CN102079790A - Recombination fusion protein PACAP-PTD (Pituitary Adenylate Cyclase Activating Peptide-Protein Transduction Domain) as well as expression method and application thereof - Google Patents
Recombination fusion protein PACAP-PTD (Pituitary Adenylate Cyclase Activating Peptide-Protein Transduction Domain) as well as expression method and application thereof Download PDFInfo
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Abstract
The invention discloses a recombination fusion protein PACAP-PTD (Pituitary Adenylate Cyclase Activating Peptide-Protein Transduction Domain) as well as an expression method and application thereof. The amino acid sequence of the recombination fusion protein PACAP-PTD provided by the invention is shown as SEQ ID NO: 1 and the nucleotide sequence encoding the protein is shown as SEQ ID NO: 2. The recombination fusion protein PACAP-PTD provided by the invention has biological activity of PACAP and gives a function of transcellular barrier transport of the PTD to the PACAP to ensure that the function of cellular barrier traversing transport can be provided, therefore, blood brain barrier, cornea, qi-blood barrier, testis barrier, endothelium and mucosa organization and the like can be effectively traversed; the application value of the PACAP-PTD in diseases related to parts of brain, eyes, lung, genitals and the like can be greatly enhanced; the route of administration can be improved; and the application range can be extended.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to a kind of recombination fusion protein PACAP-PTD and expression method and application.
Background technology
Pituitary adenylate cyclase-activating polypeptide. (pituitary adenylate cyclase activating polypeptide, PACAP) be discovery in 1989, by pituitary secretion or purpose organize the nerve polypeptide that important biomolecule is learned function that has of autocrine and paracrine, to belong to be secretin/glucagon/ newcomer in the VIP family.PACAP has a special acceptor that 3 acceptor: PAC1 are PACAP; VPAC1 and VPAC2 are the coreceptors of PACAP and vasoactive intestinal peptide (VIP); PACAP has identical exciting function to 3 acceptors.PACAP and acceptor thereof are widely distributed in vivo, mainly are distributed in the non-nervous tissues such as central nervous system, peripheral nervous system and testis, ovary, respiratory tract, lung, pancreas etc.Having confirmed is at present had by the biological function of PACAP mediation: neurotransmitter/modified, neuroprotective cell; Nerve injury is repaired; Regulate vascular function; Promote hormone secretion, the endocrine regulation balance; Regulate the generation of gonad function and sexual cell; Participate in the digestion activity, regulate the energy metabolism balance; Participate in regulating immunity system; Regulate cell proliferation and differentiation; Sleep is relevant with ingesting; Relevant with learning and memory; Relevant with some psychological feelings; Relevant with the physiological clock of animal; Or the like.Have and studies show that PACAP by activating its special acceptor, effectively promotes proliferation of neural stem cells and differentiation.Therefore, PACAP has prevention, treatment nerve injury, the potential of relevant nerve diseases such as neural reparation, nervous disorder.
PACAP is that 38 amino acid are formed, do not possess and initiatively passes through various biological barriers, as: the function of hemato encephalic barrier, blood-testis barrier, blood vessel endothelium barrier, ABB, placental barrier, cornea, inner each confluent monolayer cells of eye etc., can only propagate by diffusion.
PTD (Protein transduction domain) is the core area of wearing the film function that has that is found at first in the HIV virus TAT albumen; Form by 11 amino acid YGRKKRRQRRR, contain 8 basic aminoacidss, with higher positive charge, and form stable a spirane structure under the physiological condition.PTD has powerful delivery potential, exogenous protein, DNA, RNA, chemicals etc. can be transported in the cell, even can pass through hemato encephalic barrier.This process is not subjected to the restriction of compound/complex molecule type and size, and host cell is not had remarkable toxic side effect.
The someone utilizes the delivery function of PTD that the PACAP polypeptide is transported and enters target site as yet at present, thereby realizes the healing of relative disease, and also not seeing has relevant report.
