent protein kinase A (PKA) thereupon, PKA can make multiple proteins phosphorylation in cell, produce physiological effect, especially play an important role promoting genes involved to transcribe.PACAP mainly contains by the receptor-mediated biological function of VPAC2: neurotransmitter/modified, promote hormone secretion, endocrine regulation balance; Regulate the generation of gonad function and sexual cell; Adjusting energy metabolic balance etc.

Studies show that, VPAC2 receptor specific agonist can effectively promote the insulin secretion of dependence on the glucose, and the raising body effectively reduces blood sugar concentration to the emergency capability of high density blood sugar, protection Pancreatic beta cells function and increase β cell quantity.But PACAP and many derivatives thereof, as BAY etc., very fast by DPP IV (DPPIV) degraded in vivo, its body internal stability is relatively poor; And due to the metabolism of self and the filtration of kidney, many medicine peptides are removed rapidly in vivo.Therefore, the transformation period that extends some peptide class equimolecular medicine is target and the difficult point of present many novel molecular medicament research and development, is also its clinical application problem anxious to be resolved.Therefore, research and development have important pharmaceutical use as the derivative polypeptide of the high stability PACAP of VPAC2 receptor specific agonist.

The VPAC2 receptor specific agonist of having reported at present, as: polypeptide BAY55-9837, polypeptide Ro25-1553 etc., the stability in the aqueous solution or body fluid environment has much room for improvement, and, due to the metabolism of self and the filtration of kidney, in vivo by clean-up effect is to be improved rapidly.

Summary of the invention

The shortcoming that primary and foremost purpose of the present invention is to overcome prior art provides a kind of have more high stability and more the high stability VPAC2 receptor specific agonist MHDBAY of high biological activity with not enough.

Another object of the present invention is to provide the preparation method of described high stability VPAC2 receptor specific agonist MHDBAY.This preparation method is according to the aminoacid sequence of the derivative polypeptide MHDBAY of PACAP and the Preference of e. coli codon, design MHDBAY is in the gene order of prokaryotic expression system, adopt genetic engineering technique, realize the efficient preparation of desired polypeptides MHDBAY in conjunction with the induced shearing function of intein (intein); This preparation method's production efficiency is high, and production cost is low.

A further object of the present invention is to provide the application of described high stability VPAC2 receptor specific agonist MHDBAY.

Purpose of the present invention is achieved through the following technical solutions: a kind of high stability VPAC2 receptor specific agonist MHDBAY, and its aminoacid sequence is as follows:

MWQRPSSWIEGRFPHSDAVFTDQYTRLRKQLAAKKYLQSLKQKRY

It consists of: the human serum albumin (HSA) of methionine(Met) (M)-high-affinity is in conjunction with seven peptide WQRPSSW-factor Xa enzyme recognition sequence IEGR-dipeptidyl peptidase DPPIV recognition sequence FP-active treatment polypeptide MBAY (its aminoacid sequence is: HSDAVFTDQYTRLRKQLAAKKYLQSLKQKRY, specific action is in the VPAC2 acceptor)

The coding nucleotide sequence of described high stability VPAC2 receptor specific agonist MHDBAY is:

ATG?TGG?CAG?CGC?CCG?AGC?AGC?TGG?ATT?GAA?GGT?CGC?TTT?CCG?CAT?AGC?GATGCG?GTG?TTT?ACC?GAT?CAG?TAT?ACC?CGT?CTG?CGT?AAA?CAG?CTG?GCG?GCG?AAA?AAATAT?CTG?CAG?AGC?ATT?AAA?CAG?AAA?CGT?TAT

The preparation method of described high stability VPAC2 receptor specific agonist MHDBAY comprises the following steps:

(1) design and synthesize the MHDBAY gene:

Adopt 3 primer two step synthesis MHDBAY genes:

Primers F 1:

5’- GGT?GGT?CAT?ATG?TGG?CAG?CGC?CCG?AGC?AGC?TGG?ATT?GAA?GGT?CGC?TTT?CCGCAT?AGC?GAT-3’；

Primers F 2:

5’-CGC?CAG?CTG?TTT?ACG?CAG?ACG?GGT?ATA?CTG?ATC?GGT?AAA?CAC?CGC?ATC?GCTATG?CGG?AAA-3’；

Primers F 3:

5’- CCA?CCA?TGC?TCT?TCC?GCA?ATA?ACG?TTT?CTG?TTT?A?AT?GCT?CTG?CAG?ATA?TTTTTT?CGC?CGC?CAG?CTG?TTT-3’

Wherein, GGT GGTOr CCA CCABe the protection base, CATATG is the NdeI restriction enzyme site, and TGC TCTTCC GCA is the SapI restriction enzyme site;

At first primers F 1 and primers F 2 are carried out chain extension reaction, then take its product as DNA profiling, take F1 and F3 as primer, the PCR reaction makes the MHDBAY gene;

(2) build recombinant vectors pKY-MHDBAY:

