CN1557835A - Pituitary adenylate cyclase activated polypeptide derivatives and process for preparing the same - Google Patents
Pituitary adenylate cyclase activated polypeptide derivatives and process for preparing the same Download PDFInfo
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- CN1557835A CN1557835A CNA2004100151366A CN200410015136A CN1557835A CN 1557835 A CN1557835 A CN 1557835A CN A2004100151366 A CNA2004100151366 A CN A2004100151366A CN 200410015136 A CN200410015136 A CN 200410015136A CN 1557835 A CN1557835 A CN 1557835A
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- adenylate cyclase
- pdt
- derivative
- activating polypeptide
- pituitary adenylate
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Abstract
The present invention belongs to the field of gene engineering technology, and relates to one kind of adenylate cyclase activating polypeptide derivative. The derivative has amino acid sequence as shown in the sequence list and features one added methionine molecule in the amino terminal and one beta-mercaptoethanol molecule connected via thioester bond to the carboxyl terminal. Compared with natural and chemically synthesized adenylate cyclase activating polypeptide, the adenylate cyclase activating polypeptide derivative and its serial hydrolytic products have the same activity but raised stability. The present invention also provides the production process of the derivative.
Description
Technical field
The invention belongs to gene engineering technology field, relate to a kind of adenylate cyclase activating polypeptide derivative, and the production method of this derivative.
Background technology
Pituitary adenylate cyclase activating peptide (English name pituitary adenylate cyclase activatingpolypeptide, hereinafter to be referred as PACAP) and acceptor be distributed widely in central nervous system, peripheral nervous system and the non-nervous tissue, as brain, spinal cord, suprarenal gland, testis, ovary, liver, kidney, pancreas, pineal gland, heart, vertebra, neuroganglion, respiratory system and Digestive tract etc.PACAP regulates the second trustworthy historical record cAMP, IP3 and Ca in the target cell by activating special g protein coupled receptor on the target cell cytolemma
2+Concentration, thereby bring into play important biological function.Confirmed that at present PACAP has neurotransmitter, neuromodulator, neurotrophy and protection, regulated the secretion of Regular Insulin and other hormone, protection blood vessel epithelial cell prevents arteriosclerosis, regulates the effects such as generation of sexual cell.Natural PACAP is made up of 38 amino acid, adopts the method for chemosynthesis at present mostly, and cost costliness and yield rate are low.Because the derivative of PACAP also has identical biologic activity.Can reduce production costs so adopt engineered method to obtain the corresponding derivative of PACAP, and, can improve yield rate and indexs such as activity, stability thereof effectively by to the groping of production method.
Summary of the invention
The object of the present invention is to provide a kind of recombinant adenosine cyclase of acid activating polypeptide derivative;
Another object of the present invention is to provide the production method of recombinant adenosine cyclase of acid activating polypeptide derivative;
Still a further object of the present invention is to provide the application of recombinant adenosine cyclase of acid activating polypeptide derivative in the medicine of preparation treatment relative disease.
The present invention utilizes engineered method, by albumen intein (intein) shear derived peptide-PACAP that function prepares PACAP thioesters (PDT) of deriving certainly, it is that carboxyl terminal at natural PACAP has connected a beta-mercaptoethanol molecule (CH by thioester bond
2SHCH
2OH, β-mercaptoethanol, BME), itself and difference natural or the PACAP polypeptide that chemosynthesis produces are that PDT is 39 amino acid, first amino acid is methionine(Met), amino acid whose sequence is identical with natural PACAP since the 2nd to the 39th, but the PACAP with natural on the carboxyl that a last amino acid is Methionin (Lys) is different, see for details and see Fig. 2, Fig. 3 and Fig. 4, promptly wherein the molecular structure of last amino-acid residue of carboxyl terminal of PDT provided by the invention is Fig. 2, and its carboxyl terminal is a beta-mercaptoethanol molecule; And natural PACAP sees Fig. 3 at the amino-acid residue molecular structure of corresponding position, and its carboxyl terminal is a hydroxyl; The PACAP of chemosynthesis sees Fig. 4 at the amino-acid residue molecular structure of corresponding position in addition, and its carboxyl terminal is an amino.Can learn the derived peptide of PACAP of the present invention from above result--be inequality PDT) with the natural PACAP or the PACAP derivative of chemosynthesis.