Summary of the invention
The objective of the invention is to the deficiency that exists according to prior art, provide a kind of and both can and wear the cytolemma transportation, possess the bioactive recombination fusion protein PACAP-PTD of PACAP again by hemato encephalic barrier.
Another purpose of the present invention is to provide the nucleotide sequence of a kind of above-mentioned recombination fusion protein PACAP-PTD that encodes.
Another purpose of the present invention is to provide a kind of recombinant plasmid that contains the nucleotide sequence of the above-mentioned recombination fusion protein PACAP-PTD of above-mentioned coding.
Another purpose of the present invention is to provide a kind of expression engineering bacteria that contains above-mentioned recombinant plasmid.
Another purpose of the present invention is to provide the expression method of above-mentioned recombination fusion protein PACAP-PTD.
A further object of the invention is to provide the application of above-mentioned recombination fusion protein PACAP-PTD.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
A kind of recombination fusion protein PACAP-PTD is made up of 55 amino acid, and its aminoacid sequence is shown in SEQID NO:1.Encode the nucleotide sequence of gene of this proteic recombination fusion protein PACAP-PTD shown in SEQ ID NO:2.
A kind of recombinant plasmid contains the gene of the described recombination fusion protein PACAP-PTD of claim 2 in this recombinant plasmid.
A kind of expression engineering bacteria, this project bacterium are that the described recombinant plasmid transformed e. coli host bacteria of claim 3 is made up and gets.
Recombination fusion protein PACAP-PTD of the present invention adopts conventional gene recombination method preparation and gets, and comprises the steps:
(1) adopt primer to carry out pcr amplification, obtain the gene of nucleotide sequence shown in SEQ ID NO:2, described primer sequence is shown in SEQ ID NO:4～6;
(2) the gained gene is obtained recombinant plasmid through double digestion, clone's structure;
(3) with the recombinant plasmid transformed e. coli host bacteria, the construction expression engineering bacteria;
(4) with inductor abduction delivering engineering bacterium expression reorganization target protein;
(5) cutting of purified, the mercaptan of reorganization target protein and hydrolysis obtain recombinant protein PACAP-PTD.
As a kind of preferred version, double digestion described in the expression method step of the present invention (2) is NdeI and XhoI double digestion.
As a kind of preferred version, inductor described in the expression method step of the present invention (4) is sec.-propyl-β-D thiogalactoside.
As a kind of preferred version, purifying is to use the chitin column purification described in the expression method step of the present invention (5); The N end-grain cutting that described mercaptan cutting is a mercaptan inducible protein intein is cut; Peptide C end thioesters hydrolysis during described hydrolysis.
As a kind of most preferably scheme, the step of expression method of the present invention is as follows:
(1) amplification PACAP-PTD gene
The present invention designs synthetic 3 primers, adopts two-step pcr method amplification PACAP-PTD gene:
In above-mentioned three primers, N representative protection base,
CATATGBe the NdeI restriction enzyme site,
CTCGAGBe the XhoI restriction enzyme site.
Two-step pcr: be template with existing P ACAP gene at first, primer 1 and 2 carries out first round PCR; Being template with first round PCR product then, is primer with primer 1 and 3, carries out second and takes turns PCR, obtains the PACAP-PTD gene, and described its nucleotide sequence of PACAP-PTD gene is shown in SEQ ID NO:2.
(2) construction recombination plasmid
Carry out enzyme with NdeI and XhoI and cut, with PACAP-PTD gene clone construction recombination plasmid in the plasmid;
(3) construction expression engineering bacteria
Above-mentioned gained recombinant plasmid transformed is expressed e. coli host bacteria, construction expression engineering bacteria; E. coli host bacteria is selected E.coli.ER2566 for use.
(4) with inductor sec.-propyl-β-D thiogalactoside abduction delivering reorganization target protein;
(5) target protein of Biao Daing is through the chitin column purification, and the N end-grain cutting of mercaptan inducible protein intein is cut, and discharges desired polypeptides C end thioesters, and the hydrolysis of peptide C end thioesters obtains recombination fusion protein PACAP-PTD.