Respectively the MHDBAY gene that plasmid pKYB and step (1) make is carried out double digestion with Ndel enzyme and Sapl enzyme, then the plasmid pKYB that the MHDBAY gene after double digestion is connected with double digestion is connected, obtain recombinant vectors pKY-MHDBAY;

(3) preparation table expression engineered bacteria pKY-MHDBAY/ER2566:

Transform with recombinant vectors pKY-MHDBAY and express Host Strains intestinal bacteria Ecoli Strain ER2566, obtain expressing engineering bacteria pKY-MHDBAY/ER2566;

(4) expression and purifying:

1. abduction delivering engineering bacteria pKY-MHDBAY/ER2566 expresses " ternary " fusion rotein that is comprised of desired polypeptides, intein and chitin binding domain (Chitin binding domain, CBD);

2. " ternary " fusion rotein that obtains carries out purifying with the chitin post, and " ternary " fusion rotein is adsorbed in the chitin post, and the solution that then contains mercaptan is crossed post and is full of in the chitin band of column border reaction.The effect of mercaptan is that the N end-grain cutting of inducible protein intein is cut, and discharges desired polypeptides C end thioesters; Then wash-out chitin post, obtain desired polypeptides C end thioesters, dialysis desired polypeptides C end thioesters, thus produce stable hydrolysate;

3. utilize high-efficient liquid phase chromatogram technology purifying desired polypeptides, obtain high stability VPAC2 receptor specific agonist MHDBAY.

Described in step (1), the condition optimization of chain extension reaction is 94 ℃, 10min; Be down to 58 ℃ of 5min; 72 ℃ of 10min;

The condition optimization of the reaction of PCR described in step (1) is that reaction conditions is: 95 ℃ of 5min; 95 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 30 circulations; 72 ℃ are extended 10min;

Step (4) contains solution composed as follows of mercaptan described in 2.: 20mM Tris-HCI, and 0.5M NaCl, 1mM EDTA, the 50mM beta-mercaptoethanol, pH 8.0;

Step (4) 2. described in the reaction condition optimization be 16 ℃ the reaction 24 hours;

Step (4) 2. described in the composition of solution of wash-out chitin post be preferably as follows: 20mM Tris-HCl, 500mM NaCI, 1mM EDTA, pH 8.0;

Step (4) utilizes the step of high-efficient liquid phase chromatogram technology purifying desired polypeptides MHDBAY as follows described in 3.:

A, mobile phase A are at volume percent 10% acetonitrile (CNCH ₃, solute is pure water) in add trifluoroacetic acid (TFA) and obtain, the final concentration of TFA is volume percent 0.1%; Mobile phase B is 100% acetonitrile (CNCH ₃) the middle trifluoroacetic acid (TFA) that adds, the final concentration of TFA is volume percent 0.1%; Flow velocity 1mL/min, the 20min linear gradient elution;

In B, linear gradient elution, Mobile phase B by 0～60% (v/v), is collected the desired polypeptides elution peak, and it is 218nm that photoabsorption detects wavelength; And carry out Mass Spectrometric Identification.

Described high stability VPAC2 receptor specific agonist MHDBAY is applied to prepare the medicine for the treatment of following disease: diabetes and complication thereof, hyperglycemia, hypertension, thrombosis, obesity, cardiovascular disorder, nervous system disorders, heart and renal failure.

The present invention has following advantage and effect with respect to prior art:

(1) the prepared high stability VPAC2 receptor specific agonist MHDBAY of the present invention, it is the derivative polypeptide of PACAP, PACAP compares with wild-type, how held a methionine(Met) at its N, and have special aminoacid sequence, and namely pass through the mercapto ester bond in conjunction with mercaptan at the C end, become polypeptide thioesters, polypeptide thioesters can be hydrolyzed and produce natural carboxyl terminal, also can special hydrolysis become thiocarboxylic acid.

(2) aminoacid sequence of this VPAC2 receptor specific agonist MHDBAY is different from other and PACAP homology or nonhomologous VPAC2 receptor specific agonist, be with the distinctive points of BAY55-9837: at first, the N of recombinant polypeptide MHDBAY end has the human serum albumin (HSA) of a high-affinity in conjunction with seven peptide WQRPSSW and factor Xa enzyme recognition sequence IEGR and DPPIV recognition sequence FP; Secondly, the 9th amino acids l-asparagine (N) of Mutagen BAY55-9837 is glutamine (Q), the 17 amino acids α-amino-isovaleric acid (V) of Mutagen BAY55-9837 is leucine (L), and the 28 amino acids l-asparagine (N) of Mutagen BAY55-9837 is glutamine (Q).

