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Recombination fusion protein PACAP-PTD (Pituitary Adenylate Cyclase Activating Peptide-Protein Transduction Domain) as well as expression method and application thereof

A PACAP-PTD and fusion protein technology, which is applied in the field of recombinant fusion protein PACAP-PTD and its expression, can solve the problems of no related reports, PACAP polypeptide delivery, etc., and achieve the effect of improving drug access and expanding the scope of application

Inactive Publication Date: 2011-06-01
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, no one has used the delivery function of PTD to transport PACAP polypeptide into the target site, so as to achieve the cure of related diseases, and there are no related reports

Method used

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  • Recombination fusion protein PACAP-PTD (Pituitary Adenylate Cyclase Activating Peptide-Protein Transduction Domain) as well as expression method and application thereof
  • Recombination fusion protein PACAP-PTD (Pituitary Adenylate Cyclase Activating Peptide-Protein Transduction Domain) as well as expression method and application thereof
  • Recombination fusion protein PACAP-PTD (Pituitary Adenylate Cyclase Activating Peptide-Protein Transduction Domain) as well as expression method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1 Preparation of recombinant fusion protein PACAP-PTD

[0056] The preparation of the recombinant fusion protein PACAP-PTD in this embodiment includes the following steps:

[0057] 1. Amplification and cloning of PACAP-PTD gene:

[0058] First design and synthesize 3 primers:

[0059] Primer 1, the nucleotide sequence of which is shown in SEQ ID NO: 4;

[0060] Primer 2, the nucleotide sequence of which is shown in SEQ ID NO: 5;

[0061] Primer 3, the nucleotide sequence of which is shown in SEQ ID NO:6.

[0062] In the above three primers, N represents the protected base, CATATG is the NdeI restriction site, CTCGAG It is the XhoI restriction site.

[0063] Two-step PCR:

[0064] (1) The first step of PCR, using the existing PACAP gene as a template, the PCR reaction system is: primer 2 (0.01A / μL) 1 μL, primer 3 (0.01A / μl) 1 μL, plasmid containing PACAP gene 1 μL, dNTP 10μL, 10×TaKaRa Ex Buffer 10μL, TaKaRa Ex Taq Enzyme 1μL and H 2 O 76μL; PCR reac...

Embodiment 2

[0079] Example 2 Recombinant fusion protein PACAP-PTD promotes PAC1-CHO cell proliferation

[0080] PAC1-CHO cells specifically expressing the PACAP receptor PAC1 were plated in a 96-well plate, and when the cell density reached about 80%, serum-free culture (starvation culture) was used for overnight culture, and gradient concentrations of PACAP-PTD (1nM, 10nM, 100nM, 1000nM) were incubated, and the cell viability was measured by MTT after 24 hours.

[0081] The result is as Figure 4 As shown, PACAP-PTD has the activity of significantly (P<0.01) promoting the proliferation of PAC1-CHO, indicating that PACAP-PTD can effectively activate the PAC1 receptor in vivo.

Embodiment 3

[0082] Example 3 Functional Detection of the Recombinant Fusion Protein PTD-PACAP Penetrating the Blood-Brain Barrier Membrane

[0083] Use the FITC (fluorescein isothiocyanate) labeling kit to fluorescently label PACAP-PTD and PACAP, measure the concentration of the labeled protein and the fluorescence value of the labeled protein, and calculate the polypeptide labeling efficiency (that is, the value of FITC per mol polypeptide).

[0084] KM mice were randomly divided into two groups according to body weight, and were injected intraperitoneally: PACAP-FITC 0.1nmol / kg; and PACAP-PTD-FITC 0.1nmol / kg; 6 hours after injection, the mice were killed by cervical dislocation, and the brain tissues of the mice were collected. Rinse several times with PBS. Dispense into EP tubes at a ratio of 150mg wet brain tissue / 1ml PBS, ultrasonically break for 45s, centrifuge at 16000g for 30min at 4°C, take 200ul of the supernatant and add it to a 96-well plate, use a fluorescence spectrophotomet...

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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 [0001] The invention relates to the field of genetic engineering, in particular to a recombinant fusion protein PACAP-PTD and its expression method and application. Background technique [0002] Pituitary adenylate cyclase activating polypeptide (pituitary adenylate cyclase activating polypeptide, PACAP) was discovered in 1989. It is a neuropeptide with important biological functions secreted by the pituitary gland or autocrine and paracrine in the target tissue. A new member of the secretin / glucagon / VIP family. PACAP has three receptors: PAC1 is the specific receptor of PACAP; VPAC1 and VPAC2 are the co-receptors of PACAP and vasoactive intestinal peptide (VIP); PACAP has the same agonistic function on the three receptors. PACAP and its receptors are widely distributed in the body, mainly in the central nervous system, peripheral nervous system, testis, ovary, respiratory tract, lung, pancreas and other non-nervous tissues. It has been confirmed that the b...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21A61K38/17A61K47/48A61P1/04A61P3/00A61P3/04A61P3/10A61P7/02A61P9/00A61P9/04A61P9/10A61P9/12A61P13/12A61P15/00A61P15/10A61P15/16A61P15/18A61P21/00A61P25/00A61P25/04A61P25/18A61P25/22A61P25/28A61P27/02A61P29/00A61P31/00A61P35/00A61P43/00A61K47/64
Inventor 余榕捷陈建苏丁勇李娟黄霖赵秀丽汤翠翠
Owner JINAN UNIVERSITY
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