The present invention utilizes Principles of Gene Engineering and technology to realize PACAP and the preparation of the new fusion protein PACAP-PTD that PTD merges and the research of biological function of reorganization first.The activity of the existing PACAP of PACAP-PTD can be striden the barrier cell transportation again, passes hemato encephalic barrier, blood-testis barrier, cornea, each confluent monolayer cells of intraocular part; Make its route of administration obtain to optimize, range of application is expanded.Must inject by encephalocoele as polypeptide such as former PACAP, VIP, just can enter brain; But PACAP-PTD abdominal injection or intravenous injection can enter the brain effect; PACAP-PTD also can enter the intraocular part aqueous humor by cornea, acts on the eyeground nerve.And the function of passing through barrier cell can make the PACAP-PTD can be by transdermal or saturating mucosal absorption, thereby improves application efficiency and the range of application of PACAP-PTD.
Recombination fusion protein PACAP-PTD of the present invention can be used for diagnosis; treatment and PACAP or the relevant disease of its acceptor; as the energy Metabolic disorder; diabetes (comprise the I type; the II type); fat; obesity-related disease; metabolic disturbance; the metabolism syndromes of being correlated with; brain injury; cerebral ischemia; prevent nerve cell death; injured nerve cytoprotective and regeneration; anti-inflammatory; impotence; hyposexuality; infertile; Female sexual dysfunction; neurodynia; neuropathy; dizzy; anxiety disorder; psychosis; memory impairment; dull-witted; Cog Disorg; central nervous system disease; migraine; nerve is shunk; myocardial ischemia; myocardial infarction/fibrosis; arteriosclerosis; the flesh disease of shrinking; stomach ulcer; hypertension; interior toxicogenic shock; thrombosis; retinopathy; neuratorphy; neuroprotective; nerve injury is repaired; cardiovascular disorder; renal failure; heart failure; promote apoptosis of tumor cells; suppress tumor cell proliferation; antitumor; and any and PACAP/VIP diseases associated, or the like.
Compared with prior art, the present invention has following beneficial effect:
1. recombination fusion protein PACAP-PTD of the present invention had both had the biologic activity of PACAP, because of containing PTD, strode the function that biological barrier transports and possess again;
2. cell levels discovers that recombination fusion protein PACAP-PTD of the present invention has the function that promotes PAC1-CHO cell proliferation, illustrates that PACAP-PTD has the activity of PACAP;
3. experimentation on animals found that, PACAP-PTD can not only effectively pass through hemato encephalic barrier, and can pass cornea tissue and enter aqueous humor.The medicinal function of PACAP is determined by numerous institutes, and the function of passing through the biological barrier transportation that PACAP-PTD gives will make it more easily pass biological barriers such as hemato encephalic barrier, cornea, ABB, blood-testis barrier and endothelial barrier, greatly improve it at brain, the using value at eye, lung and sexual organ official rank position.
4. recombination fusion protein PACAP-PTD of the present invention in Transdermal absorption, mucosal absorption, wear aspect such as cornea dispenser the research basis all be provided, and in view of the activity of the existing PACAP of this recombination fusion protein PACAP-PTD, can stride the biological barrier transportation again, can improve its route of administration and enlarge its range of application.