The present invention can be used for preparing the medicine for the treatment of relative disease, and such medicine can be used for brain injury, cerebral ischemia, prevent nerve cell death, anti-inflammatory, impotence, hyposexuality, infertile, neurodynia, neuropathy, dizzy, anxiety disorder, memory impairment, dull-witted, Cog Disorg, central nervous system disease, migraine, nerve is shunk, myocardial ischemia, myocardial infarction/fibrosis, arteriosclerosis, diabetes, the flesh disease of shrinking, stomach ulcer, hypertension, interior toxicogenic shock, thrombosis, retinopathy, cardiovascular disorder, renal failure, treatment of diseases such as heart failure.
Below describe technical scheme of the present invention in detail:
The derived peptide of PACAP provided by the present invention--the PACAP thioesters (PDT) of deriving, it specifically prepares route and is:
At first, use method well known to those skilled in the art, design primer, the method synthetic Pituitary adenylate cyclase-activating polypeptide. gene of employing PCR;
Secondly, the gene clone of synthetic Pituitary adenylate cyclase-activating polypeptide. in plasmid vector, is successfully constructed recombinant expression vector, wherein plasmid vector is preferably pTYB1, make up and recombinant expression vector called after pTY-PDT.
Express the host bacterium again that (wherein said host bacterium is preferably intestinal bacteria, preferably intestinal bacteria ER2566 for chitin binding domain, CBD) " ternary " fusion rotein of Gou Chenging by PACAP, intein and chitin binding domain;
Again expression product and affinity column are carried out affinity chromatography, this affinity column is the chitin post preferably; " ternary " fusion rotein is by CBD and affinity column specific combination, behind the flush away foreign protein, induce intein that target protein PACAP is cut down from fusion rotein with inductor, this inductor preferably beta-mercaptoethanol (β-mercaptoethanol, BME);
At last, prepared derived peptide protein carboxyl terminal connects a β mercaptoethanol molecule by thioester bond, becomes the albumen thioesters, is a kind of PACAP derivative, claims PACAP the thioesters (English name PACAPderived thioester is hereinafter to be referred as PDT) of deriving.
Above-described concrete preparation route can be with reference to figure 5.In addition, target protein PACAP cuts down from fusion rotein, and promptly the synoptic diagram of the generation mechanism of PDT is seen Fig. 6.
The activity of PDT is identical with native protein after testing, but the external activity transformation period than native protein to 10 times of the youthful and the elderlys, illustrate that PDT is higher than the stability of native protein.
What deserves to be explained is, hydrolysis reaction can take place in PDT in water, its further hydrolysis formation-SH of β mercaptoethanol molecular radical meeting that connects by thioester bond ,-SOH ,-OH ,-groups such as H, but activity test proves that these hydrolysed mix do not influence its activity.
The Pituitary adenylate cyclase-activating polypeptide. derivative PDT that obtains is owing to have neurotransmitter; neuromodulator; neurotrophy and protection; regulate the secretion of Regular Insulin and other hormone; protection blood vessel epithelial cell; prevent arteriosclerosis; regulate the effects such as generation of sexual cell; therefore it also can be used for the further medicine of preparation treatment relative disease, and such medicine can be used for treatment and comprises brain injury; cerebral ischemia; prevent nerve cell death; anti-inflammatory; impotence; hyposexuality; infertile; neurodynia; neuropathy; dizzy; anxiety disorder; memory impairment; dull-witted; Cog Disorg; central nervous system disease; migraine; nerve is shunk; myocardial ischemia; myocardial infarction/fibrosis; arteriosclerosis; diabetes; the flesh disease of shrinking; stomach ulcer; hypertension; interior toxicogenic shock; thrombosis; retinopathy; cardiovascular disorder; renal failure; diseases such as heart failure.