These differences make MHDBAY have higher stability and to the special Activation Activity of VPAC2 receptor.After entering blood, MHDBAY is combined with human serum albumin and is formed HSA-MHDBAY mixture (MW:67.03kDa.), and with blood circulation, this both can prevent due to little (the MHDBAY molecular weight: 5544.2Da.) removed fast by kidney of molecular weight; Again owing to containing factor Xa enzyme and DPPIV recognition sequence in mixture, can be by two kinds of orderly enzymolysis of enzyme, discharge at last the active treatment polypeptide MBAY (HSDAVFTDQYTRLRKQLAAKKYLQ SLKQKRY) that carries out the key amino acid sudden change, be combined with the VPAC2 receptor-specific, thus the effect of performance treatment type ii diabetes.

(3) the prepared high stability VPAC2 receptor specific agonist MHDBAY of the present invention is treating diabetes and complication thereof, hyperglycemia, hypertension, thrombosis has significant curative effect in obesity, cardiovascular disorder, nervous system disorders, heart and renal failure disease.

(4) utilize the stability of the recombinant polypeptide MHDBAY that preparation method of the present invention obtains to improve approximately 8 times than chemosynthesis MHDBAY polypeptide, improve approximately 25 times than the stability of chemosynthesis BAY55-9837 polypeptide.Expression method efficient of the present invention is high, cost is low, has a extensive future.

Description of drawings

Fig. 1 contains the phage of selected 7-mer peptide and the affinity interaction curve of human serum albumin.

Fig. 2 is the preparation schematic diagram of recombinant polypeptide MHDBAY.

Fig. 3 is that the synthetic recombinant expression plasmid pKY-MHDBAY of reaching of MHDBAY gene builds schematic diagram;

In figure: the MCS-multiple clone site; CBD-chitin binding domains; The intein-intein.

Fig. 4 is that SDS-PAGE detects the evaluation figure that fusion rotein Intein-CBD-MHDBAY expresses; Wherein, swimming lane 1 is that IPTG induces rear bacterial cell disruption supernatant liquor; Swimming lane 2 is that IPTG induces front bacterial cell disruption supernatant liquor.

Fig. 5 identifies figure for the flight mass spectrum of the recombinant polypeptide MHDBAY of preparation.

Fig. 6 is that the stability of recombinant polypeptide MHDBAY and BAY55-9837 compares and detected result figure.

Fig. 7 be recombinant polypeptide MHDBAY/BAY55-9837 and [ ¹²⁵I] PACAP38 is to the competitive binding detected result figure of VPAC2 acceptor.

Fig. 8 is that recombinant polypeptide MHDBAY and BAY55-9837 promote determination of activity that CHO-VPAC2 cell cAMP generates figure as a result.

Fig. 9 is that recombinant polypeptide MHDBAY and BAY55-9837 are to the detected result figure of the acute carbohydrate tolerance test of ICR mouse.

Embodiment

The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.

Embodiment 1

The elutriation of Trp-Gln-Arg-Pro-Ser-Ser-Trp

Utilize New England Biolabs, the high affine 7-mer peptide of random seven peptide phage display libraries (Ph.D.-7 phage display peptide library) screening human serum albumin of Inc company.

The Ph.D.-7 phage display peptide library is that random seven peptides are fused on the less important capsid protein of M13 phage (pIII), is a combinatorial library, and the random seven peptide sequence numbers that Ben Wenku comprises are more than 10, and the titre of Ben Wenku is 2 * 10 ¹³Pfu/mL.

Carry out the four-wheel elutriation with fixed target molecule direct coated flat band method.

First round elutriation:

(1) human serum albumin (Sigma-Aldrich company) is dissolved in the NaHCO of 0.1M ₃Solution (pH8.6), compound concentration are the human serum albumin solution (pH 8.6) of 100 μ g/mL;

(2) at the dull and stereotyped (60 * 15mm of single sterilization polystyrene culture dish, Coring Incorporated company) add the above-mentioned human serum albumin solution's solution of 1.5mL in, rotation is until the surface is fully moistening repeatedly, 4 ℃ of slight concussions in humidifier vessel, overnight incubation, flat board can be in this container 4 ℃ of storages standby;

(3) with the TBST damping fluid of 1mL (contain 0.1%[v/v in the TBS damping fluid] tween 20, pH 7.5) dilution 2 * 10 ¹¹Phage (i.e. the original library of 10 μ L), then be added to coated and flat board that sealing is good on, the room temperature gentleness is shaken 50min;

(4) topple over and remove not in conjunction with phage and clap to get rid of and remove residual solution, wash plate 10 times with above-mentioned TBST damping fluid; Human serum albumin is dissolved in TBS damping fluid (pH 7.5), and compound concentration is the solution of 100 μ g/mL, with this solution 1mL in connection with phage competitiveness elute, elution time is 40min;

(5) the take a morsel phage of wash-out carries out titer determination according to test kit specification sheets method, and the phage of all the other wash-outs is increased and measures titre according to test kit specification sheets method;

Second takes turns elutriation:

According to first round elutriation method, get coated good polystyrene culture dish flat board, add the phage after first round elutriation is increased after sealing, adding the phagocytosis scale of construction is 2 * 10 ¹¹Pfu carries out second and takes turns elutriation, and method is identical with first round elutriation;