Description of drawings
Fig. 1 is a recombination fusion protein PACAP-PTD structural representation;
Fig. 2 is the SDS-PAGE electrophorogram of recombination fusion protein PACAP-PTD purifying;
Wherein, M is albumen marker, 1 for before thalline IPTG induces, 2 for after thalline IPTG induces, 3 for thalline IPTG induces the broken precipitation in back, and 4 for thalline IPTG induces broken supernatant afterwards, and 5 cross chitin post effluent liquid for supernatant, 6 for washing the post effluent liquid, and 7/8/9 is mercaptan cutting back elutriant (containing target protein); 10 for conjugated protein on the column packing of mercaptan cutting back;
Fig. 3 is a PACAP-PTD flight mass spectrum mass spectrum as a result;
Fig. 4 measures for PACAP-PTD promotes the PAC1-CHO proliferation activity;
Fig. 5 passes through the efficiency test of mouse brain barrier for PACAP-PTD;
Fig. 6 passes through the efficiency test of mouse cornea for PACAP-PTD;
Fig. 7 passes through the efficiency test of rabbit corneal for PACAP-PTD.
Embodiment
Below in conjunction with specific embodiment the present invention is done description further, but specific embodiment is not done any qualification to the present invention.
The preparation of embodiment 1 recombination fusion protein PACAP-PTD
The preparation of present embodiment recombination fusion protein PACAP-PTD comprises the steps:
1.PACAP-PTD the amplification of gene and clone:
At first design and synthesize 3 primers:
In above-mentioned three primers, N representative protection base,
CATATGBe the NdeI restriction enzyme site,
CTCGAGBe the XhoI restriction enzyme site.
Two-step pcr:
(1) the first step PCR, with existing P ACAP gene is template, and the PCR reaction system is: primer 2 (0.01A/ μ L) 1 μ L, primer 3 (0.01A/ μ l) 1 μ L, the plasmid 1 μ L that contains the PACAP gene, dNTP 10 μ L, 10 * TaKaRa Ex Buffer, 10 μ L, TaKaRa Ex Taq enzyme 1 μ L and H
2O 76 μ L; The PCR reaction conditions is: 94 ℃, and 4min; 94 ℃, 45sec, 55 ℃, 45sec, 72 ℃, 60sec, 35 circulations; 72 ℃, 10min;
(2) second step PCR, with the first step PCR reaction product is template, and the PCR reaction system is: primer 1 (0.01A/ μ L) 1 μ L, primer 3 (0.01A/ μ l) 1 μ L, the first step PCR reaction solution 1 μ L, dNTP10 μ L, 10 * TaKaRa Ex Buffer, 10 μ L, TaKaRa Ex Taq enzyme 1 μ L and H
2O 76 μ L; The PCR reaction conditions is: 94 ℃, and 4min; 94 ℃, 45sec, 58 ℃, 45sec, 72 ℃, 60sec, 35 circulations; 72 ℃, 10min.
The involved reagent of above-mentioned two-step pcr is commercially available or adopts all can that test kit wears.
2. construction of recombinant plasmid:
With NdeI and XhoI, the PACAP-PTDX gene clone is arrived between the NdeI and XhoI site of plasmid pKYB (commercially available) construction recombination plasmid pKY-PACAP-PTD;
3. express the structure of engineering bacteria:
Recombinant plasmid pKY-PACAP-PTD transforms expressive host bacterium E.coli Strain ER2566 (commercially available), construction expression engineering bacteria pKY-PACAP-PTD-ER;
Inductor IPTG induce by desired polypeptides, albumen intein and chitin binding domain (Chitin binding domain, CBD) " ternary " Expression of Fusion Protein of Zu Chenging:
The picking mono-clonal contains the LB substratum of 50 μ g/mL kantlex in 5mL, shakes the bacterium overnight incubation, is connected to the LB substratum that 1L contains the 50mg/L kantlex with 1: 20 (volume ratio), and 37 ℃ are shaken bacterium to OD
600Be 0.5-0.8, add inductor sec.-propyl-β-D thiogalactoside (IPTG), induce expressing fusion protein 4h for 30 ℃ to final concentration 1mmol/L, centrifugal collection thalline, be resuspended in solution A (20mMTris.HCl, 500mM NaCl, the 1mM EDTA of 60mL, pH8.0), ultrasonication then, 20, the centrifugal 30min of 000g, collect supernatant, as last all product of subsequent purification.
