High-stability PAC1 type receptor specific agonist MPAPO and preparation method and application thereof

A receptor-specific, high-stability technology, applied in chemical instruments and methods, DNA/RNA fragments, pharmaceutical formulations, etc., can solve problems such as loss of activity and poor stability, and achieve a low-cost, high-efficiency solution for promoting nerve regeneration. Effect

Active Publication Date: 2013-01-16
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The wild-type PACAP currently used as a PAC1 receptor agonist is a polypeptide composed of 27 or 38 amino acids. The stability of wild-type PACAP 27 or PACAP 38 in aqueous or body fluid environments is poor, especially for some enzymes such as dipeptidyl Peptidase (DPPIV) can also degrade its N-terminus, thereby making it lose the activity of binding to the receptor

Method used

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  • High-stability PAC1 type receptor specific agonist MPAPO and preparation method and application thereof
  • High-stability PAC1 type receptor specific agonist MPAPO and preparation method and application thereof
  • High-stability PAC1 type receptor specific agonist MPAPO and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The preparation process of high stability PAC1 type receptor specific agonist MPAPO is as follows: figure 1 As shown, the specific steps are as follows:

[0061] (1) Obtaining of MPAPO coding sequence:

[0062] Design the cDNA encoding MPAPO according to the coding preference of Escherichia coli, design 3 primers, and adopt the two-step method to obtain the sequence (such as figure 2 shown):

[0063] Primer F1: 5'- GGT GGT CATATG CAT AGC GAT GGC ATT TTT ACC GAT AGC TAT AGC-3';

[0064] Primer F2: 5'-TTT TTT CAC CGC CAG CTG TTT GCG ATA GCG GCT ATA GCT ATC GGT-3';

[0065] Primer F3:

[0066] 5'- CCACCA TGCTCTTCCGCA TTT TTT CAC CGC CGC CAG ATA TTT TTT CAC CGC CAG-3’

[0067] in, GGT GGT or CCACCA To protect the base, CATATG is the NdeI restriction site, and TGCTCTTCCGCA is the SapI restriction site;

[0068] ① Chain extension reaction:

[0069] The reaction system is: 2 μL of primer F1 (250 μM / L), 2 μL of primer F2 (250 μM / L), 10×TaKaRaBuffer (containing 4...

Embodiment 2

[0085] The in vitro stability determination of recombinant peptide MPAPO (the MPAPO that is embodiment 1 preparation)

[0086] Recombinant peptide MPAPO, chemically synthesized MPAPO (compared with recombinant peptide MPAPO, its N-terminal has no methionine) and wild-type PACAP38 (product of US Biological Company) were dissolved in 20mM sodium phosphate buffer (pH 8.0, containing 150mM sodium chloride), incubated in a 37°C water bath, sampling at different time points, and using liquid chromatography-mass spectrometry (LC-MS) to detect the retention of the polypeptide in the aqueous environment over time, and Determine the stability of the recombinant peptide MPAPO in an aqueous solution environment in vitro, the results are as follows Figure 5 Shown: When incubated for 1 week, wild-type PACAP38 was reduced by 53.2%, when incubated for 2 weeks, wild-type PACAP38 was reduced by 79.1%, when incubated for 3 weeks and 4 weeks, wild-type PACAP38 was reduced by 85.5% and 96.0%, res...

Embodiment 3

[0088] Determination of Competitive Binding Activity of Recombinant Peptide MPAPO to PAC1 Receptor

[0089] PAC1-CHO cells (Invitrogen Company) were inoculated into 12-well plates for culture, and 2 h before the experiment, the cells were washed twice with 0.5 mL of serum-free Ham's F-12 medium. Then, the cells were cultured overnight at 4°C with Ham’s F-12 medium containing 2% (mg / ml) bovine serum albumin (BSA) and 10 mM glucose, and 0.1 nM [ 125 I] PACAP38 and the polypeptide to be tested (being wild-type PACAP38 and recombinant peptide MPAPO), and gradually increase the polypeptide concentration to be tested (10 -12 M~10 -5 M). After culturing, discard the supernatant, wash the cells 3 times with ice-cold PBS (0.01M, pH 7.4), mix the cells with 0.5mL 0.5M NaOH and 0.1% (mg / ml) SDS at room temperature for 10min, and then The radioactivity of cell lysates was measured by gamma counting method, and the half-inhibitory concentration (IC50, half-maximal inhibitory concentrati...

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Abstract

The invention discloses a high-stability PAC1 type receptor specific agonist MPAPO and a preparation method and an application thereof. Compared with wild type PACAP, the agonist MPAPO has the following different points: the 17th changes from wild type methionine to leucine by mutation; the 27th changes from wild type leucine to lysine by mutation; the 28th changes from wild type glycin to lysineby mutation; and one methionine is added at the N end. The differences lead the MPAPO to have higher stability and specific activation activity to a PAC1 type receptor; furthermore the stability is about 30 times higher than that of the wild type PACAP. Experiments prove that the agonist MPAPO can be applied in the preparation of drugs with the following functions: promoting nerve regeneration and repair after nerve injuries, such as promoting corneal neuritogenesis after corneal injuries, promoting corneal epithelial cell repair, improving corneal sensitivity and improving the lacrimal glandsecretion function.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a highly stable PAC1 receptor-specific agonist MPAPO and its preparation method, and its use in the preparation of drugs with the following functions: promoting nerve regeneration and nerve repair after injury, such as: After corneal injury, it can promote the formation of corneal neurite, promote the repair of corneal epithelial cells, improve corneal sensitivity, and improve the secretion function of lacrimal gland. Background technique [0002] Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide with important biological functions discovered in 1990 and secreted by the pituitary gland. It is a new member of the secretin / glucagon / VIP family. PACAP exists in two forms: PACAP38, which consists of 38 amino acids; PACAP27, which consists of 27 amino acids at the N-terminal of PACAP38. PACAP exerts extensive and important biological function...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C07K1/20C07K1/14C12N15/11C12N15/70C12N1/21A61K38/16A61P25/00A61P27/02
Inventor 洪岸马义
Owner JINAN UNIVERSITY
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