The third round elutriation:

According to first round elutriation method, get coated good polystyrene culture dish flat board, add the second phage of taking turns after elutriation is increased after sealing, adding the phagocytosis scale of construction is 2 * 10 ¹¹Pfu carries out the third round elutriation, and method and first round elutriation are slightly changed: the concentration with tween 20 in the TBST damping fluid in cleaning step increases to 0.3%[v/v];

The fourth round elutriation:

According to first round elutriation method, get coated good polystyrene culture dish flat board, add the phage after the third round elutriation is increased after sealing, adding the phagocytosis scale of construction is 2 * 10 ¹¹Pfu carries out the fourth round elutriation, and method and first round elutriation have part to change: the concentration with tween 20 in the TBST damping fluid in cleaning step increases to 0.3%[v/v]; During coated flat board, human serum albumin solution's used concentration is reduced to 10 μ g/mL; The 50min when binding time of phage and flat board is screened by front three-wheel shortens to 20min, and the 40min when elution time is screened by front three-wheel increases to 60min.

10 plaques of random selection carry out DNA sequencing in the phage that the fourth round elutriation goes out, sequencing primer for-(its DNA sequence dna is the 96gIII sequencing primer: 5 '-CCC TCA TAG TTA GCG TAACG-3 '), deduce according to the DNA sequencing result and be aminoacid sequence, experimental result shows, with the aminoacid sequence of Trp-Gln-Arg-Pro-Ser-Ser-Trp is: WQRPSSW.

Embodiment 2

The human serum albumin parent measures with the avidity of the 7-mer peptide of institute's elutriation

Utilize enzyme-linked immunosorbent assay (ELISA) method to detect 7-mer peptide that elutriation obtains and the avidity of human serum albumin, concrete steps are as follows:

(its aminoacid sequence is: plaque clone WQRPSSW) carries out pure culture and measures titre with Trp-Gln-Arg-Pro-Ser-Ser-Trp to containing of will identifying.The intestinal bacteria ER2738 (New England Biolabs company) of incubated overnight is diluted in 20mLLuria-Bertani (broth) substratum (LB) by the volume ratio of 1: 100, add 5 μ L phage supernatants in every pipe ER2738 nutrient solution, 37 ℃ of aerated culture 4.5h.

above-mentioned culture changes in centrifuge tube, 10, the centrifugal 10min of 000rpm, supernatant moves into fresh centrifuge tube, centrifugal again, get volume percent 80% supernatant in fresh centrifuge tube, add 1/6 volume PEG-8000/NaCl (20%[w/v] PEG-8000, 2.5M NaCl), after 4 ℃ of precipitations are spent the night, 10, 000rpm centrifugation 15min, abandon supernatant, precipitation is resuspended in 1mL TBS damping fluid, suspension changes Eppendorf tube over to, 4 ℃ of centrifugal 5min, supernatant changes fresh Eppendorf tube over to, the PEG-8000/NaCl redeposition that adds 1/6 volume, act on 30min on ice, 4 ℃ of centrifugal 10min, abandon supernatant, precipitation is resuspended in 50 μ L TBS damping fluids, measures phage titre, 4 ℃ of storages.

Human serum albumin (Sigma-Aldrich company) is dissolved in the NaHCO of 0.1M ₃Solution (pH 8.6), compound concentration are the human serum albumin solution (pH 8.6) of 100 μ g/mL, emptying with the coated elisa plate of this solution, and every sky adds 200 μ L, as positive experimental group; With the continuous four times of dilution phages of another elisa plate, add 10 by the 12nd hole ¹²Individual virion, carrying out 4 times of serial dilution to the 1 holes is 2.4 * 10 ⁵Individual virion; These two porous plates are all used 1%[w/v] sealing of enzymic hydrolysis casein food grade.

To dilute good phage with Multi-channel liquid transfer device and add in the plate that is coated with human serum albumin, room temperature concussion effect 2h; With the TBST damping fluid (contain 0.3%[v/v in the TBS damping fluid] tween 20, pH 7.5) wash plate 6 times.

In the liquid of blockading with 1: 5,000 dilution proportion HRP mark anti--M13 antibody (Pharmacia#27-9411-01).Every hole adds 200 μ L dilution antibody, room temperature concussion effect 1h, with the TBST damping fluid (contain 0.3%[v/v in the TBS damping fluid] tween 20, pH 7.5) wash plate 6 times, then add ABTS/H ₂O ₂(Sigma#A1888) substrate solution 200 μ L, room temperature effect 40min records the light absorption value at 405nm place with microplate reader, and the experiment triplicate is got its mean value; The control experiment group does not add phage, and other are with positive experimental group, to measure substrate background light absorption value.