5. the purifying of fusion rotein:
Get 10mL chitin pearl filling 2.5 * 10cm chromatography column, solution A (1mM EDTA, pH 8.0 for 20mM Tris-HCl, 0.5MNaCl) is washed post; Get above-mentioned all product with sample on the flow velocity of 0.5mL/min; Solution A is with 2mL/min flush away foreign protein, 30mL solution B (20mM Tris-HCl, 0.5M NaCl, 1mM EDTA, the 50mM beta-mercaptoethanol, pH 8.0) the quick post of crossing, make the beta-mercaptoethanol uniform distribution and soak filler in the post, 16 ℃ of inducible protein peptide cutting 16h; Solution A wash-out desired polypeptides is in charge of collection, and every 2mL is a pipe, and desired polypeptides is present in preceding 10 pipes mostly.
SDS-PAGE identifies desired polypeptides, electrophoresis result as shown in Figure 2, as can be seen from the figure the fusion rotein part is present in broken supernatant with solvable state, and can effectively combine (the purpose band disappears after crossing post) with the chitin post, after the mercaptan cutting, fusion rotein can effectively discharge desired polypeptides PACAP-PTD, and its electrophoresis position and expection match; And can detect former fusion rotein on the column packing and cut two kinds of albumen of rear fusion protein.
4 ℃ of dialysed overnight are removed beta-mercaptoethanol.
Recombinant polypeptide PACAP-PTD send Beijing Military Medical Science Institute to carry out flight mass spectrum and detects, and molecular weight is 6681.81 (Fig. 3), conforms to theoretical value.
The used LB substratum of present embodiment adopts those skilled in the art to prepare the conventional formulation that the LB substratum is adopted.
Adopt the PAC1-CHO cell of specifically expressing PACAP acceptor PAC1, be plated on 96 orifice plates, treat that cell density reaches about 80%, using serum-free culture (the hungry cultivation) instead spends the night, the PACAP-PTD (1nM, 10nM, the 100nM that add gradient concentration, 1000nM) hatch, measure cytoactive with MTT after 24 hours.
The result as shown in Figure 4, PACAP-PTD has significantly the activity that (P＜0.01) promotes PAC1-CHO propagation, illustrates that PACAP-PTD can effectively activate intravital PAC1 acceptor.
The Function detection that embodiment 3 recombination fusion protein PTD-PACAP wear the hemato encephalic barrier film
Utilize FITC (fluorescein isothiocyanate) labelling kit, to PACAP-PTD, PACAP carries out fluorescent mark, measures labelled protein concentration and labelled protein fluorescent value, calculates polypeptide marker efficient (being the value of the entrained FITC of every mol polypeptide).
The KM mouse is divided two groups at random by body weight, respectively abdominal injection: PACAP-FITC 0.1nmol/kg; With PACAP-PTD-FITC 0.1nmol/kg; Injection back 6h, mouse is put to death in the cervical vertebra dislocation, gets the cerebral tissue of mouse, with the PBS rinsing for several times.Ratio branch with 150mg weight in wet base cerebral tissue/1ml PBS is filled in the EP pipe, ultrasonication 45s, 4 ℃ of centrifugal 30min of following 16000g, getting supernatant 200ul adds in 96 orifice plates, use spectrophotofluorometer, exciting light 495nm detects the emissive porwer of each sample at 520nm.Polypeptide passes through hemato encephalic barrier efficient (%)=(sample fluorescence value-blank fluorescent value)/(labeling effciency W * albumen applied sample amount).
If the efficient of wearing hemato encephalic barrier of PACAP-FITC is 1, the ratio of the efficient of wearing hemato encephalic barrier of PACAP-PTD-FITC group and PACAP-FITC group is the ordinate zou mapping, result's PACAP-PTD-FITC group efficient of wearing hemato encephalic barrier as shown in Figure 5 is 5.26 ± 0.17 times of PACAP-FITC group, this explanation PACAP-PTD effectively passes through hemato encephalic barrier than PACAP, enters cerebral tissue.