Below the present invention is described in further detail by specific embodiments and the drawings explanations, but the content of embodiment and accompanying drawing is not used in qualification the present invention.
Description of drawings
What Fig. 1 showed is a beta-mercaptoethanol molecule;
What Fig. 2 showed is the last molecular structure that amino acid is Methionin (Lys) of carboxyl terminal of the derived peptide (PDT) of PACAP, and its carboxyl terminal has connected a beta-mercaptoethanol molecule;
What Fig. 3 showed is the last molecular structure that amino acid is Methionin (Lys) of carboxyl terminal of natural PACAP, and its carboxy terminal group is a hydroxyl;
What Fig. 4 showed is the last molecular structure that amino acid is Methionin (Lys) of carboxyl terminal of chemosynthesis PACAP, and its carboxy terminal group is amino;
Fig. 5 utilizes intein to prepare the synoptic diagram of PDT;
The generation mechanism of Fig. 6 PDT;
Fig. 7 PACAP gene synthesizes synoptic diagram;
The structure synoptic diagram of Fig. 8 recombinant expression plasmid pTY-PDT;
The PCR of Fig. 9 PACAP gene amplification and recombinant plasmid PTY-PDT identifies.1.PACAP gene wherein; 2.3. negative clone's PCR result; 4.5. the result of positive products PCR;
The sequencer map of Figure 10 PACAP gene;
Figure 11 contains the fusion rotein of the PDT that recombinates and the SDS-PAGE of reorganization PDT analyzes.M. protein relative molecular mass standard wherein; 1. inductive PTY-PDT/ER2566 expression system not; 2. inductive PTY-PDT/ER2566 expression system; 3. PDT recombinates;
The recombinate Westernblot of PDT of Figure 12.M. protein relative molecular mass standard wherein; 1. PDT recombinates;
The recombinate mass spectroscopy figure of PDT of Figure 13;
Figure 14 recombinates specific activity that the short S19901 cell cAmp of PDT and PACAP38 generates;
Figure 15 recombinate PACAP38 and reorganization PDT vitro stability relatively.
Specific embodiment
The amplification of embodiment 1PACAP gene and the structure of PTY-PDT plasmid
According to the aminoacid sequence of PACAP mature peptide, by its nucleotide sequence of colibacillary password preference design.The salvage primer, by shown in Figure 3, by PCR, external synthetic PACAP gene.3 primers of synthetic PACAP:
F1 5’ATG?CAC?TCT?GAC?GGC?ATC?TTC?ACA?GAC?TCT?TAT?TCC?CGCTAC?CGA?AAA?CAA?ATG3’；
F2 5’TCC?CGC?TAC?CGA?AAA?CAA?ATG?GCT?GTC?AAG?AAA?TAC?TTGGCG?GCC?GTG?CTA?GGG?AAA?AGG3’；
F3 5’TTA?CTA?TTT?GTT?TTT?AAC?CCT?CTG?TTT?ATA?CCT?TTT?CCC?TAGCAC?GGC?CGC3’
The structure of expressed fusion protein plasmid is seen Fig. 8.The design primer is introduced the Ndel recognition site in the upstream of PACAP gene, introduces the Sapl recognition site in the downstream; The PACAP gene is inserted among the PTYB1, obtain expression vector PTY-PDT.The PCR of expression plasmid identifies and sees Fig. 9 that sequencing result is seen Figure 10.