Learn to process by statistics, the positive experimental group of table 1 and blank group are at each empty light absorption value of 405nm place; Fig. 1 shown contain affine in conjunction with the 7-mer peptide (its aminoacid sequence is: phage WQRPSSW) with the avidity curve of human serum albumin, experimental result shows, institute's elutriation to 7-mer peptide and human serum albumin have higher avidity.

Table 1 utilizes the affinity interaction of the selected 7-mer peptide of Enzyme-linked Immunosorbent Assay technology for detection and human serum albumin

Annotate: in 1 table, numeral 1,2,3......12 represents the phage number that raises successively after 4 times of serial dilutions. as 1 expression 2.4 * 10 ⁵, 12 expressions 10 ¹².

2 experiments repeat 3 times. and in table, numerical value is the mean value of 3 times.

Embodiment 3

The preparation of recombinant VPAC 2 receptor specific agonist MHDBAY

The preparation process of high stability VPAC2 receptor specific agonist MHDBAY as shown in Figure 2, concrete steps are as follows:

(1) acquisition of MHDBAY encoding sequence:

CDNA according to colibacillary password Preference design coding MHDBAY designs 3 primers,

Adopt two-step approach to obtain this sequence (as shown in Figure 2):

Primers F 1:

5’- GGT?GGT?CAT?ATG?TGG?CAG?CGC?CCG?AGC?AGC?TGG?ATT?GAA?GGT?CGC?TTT?CCGCAT?AGC?GAT-3’；

Primers F 2:

5’-CGC?CAG?CTG?TTT?ACG?CAG?ACG?GGT?ATA?CTG?ATC?GGT?AAA?CAC?CGC?ATC?GCTATG?CGG?AAA-3’；

Primers F 3:

5’- CCA?CCA?TGC?TCT?TCC?GCA?ATA?ACG?TTT?CTG?TTT?A?AT?GCT?CTG?CAG?ATA?TTTTTT?CGC?CGC?CAG?CTG?TTT-3’

Wherein, GGT GGTOr CCA CCABe the protection base, CATATG is the NdeI restriction enzyme site, and TGC TCTTCC GCA is the SapI restriction enzyme site;

1. chain extension reaction:

Reaction system is: primers F 1 (250 μ M/L) 2 μ L, and primers F 2 (250 μ M/L) 2 μ L, 10 * TaKaRaBuffer (contains 4mmol/L MgCl ₂) 2 μ L, dNTP 2 μ L, H ₂O 11.5 μ L, TaKaRa Taq enzyme 0.5 μ L;

Reaction conditions is: 94 ℃, and 10min; Be down to 58 ℃ of 5min; 72 ℃ of 10min;

Obtain extension liquid.

2. PCR reaction:

Reaction system is: take extension liquid as DNA profiling, and primers F 1 (25 μ M/L) 2 μ L, F3 (25 μ M/L) 2 μ L, extension liquid 1.5 μ L, dNTP 2 μ L, 10 * TaKaRa Ex Buffer, 2 μ L, TaKaRa Taq enzyme 0.5 μ L, H ₂O 10 μ L;

Reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ are extended 10min;

Obtaining length is the PCR product of 162bp

(2) build recombinant vectors pKY-MHDBAY, as shown in Figure 3:

Carry out enzyme with NdeI and SapI and cut the PCR product that step (1) obtains, utilize PCR product purification test kit to carry out purifying (operating according to specification sheets).PCR product after purifying first carries out LguI (isozyme of SapI, Fermatas product) single endonuclease digestion, and the enzyme tangent condition is: at 1 * Tango ^TMIn the buffer reaction system, 1 μ g PCR product adds 15unit LguI (3 μ L), and 37 ℃ of enzymes are cut 4h; After the LguI single endonuclease digestion is completed, add 10 * Tango of appropriate volume ^TMBuffer makes reaction system become 2 * Tango ^TMThe buffer system adds 20unit NdeI (2 μ L, Fermatas product), and 37 ℃ of enzymes are cut 4h;

The double digestion of plasmid pKYB (New England Biolabs (NEB) company): the double digestion condition of LguI (isozyme of SapI) and NdeI is the same.

The plasmid pKYB that MHDBAY gene after double digestion is connected with double digestion connects, making, conversion and the screening (kantlex) of linked system and tie-time, bacillus coli DH 5 alpha (available from the precious biotech firm in Dalian) competent cell operate according to " molecular cloning ".The MHDBAY gene clone between the NdeI of plasmid pKYB and SapI site, is obtained recombinant vectors pKY-MHDBAY.