The Function detection that embodiment 4 recombination fusion protein PACAP-PTD wear mouse cornea barrier
Utilize FITC (fluorescein isothiocyanate) labelling kit, to PACAP-PTD, PACAP carries out fluorescent mark, measures labelled protein concentration and labelled protein fluorescent value, calculates polypeptide marker efficient (being the value of the entrained FITC of every mol polypeptide).
The KM mouse is divided into three groups at random by body weight, 8 every group: blank (PBS), PACAP-FITC group, PACAP-PTD-FITC group.During mouse experiment:, drip the corresponding laboratory sample of each group of 50 μ L at eye earlier with 10% chloral hydrate anesthesia mouse.Placed after one hour the dark place, and mouse is put to death in the cervical vertebra dislocation, gets the complete eyeball of mouse, puts into the EP pipe, adds the PBS of 1ml, in ultrasonication 30s, repeats twice.15000g in refrigerated centrifuge, centrifugal 30min gets supernatant 200 μ L and adds in 96 orifice plates, uses spectrophotofluorometer, and exciting light 495nm detects the fluorescent value of each sample at 520nm.Calculate polypeptide and pass through cornea efficient (%)=(sample fluorescence value-blank fluorescent value)/(labeling effciency W * albumen applied sample amount).
If the efficient of wearing the mouse cornea of PACAP-FITC is 1, the ratio of the efficient of wearing the mouse cornea of PACAP-PTD-FITC group and PACAP-FITC group is the ordinate zou mapping, result's efficient of PACAP-PTD-FITC group mouse cornea as shown in Figure 6 is 6.67 ± 0.19 times that PACAP-FITC organizes, this explanation PACAP-PTD effectively passes through the mouse cornea than PACAP, enters inside ofeye.
The Function detection that embodiment 5 recombination fusion protein PTD-PACAP wear the rabbit cornea barrier
Utilize FITC (fluorescein isothiocyanate) labelling kit, to PACAP-PTD, PACAP carries out fluorescent mark.Measure labelled protein concentration and labelled protein fluorescent value, calculate polypeptide marker efficient (being the value of the entrained FITC of every mol polypeptide).
New zealand white rabbit is divided into three groups at random by body weight, 5 every group: blank (PBS), PACAP-FITC group, PACAP-PTD-FITC group.Rabbit when experiment: splash into the laboratory sample of 100 μ L correspondences at eye, make sample fully soak into whole eyeball.Placed after one hour the dark place, with the syringe eye that punctures, draws aqueous humor, in the EP pipe of packing into, 15000g in refrigerated centrifuge, centrifugal 5min gets supernatant 200 μ L and adds in 96 orifice plates, use spectrophotofluorometer, exciting light 495nm detects the fluorescent value of each sample at 520nm.
Polypeptide passes through cornea efficient (%)=(sample fluorescence value-blank fluorescent value)/(labeling effciency W * albumen applied sample amount).
If the efficient of wearing rabbit corneal of PACAP-FITC is 1, the ratio of the efficient of wearing rabbit corneal of PACAP-PTD-FITC group and PACAP-FITC group is the ordinate zou mapping, result's efficient of PACAP-PTD-FITC group rabbit corneal as shown in Figure 7 is 6.17 ± 0.23 times that PACAP-FITC organizes, this explanation PACAP-PTD effectively passes through rabbit corneal than PACAP, enters aqueous humor.
Claims (10)
1. recombination fusion protein PACAP-PTD, its aminoacid sequence is shown in SEQ ID NO:1.
2. the gene of coding claim 1 described recombination fusion protein PACAP-PTD, the nucleotide sequence that it is characterized in that described gene is shown in SEQ ID NO:2.
3. recombinant plasmid is characterized in that containing in the described recombinant plasmid gene of the described recombination fusion protein PACAP-PTD of claim 2.