The expression of embodiment 2 recombination fusion proteins and the cutting of target protein
Expression plasmid transforms expressive host bacterium E.coli Strain ER2566, the picking mono-clonal shakes the bacterium overnight incubation, being connected to the LB substratum that 1L contains the 50mg/L kantlex at 1: 20,37 ℃ shake bacterium to OD be 0.5-0.8, add IPTG to final concentration 0.3-0.5mmol/L, induce 3h for 30 ℃.Centrifugal collection thalline, and BufferA (20mMTris.HCl, 500mM NaCl, 1mM EDTA, pH7.5) resuspended, in 4 ℃ of following ultrasonications, centrifuging and taking supernatant.
Use 60mL Buffer A balance after getting 6mL chitin beads slurry dress post; With the flow velocity of＜=0.5mL/min with sample on the bacterial cell disruption supernatant; Wash post by the 1mL/min flow velocity with the Buffer A that is no less than 100mL, remove foreign protein.With the Buffer B of 18mL (20mM Tris.HCl, 500mM NaCl, 1mM EDTA, 50mM BME, pH7.5) wash post fast after, place 16h for 4 ℃.18mL Buffer A washes target protein PDT come.PDT identifies through SDS-PAGE and westernblot, and with its molecular weight of laser flying mass spectroscopy.The SDS-PAGE protein electrophoresis the results are shown in Figure 11, and the westernblot qualification result is seen Figure 12, and mass spectrum the results are shown in Figure 13.
The activity of embodiment 3PDT and PACAP38 and stability are relatively
After the S1990 cell dissociation of taking the logarithm vegetative period took off wall, adjusting concentration of cell suspension was 1 * 10
7Individual/mL, add the PDT and the PACAP38 of same concentrations respectively, be control group with the nutrient solution; After cultivating 5min, take out 200 μ L suspensions from experimental group and control group respectively, add Tricholroacetic Acid, fully shake mixing, centrifugal, get supernatant 0.5mL, after with the water saturation ether supernatant being washed, evaporate to dryness.Use Cyclic AMP EnzymeImmunoassay Kit and measure cAMP concentration.
Measure the activity data that cAmp generates in PDT and the short S1990 pancreatic cell of PACAP38 by as above method and see Table 1, graphic representation is seen Figure 14, and the result shows that PDT and PACAP38's is quite active.
Table 1
The stability of embodiment 4PDT and PACAP38 is measured
With the PDT and the PACAP of physiological saline configuration same concentrations, 4 ℃ of placements, per 5 days its activity of sampling and measuring.With 0 o'clock activity was 100%, drew active time history plot, and relatively both external activity transformation period, data see Table 2, and graphic representation is seen Figure 15.
By the as above method vitro stability of PDT and PACAP38 relatively, find to place about 5 days in room temperature, synthetic PACAP38 decay of activity 50%, the PDT room temperature was placed 30 days, and activity still keeps 75%, places about 50 days, and decay of activity is 50%.Therefore PDT has the external activity transformation period that is about 10 times at least than PACAP.
Table 2
Sequence table
＜110〉Bio-engineering Institute of Jinan University
<120>pituitary?adenylate?cyclase?activating?polypeptide?derivedthioester(PDT)
<130>no
<160>2
<170>PatentIn?version?3.2
<210>1
<211>117
<212>DNA
<213>Unknown
<220>
<223>The?codons?of?PDT?gene?were?modified
<400>1
atgcactctg?acggcatctt?cacagactct?tattcccgct?accgaaaaca?aatggctgtc 60
aagaaatact?tggcggccgt?gctagggaaa?aggtataaac?agagggttaa?aaacaaa 117
<210>2
<211>39
<212>PRT
<213>Unknown
<220>
<223>The?last?amino?acid?is?modified
<220>
<221>MISC_FEATURE
<222>(39)..(39)
<400>2
Met?His?Ser?Asp?Gly?Ile?Phe?Thr?Asp?Ser?Tyr?Ser?Arg?Tyr?Arg?Lys
1 5 10 15
Gln?Met?Ala?Val?Lys?Lys?Tyr?Leu?Ala?Ala?Val?Leu?Gly?Lys?Arg?Tyr
20 25 30
Lys?Gln?Arg?Val?Lys?Asn?Lys
35
Claims (9)
1. Pituitary adenylate cyclase-activating polypeptide. derivative PDT, its aminoacid sequence is characterized in that its carboxyl terminal connects a beta-mercaptoethanol molecule by thioester bond shown in sequence table, this molecular structure is seen accompanying drawing 1.