(3) expression and purifying:

1. recombinant plasmid pKY-MHDBAY is transformed and express Host Strains E.coli Strain ER2566 (NewEngland Biolabs (NEB) company), obtain expressing engineering bacteria pKY-MHDBAY/ER2566; The picking mono-clonal contains Luria-Bertani (broth) substratum (being called for short the LB substratum) of 50 μ g/mL kantlex in 5mL, shake the bacterium overnight incubation, enlarge, be connected to take volume ratio as the ratio of 1: 100 the LB substratum that 1L contains the 50mg/L kantlex, 37 ℃ are shaken bacterium to OD ₆₀₀Be 0.6, add isopropyl-β-D-thiogalactoside(IPTG) (IPTG)) to final concentration 0.6mmol/L, 37 ℃ of abduction delivering 6h.The centrifugal 20min of 8000rpm collects thalline, thalline is resuspended in 60mL Buffer A solution (20mM Tris-HCl, 500mM NaCI, 1mM EDTA, pH 8.0) in, then carrying out fragmentation with the broken instrument of low-temperature ultrahigh-pressure continuous flow cell, broken condition is: the concentration of BufferA solution dilution thalline is 18% (m/v), cracking pressure is 1700bar, and refrigeration temperature is 3 ℃.

Breakdown products is at 4 ℃, and the centrifugal 30min of 10000rpm collects supernatant, and this supernatant is called broken supernatant.SDS-PAGE detects the expression of fusion rotein Intein-CBD-MHDBAY, and qualification result as shown in Figure 4.

Get 25mL chitin pearl (the NEB product #S6651L of company) filling 4.5 * 20cm chromatography column, wash post with the BufferA solution of 10 times of column volumes; Annotate on the flow velocity of broken supernatant with 0.5mL/min; Buffer A solution with 10 times of column volumes is crossed post with 2mL/min, to wash away foreign protein or the fusion rotein of affine combination not, with 80mL Buffer B solution (20mM Tris-HCI, 0.5M NaCl, 1mM EDTA, the 50mM beta-mercaptoethanol, pH 8.0) naturally cross post, make beta-mercaptoethanol evenly distribute and soak filler in post, then seal the import and export end, above-mentioned reaction system is cut with the N end-grain cutting of inducible protein intein at 16 ℃ of reaction 24h, discharges desired polypeptides C end thioesters.Collect desired polypeptides solution C end thioesters with Buffer A eluant solution and with clean beaker, 4 ℃ of dialysed overnight are removed beta-mercaptoethanol and are made the hydrolysis of desired polypeptides C end thioesters, again through high performance liquid chromatography (HPLC) purifying, to prepare purity be the VPAC2 receptor specific agonist MHDBAY of mass percent more than 95%, the preparation condition of desired polypeptides is: mobile phase A is [at volume percent 10% acetonitrile (CNCH ₃, solute is pure water) in add trifluoroacetic acid (TFA) and obtain, the final concentration of TFA is volume percent 0.1%], Mobile phase B [100% acetonitrile (CNCH ₃) the middle trifluoroacetic acid (TFA) that adds, the final concentration of TFA is volume percent 0.1%], flow velocity 1mL/min, the 20min linear gradient elution, in linear gradient elution, Mobile phase B by 0～60% (v/v), is collected the desired polypeptides elution peak, and it is 218nm that photoabsorption detects wavelength.The purity of desired polypeptides MHDBAY is measured (condition is with the preparation condition of desired polypeptides) by analytical HPLC.The high stability VPAC2 receptor specific agonist MHDBAY of preparation utilizes mass-spectrometric technique to identify (as shown in Figure 5), and the mass spectrometric detection molecular weight is 5544.2kDa., is consistent with its theoretical value.

Embodiment 4

The vitro stability of recombinant peptide MHDBAY (being the MHDBAY of embodiment 3 preparations) is measured

recombinant peptide MHDBAY, (MHDBAY compares chemosynthesis MHDBAY with recombinant peptide, its N end is without methionine(Met)) and BAY55-9837 (the Tocris Bioscience product #2711 of company) (pH 8.0 to be dissolved in respectively the 20mM sodium phosphate buffer with final concentration 1mg/ml, contain 150mM sodium-chlor), hatch in 37 ℃ of water-baths, at different point in time sampling, utilize LC-MS-MS (LC-MS) to detect polypeptide in time amount retained in aqueous environment, to determine the stability of recombinant peptide MHDBAY in external aqueous environment, result is as shown in Figure 6: when hatching for 1 week, BAY55-9837 subdues 33.7%, when hatching for 2 week, BAY55-9837 subdues 73.9%, when hatching 3 weeks and 4 week, BAY55-9837 subdues respectively 86.5% and 95.9%, chemosynthesis MHDBAY polypeptide subdues 23.2% when hatching for 2 week, when hatching for 4 week, subdue 48.0%, and recombinant peptide MHDBAY only subdues 2.1% when hatching for 2 week, when hatching for 4 week, only subdues 6.7%.The experimental result statistical procedures shows, the vitro stability of recombinant peptide MHDBAY improves approximately 25 times than BAY55-9837, improves approximately 8 times than chemosynthesis MHDBAY polypeptide.