4. express engineering bacteria for one kind, it is characterized in that described engineering bacteria is that the described recombinant plasmid transformed e. coli host bacteria of claim 3 is made up and gets.
5. the expression method of the described recombination fusion protein PACAP-PTD of claim 1 is characterized in that described method comprises the steps:
(1) adopt primer to carry out pcr amplification, obtain the gene of nucleotide sequence shown in SEQ ID NO:2, described primer sequence is shown in SEQ ID NO:4～6;
(2) the gained gene is obtained recombinant plasmid through double digestion, clone's structure;
(3) with the recombinant plasmid transformed e. coli host bacteria, the construction expression engineering bacteria;
(4) with inductor abduction delivering engineering bacterium expression reorganization target protein;
(5) cutting of purified, the mercaptan of reorganization target protein and hydrolysis obtain recombinant protein PACAP-PTD.
6. according to the expression method of the described recombination fusion protein PACAP-PTD of claim 5, it is characterized in that double digestion described in the step (2) is NdeI and XhoI double digestion.
7. according to the expression method of the described recombination fusion protein PACAP-PTD of claim 5, it is characterized in that inductor described in the step (4) is sec.-propyl-β-D thiogalactoside.
8. according to the expression method of the described recombination fusion protein PACAP-PTD of claim 5, it is characterized in that purifying is to use the chitin column purification described in the step (5); The N end-grain cutting that described mercaptan cutting is a mercaptan inducible protein intein is cut; Peptide C end thioesters hydrolysis during described hydrolysis.
9. the application of the described recombination fusion protein PACAP-PTD of claim 1 in the medicine of preparation diagnosis, treatment and PACAP or its receptor associated diseases.
10. according to the application of the described recombination fusion protein PACAP-PTD of claim 9; it is characterized in that described and PACAP or its receptor associated diseases are the energy metabolism imbalance; diabetes I type; diabetes type II; fat; obesity-related disease; metabolic disturbance; the metabolism syndromes of being correlated with; brain injury; cerebral ischemia; prevent nerve cell death; injured nerve cytoprotective and regeneration; anti-inflammatory; impotence; hyposexuality; infertile; Female sexual dysfunction; neurodynia; neuropathy; dizzy; anxiety disorder; psychosis; memory impairment; dull-witted; Cog Disorg; central nervous system disease; migraine; nerve is shunk; myocardial ischemia; myocardial infarction/fibrosis; arteriosclerosis; the flesh disease of shrinking; stomach ulcer; hypertension; interior toxicogenic shock; thrombosis; retinopathy; neuratorphy; neuroprotective, the nerve injury reparation; cardiovascular disorder; renal failure; heart failure; promote apoptosis of tumor cells; suppress tumor cell proliferation; antitumor or other and PACAP/VIP diseases associated.
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| CN103044556A (en) * | 2013-01-14 | 2013-04-17 | 余榕捷 | Recombined fusion protein VIP-TAT and application thereof |
| CN103044556B (en) * | 2013-01-14 | 2014-05-28 | 余榕捷 | Recombined fusion protein VIP-TAT and application thereof |
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| CN103145851A (en) * | 2013-02-22 | 2013-06-12 | 暨南大学 | Recombinant protein PACAP38-NtA, and coding gene and application thereof |
| CN103145851B (en) * | 2013-02-22 | 2014-07-02 | 暨南大学 | Recombinant protein PACAP38-NtA, and coding gene and application thereof |
| CN111110830A (en) * | 2020-02-18 | 2020-05-08 | 暨南大学 | Application of bioactive polypeptide PACAP in preparation of medicine for improving fertility of obese men |
| CN113648418A (en) * | 2021-05-08 | 2021-11-16 | 南方医科大学 | Application of Apelin-APJ inhibitor in treating blood testis barrier injury |
| CN113648418B (en) * | 2021-05-08 | 2022-08-05 | 南方医科大学 | Application of Apelin-APJ inhibitor in preparation of medicine for treating blood testis barrier injury |
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