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Restructured fusion protein PTD-MAX and expression method and application thereof

A PTD-MAX and fusion protein technology, applied in the field of genetic engineering, can solve the problems of maxadilan polypeptide delivery and no related reports, and achieve the effects of improving drug access, promoting nerve growth, and expanding the scope of application

Inactive Publication Date: 2010-03-10
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, no one has used the delivery function of PTD to transport maxadilan 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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  • Restructured fusion protein PTD-MAX and expression method and application thereof
  • Restructured fusion protein PTD-MAX and expression method and application thereof
  • Restructured fusion protein PTD-MAX and expression method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 Preparation of recombinant fusion protein PTD-MAX

[0049] The preparation of the recombinant fusion protein PTD-MAX in this embodiment includes the following steps:

[0050] 1. Amplification and cloning of PTD-MAX gene:

[0051] First design and synthesize 3 primers:

[0052] Primer 1, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0053] Primer 2, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0054] Primer 3, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0055] In the above three primers, N represents the protected base, CATATG is the NdeI restriction site, CTCGAG It is the XhoI restriction site.

[0056] Two-step PCR:

[0057] (1) The first step of PCR, using the existing maxadilan 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 maxadilan gene 1 μL, dNTP 10μL, 10×TaKaRa Ex Buffer 10μL, TaKaRa Ex Taq Enzyme 1μL and H 2 O 76μL; PCR re...

Embodiment 2

[0072] Example 2 Effect of transient action of recombinant fusion protein PTD-MAX on blood glucose concentration in KM mice

[0073] Experimental materials: Clean grade (SPF grade) KM male mice, weighing 20±3g, were randomly divided into groups according to body weight, with 8 mice in each group.

[0074] This embodiment is divided into three groups: PTD-MAX group, maxadilan group and blank control group.

[0075] After the mice were fasted for 12 hours, intraperitoneal injections were made to KM male mice (the PTD-MAX group was injected with 50nmol / kg of recombinant fusion protein PTD-MAX, the maxadilan group was injected with 50nmol / kg of maxadilan protein, and the blank control group was injected with 50nmol / kg of physiological saline).

[0076] Before injection (0 min) and after 10 min, 30 min, and 60 min after injection, blood was collected from the tail of the mice, and blood sugar changes within 60 min were measured with an automatic blood glucose analyzer (OneTouch Ul...

Embodiment 3

[0078] Example 3 Functional Detection of Recombinant Fusion Protein PTD-MAX Penetrating Cell Membrane

[0079] Use FITC (fluorescein isothiocyanate) labeling kit to fluorescently label PTD-MAX and maxadilan. When CHO cells grow to 80% confluence, remove the original medium, add serum-free medium, and add FITC-labeled PTD-MAX-FITC and maxadilan-FITC, to a final concentration of 200nmol / L, add an equal volume of FITC labeling solution as a control, incubate the cells for 1 hour, rinse the cells with PBS 4 times, 10min each time, to remove free protein. Add 1 mL of ice-cold methanol, fix at -20°C for 5 min, then fix with 1 mL of 1% formaldehyde, fix at room temperature for 10 min, wash with PBS several times, and observe with an inverted fluorescence microscope.

[0080] The result is as Figure 5 As shown, the superimposition of the images observed by the fluorescence microscope and the ordinary microscope cell images revealed that: in the PTD-MAX-FITC group, there were a large...

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Abstract

The invention discloses a restructured fusion protein PTD-MAX, the amino acid sequence of which is shown in SEQ ID NO: 6, and the coding nucleotide sequence of which is shown in SEQ ID NO: 4. The restructured fusion protein PTD-MAX not only has the biologic activity of maxadilan of a specific agonist of PAC1 receptor, but also has the function of trans-cell membrane conveying, can effectively enter cells, penetrates a blood brain barrier, acts on the central nervous system, and has more remarkable effect of inhibiting feeding behavior for a long time than maxadilan. The restructured fusion protein PTD-MAX confers the function of trans-cell membrane conveying of PTD to Maxadilan, thus leading the Maxadilan to penetrate the blood brain barrier and a cornea more easily, greatly improving theapplication value thereof in nervous diseases of brain and eyes, and being capable of improving the ways of application and application range thereof.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to the field of recombinant fusion protein technology, in particular to a recombinant fusion protein PTD-MAX and its expression method and application. Background technique [0002] PAC1 receptor is the specific receptor of pituitary adenylate cyclase activating polypeptide (PACAP), which is involved in mediating various biological functions of PACAP. PACAP was discovered in 1989. It is a neuropeptide with important biological functions secreted by the pituitary gland or autocrine and paracrine by the target tissue. It is a new member of the secretin / glucagon / VIP family. [0003] 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, among which PAC1 receptor It is mainly distributed in the central nervous system,...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12P21/02G01N33/68A61K38/17A61K47/48A61P7/02A61P9/10A61P15/08A61P15/10A61P25/00A61P25/04A61P25/06A61P25/22A61P25/28A61P27/02A61P29/00C12R1/19
Inventor 陈建苏余榕捷丁勇
Owner JINAN UNIVERSITY
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