Embodiment 5

The competitive binding determination of activity of recombinant peptide MHDBAY to the VPAC2 acceptor

With 200 μ L PBS (PH7.4) dissolving recombinant peptide MHDBAY to final concentration be respectively 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M, 0.0001 μ M, 0.00001 μ M puts cooled on ice 20min, keep sample on ice, then add 10unit factor Xa enzyme/(every milligram of recombinant peptide MHDBAY) and 1ng people's source dipeptidyl peptidase (DPPIV)/(1 μ M recombinant peptide MHDBAY), react 24h in 37 ℃ of water-baths, to discharge active polypeptide MBAY (acting on the VPAC2 acceptor).Then adding Rivaroxaban (Toronto Research Chemicals, the Inc product #2711L28828775 of company) and DPP-IV inhibitor (Linco, St.Charles, MO) is all that 50 μ M are to stop endonuclease reaction to final concentration.

VPAC2-CHO cell (Invitrogen company) is seeded on 12 orifice plates to be cultivated, and 2h before experiment uses twice of 0.5mL serum-free Ham ' s F-12 substratum washed cell.Then, cell adds 0.1nM[with the 4 ℃ of overnight incubation of Ham ' s F-12 nutrient solution that contain 2% (mg/ml) bovine serum albumin (BSA) and 10mM glucose in nutrient solution ¹²⁵I] PACAP38 and polypeptide to be measured (being the double digestion reaction solution of BAY55-9837 or different concns group recombinant peptide MHDBAY), and progressively improve peptide concentration (10 to be measured ^-11M～10 ^-5M).Cultivate complete after, discard supernatant liquor, with ice-cold PBS (0.01M, the pH value is 7.4) wash cell 3 times, cell is at room temperature used 0.5mL 0.5M NaOH and 0.1% (mg/ml) SDS mixing 10min, then measures the cell lysate exit dose with the γ counting process, calculates the half amount of suppression (IC50 of recombinant peptide MHDBAY or BAY55-9837, half-maximal inhibitory concentration), every group of experiment repeats 3 times.

Experimental result such as Fig. 7, BAY55-9837 is 68.3 ± 8.1nM to half amount of suppression IC50 of VPAC2 acceptor; The half amount of suppression IC50 to the VPAC2 acceptor that recombinant peptide MHDBAY discharges polypeptide MBAY is 49.8 ± 7.5nM; Result shows, recombinant peptide MHDBAY to the competitive binding activity of VPAC2 acceptor higher than VPAC2 receptor specific agonist BAY55-9837.

Embodiment 6

Recombinant peptide MHDBAY discharges the determination of activity that polypeptide MBAY promotes that VPAC2-CHO cell cAMP generates

The VPAC2-CHO cell of taking the logarithm vegetative period, PBS (0.01M, the pH value is 7.4) washes 2 times, and adjusting concentration of cell suspension is 1 * 10 ⁶/ mL adds respectively polypeptide MBAY or the BAY55-9837 (the Tocris Bioscience product #2711 of company) of same concentrations, regulates the concentration to 10 of polypeptide ^-11M～10 ^-6M, 37 ℃ of incubation 20min.Again will be respectively by each 100 μ L cell suspending liquids and 200 μ L 0.2M HCl solution of polypeptide MBAY and BAY55-9837 effect, room temperature is placed 20min, tip head piping and druming dissolved cell and mixing, the centrifugal 10min of 1500g draws supernatant and presses the amount that cAMP is measured in Cyclic AMP Enzyme Immunoassay Kit explanation.

Experimental result such as Fig. 8, BAY55-9837 to the VPAC2 acceptor partly to activate concentration EC50 (half-maximal stimulatory concentration) be 1.10 ± 0.23nM; Polypeptide MBAY is 0.70 ± 0.15nM to the concentration EC50 that partly activates of VPAC2 acceptor; Result shows, polypeptide MBAY to the selectively activate activity of VPAC2 acceptor apparently higher than BAY55-9837.

Embodiment 7

The detection of recombinant polypeptide MHDBAY on normal ICR mouse oral glucose tolerance impact

Normal male ICR mouse is (available from Beijing Vital River Experimental Animals Technology Co., Ltd., license licensed licenser licence numbering SCXK[capital] 2002-0003) be divided into 4 groups by body weight, Normal group, BAY55-9837 group (0.5mg/kg), recombinant peptide MHDBAY organize (0.5mg/kg).Animal overnight fasting (12h), after getting fasting blood (0min), intravenous administration, Normal group intravenous injection physiological saline, give glucose solution (2g/kg) respectively at 15min gavage after administration, and after giving sugar measuring blood sugar of blood extracting value when 30min, 60min and 120min.

Experimental result is as shown in Figure 9: after recombinant peptide MHDBAY administration 15min, can significantly reduce 30min, 60min and 120min glucose level (p＜0.05) after the normal mouse glucose load, area under curve significantly reduces; BAY55-9837 also can reduce 30min blood sugar after the normal mouse glucose load, but can not reduce 60min and 120min glucose level after the normal mouse glucose load.Experimental result shows, recombinant peptide MHDBAY can obviously reduce the glucose level of dependence on the glucose, and successful is better than BAY55-9837.

Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, within being included in protection scope of the present invention.

Claims (8)

1. a high stability VPAC2 receptor specific agonist MHDBAY, is characterized in that aminoacid sequence is as shown in SEQ ID:NO.1.

2. high stability VPAC2 receptor specific agonist MHDBAY according to claim 1, is characterized in that its coding nucleotide sequence is as shown in SEQ ID:NO.2.

3. the preparation method of the described high stability VPAC2 receptor of claim 1 specific agonist MHDBAY is characterized in that comprising the following steps:

(1) design and synthesize the MHDBAY gene:

Adopt 3 primer two step synthesis MHDBAY genes:

Primers F 1:

5'- GGT?GGT ?CAT?ATG?TGG?CAG?CGC?CCG?AGC?AGC?TGG?ATT?GAA?GGT?CGC?TTT?CCG?CAT?AGC?GAT-3'；

Primers F 2:

5'-CGC?CAG?CTG?TTT?ACG?CAG?ACG?GGT?ATA?CTG?ATC?GGT?AAA?CAC?CGC?ATC?GCT?ATG?CGG?AAA-3'；

Primers F 3:

5’– CCA?CCA ?TGC?TCT?TCC?GCA?ATA?ACG?TTT?CTG?TTT?AAT?GCT?CTG?CAG?ATA?TTT?TTT?CGC?CGC?CAG?CTG?TTT-3’

Wherein, GGT GGTOr CCA CCABe the protection base, CATATGFor NdeThe I restriction enzyme site, TGC TCT TCC GCAFor SapThe I restriction enzyme site;

At first primers F 1 and primers F 2 are carried out chain extension reaction, then take its product as DNA profiling, take F1 and F3 as primer, the PCR reaction makes the MHDBAY gene;

(2) build recombinant vectors pKY-MHDBAY:

Respectively the MHDBAY gene that plasmid pKYB and step (1) make is carried out double digestion with Ndel enzyme and Sapl enzyme, then the plasmid pKYB that the MHDBAY gene after double digestion is connected with double digestion is connected, obtain recombinant vectors pKY-MHDBAY;

(3) preparation table expression engineered bacteria pKY-MHDBAY/ER2566:

With recombinant vectors pKY-MHDBAY transform to express express intestinal bacteria ( Escherichia coli) ER2566, obtain expressing engineering bacteria pKY-MHDBAY/ER2566;

(4) expression and purifying:

1. abduction delivering engineering bacteria pKY-MHDBAY/ER2566 expresses " ternary " fusion rotein that is comprised of desired polypeptides, intein and chitin binding domain;

2. " ternary " fusion rotein that obtains carries out purifying with the chitin post, and " ternary " fusion rotein is adsorbed in the chitin post, and the solution that then contains mercaptan is crossed post and is full of in chitin band of column border, and reaction is cut in the N end-grain cutting of inducible protein intein; Then wash-out chitin post, obtain desired polypeptides C end thioesters; Dialysis desired polypeptides C end thioesters obtains stable hydrolysate;

3. utilize high-efficient liquid phase chromatogram technology purifying desired polypeptides, obtain high stability VPAC2 receptor specific agonist MHDBAY;

Step (4) contains solution composed as follows of mercaptan described in 2.: 20mM Tris-HCI, and 0.5M NaCl, 1mM EDTA, the 50mM beta-mercaptoethanol, pH 8.0.

4. preparation method according to claim 3, it is characterized in that: described in step (1), the condition of chain extension reaction is 94 ℃, 10min; Be down to 58 ℃ of 5min; 72 ℃ of 10min.

5. preparation method according to claim 3 is characterized in that: the condition of the reaction of PCR described in step (1) is that reaction conditions is: 95 ℃ of 5min; 95 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 30 circulations; 72 ℃ are extended 10min.

6. preparation method according to claim 3 is characterized in that:

Step (4) 2. described in the reaction condition be 16 ℃ the reaction 24 hours;

Step (4) 2. described in solution composed as follows of wash-out chitin post: 20mM Tris-HCl, 500mM NaCl, 1mM EDTA, pH 8.0.

7. preparation method according to claim 3 is characterized in that:

Step (4) utilizes the step of high-efficient liquid phase chromatogram technology purifying desired polypeptides as follows described in 3.:

A, mobile phase A obtain for add trifluoroacetic acid in volume percent 10% acetonitrile, and the final concentration of TFA is volume percent 0.1%; Mobile phase B is to add trifluoroacetic acid in 100% acetonitrile, and the final concentration of TFA is volume percent 0.1%; Flow velocity 1mL/min, 20 min linear gradient elutions;

In B, linear gradient elution, Mobile phase B by volume percent 0～60%, is collected the desired polypeptides elution peak, and it is 218nm that photoabsorption detects wavelength.

8. high stability VPAC2 receptor specific agonist MHDBAY claimed in claim 1 is applied to prepare the medicine for the treatment of diabetes.

Images