2. method of producing claim 1 Pituitary adenylate cyclase-activating polypeptide. derivative PDT, step is as follows:
(1) design primer, the method synthetic Pituitary adenylate cyclase-activating polypeptide. gene of employing PCR;
(2) with the gene clone of synthetic Pituitary adenylate cyclase-activating polypeptide. in plasmid vector, successfully construct recombinant expression vector, express in the host bacterium, its expression product is the fusion rotein that is made of Pituitary adenylate cyclase-activating polypeptide., intein and chitin binding domain;
(3) collect expression product, cross chromatography column and carry out affinity chromatography, the flush away foreign protein is crossed chromatography column with inductor more again, induces the intein cleavage of fusion proteins, obtains the mixture of Pituitary adenylate cyclase-activating polypeptide. derivative or hydrolysate.
3. as the production method of Pituitary adenylate cyclase-activating polypeptide. derivative PDT as described in the claim 2, it is characterized in that synthetic Pituitary adenylate cyclase-activating polypeptide. gene, its gene order is shown in sequence table.
4. as the production method of Pituitary adenylate cyclase-activating polypeptide. derivative PDT as described in the claim 2, it is characterized in that described plasmid vector is pTYB1.
5. as the production method of Pituitary adenylate cyclase-activating polypeptide. derivative PDT as described in the claim 2, it is characterized in that described recombinant expression vector is pTY-PDT.
6. as the production method of Pituitary adenylate cyclase-activating polypeptide. derivative PDT as described in the claim 2, it is characterized in that described host bacterium is intestinal bacteria ER2566.
7. as the production method of Pituitary adenylate cyclase-activating polypeptide. derivative PDT as described in the claim 2, it is characterized in that described chromatography column is the chitin post.
8. as the production method of Pituitary adenylate cyclase-activating polypeptide. derivative PDT as described in the claim 2, it is characterized in that described inductor is a beta-mercaptoethanol.
9. the described Pituitary adenylate cyclase-activating polypeptide. derivative of claim 1 PDT is in preparation treatment brain injury, cerebral ischemia, prevent nerve cell death, anti-inflammatory, impotence, hyposexuality, infertile, neurodynia, neuropathy, dizzy, anxiety disorder, memory impairment, dull-witted, Cog Disorg, central nervous system disease, migraine, nerve is shunk, myocardial ischemia, myocardial infarction/fibrosis, arteriosclerosis, diabetes, the flesh disease of shrinking, stomach ulcer, hypertension, interior toxicogenic shock, thrombosis, retinopathy, cardiovascular disorder, renal failure, application in the medicine of heart failure disease.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100360562C (en) * | 2005-10-11 | 2008-01-09 | 暨南大学 | Recombinant VPAC2 receptor specific agonist RMROM, its expression and use |
| CN101356191B (en) * | 2005-11-22 | 2012-05-30 | 遗传工程与生物技术中心 | Neuropeptides for the culture of aquatic organisms |
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2004
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100360562C (en) * | 2005-10-11 | 2008-01-09 | 暨南大学 | Recombinant VPAC2 receptor specific agonist RMROM, its expression and use |
| CN101356191B (en) * | 2005-11-22 | 2012-05-30 | 遗传工程与生物技术中心 | Neuropeptides for the culture of aquatic organisms |
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| CN1240716C (en) | 2006-02-08 |
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Granted publication date: 20060208 Termination date: 20110115 |