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High-stability VPAC2 type receptor specific activator MHDBAY, preparation method and application thereof

A receptor-specific, high-stability technology, applied in the field of bases, can solve the problem of scavenging effect that needs to be improved, and achieve the effect of low cost, high efficiency and broad application prospects

Active Publication Date: 2013-05-15
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The VPAC2 receptor-specific agonists that have been reported so far, such as: polypeptide BAY55-9837, polypeptide Ro25-1553, etc., need to be improved in stability in aqueous solution or body fluid environment, and, due to their own metabolism and renal filtration, The rapid clearance effect in the organism needs to be improved

Method used

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  • High-stability VPAC2 type receptor specific activator MHDBAY, preparation method and application thereof
  • High-stability VPAC2 type receptor specific activator MHDBAY, preparation method and application thereof
  • High-stability VPAC2 type receptor specific activator MHDBAY, preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Panning of Human Serum Albumin Affinity Binding 7-mer Peptides

[0061] Use New England Biolabs, Inc's random heptapeptide phage display library (Ph.D.-7 phage display peptide library) to screen human serum albumin high-affinity 7-mer peptides.

[0062] Ph.D.-7 phage display peptide library is a combination library that fuses random heptapeptides to the minor capsid protein (pIII) of M13 phage. This library contains more than 10 random heptapeptide sequences. The titer is 2×10 13 pfu / mL.

[0063] Four rounds of panning were performed by direct coating plate method with immobilized target molecules.

[0064] The first round of panning:

[0065] (1) Human serum albumin (Sigma-Aldrich company) was dissolved in 0.1M NaHCO 3 Solution (pH8.6), the preparation concentration is the human serum albumin solution (pH 8.6) of 100μg / mL;

[0066] (2) Add 1.5mL of the above-mentioned human serum albumin solution to a single sterilized polystyrene petri dish (60×15mm, Coring Incor...

Embodiment 2

[0078] Affinity determination of human serum albumin to panned 7-mer peptides

[0079] The affinity between the 7-mer peptide obtained by panning and human serum albumin was detected by enzyme-linked immunosorbent assay (ELISA), and the specific steps were as follows:

[0080] The phage plaque clone to be identified containing the 7-mer peptide (whose amino acid sequence is: WQRPSSW) affinity-binding to human serum albumin was purified and its titer was determined. Dilute overnight cultured Escherichia coli ER2738 (New England Biolabs) into 20 mL of Luria-Bertani (broth) medium (LB) at a volume ratio of 1:100, add 5 μL of phage supernatant to each tube of ER2738 culture medium, and store at 37°C Aerated culture for 4.5h.

[0081] Transfer the above culture into a centrifuge tube, centrifuge at 10,000rpm for 10min, transfer the supernatant into a fresh centrifuge tube, and centrifuge again, take 80% of the supernatant by volume in a fresh centrifuge tube, add 1 / 6 volume of PEG...

Embodiment 3

[0091] Preparation of Recombinant VPAC2 Receptor Specific Agonist MHDBAY

[0092] The preparation process of high stability VPAC2 type receptor specific agonist MHDBAY is as follows figure 2 As shown, the specific steps are as follows:

[0093] (1) Obtaining of MHDBAY coding sequence:

[0094] Design the cDNA encoding MHDBAY according to the codon preference of Escherichia coli, and design 3 primers,

[0095] The sequence is obtained using a two-step method (such as figure 2 shown):

[0096] Primer F1:

[0097] 5'- GGT GGT CAT ATG TGG CAG CGC CCG AGC AGC TGG ATT GAA GGT CGC TTT CCGCAT AGC GAT-3';

[0098] Primer F2:

[0099] 5'-CGC CAG CTG TTT ACG CAG ACG GGT ATA CTG ATC GGT AAA CAC CGC ATC GCTATG CGG AAA-3';

[0100] Primer F3:

[0101] 5'- CCA CCA TGC TCT TCC GCA ATA ACG TTT CTG TTT A AT GCT CTG CAG ATA TTTTTT CGC CGC CAG CTG TTT-3’

[0102] in, GGT GGT or CCA CCA To protect the base, CATATG is the NdeI restriction site, and TGC TCTTCC GCA is the SapI rest...

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Abstract

The invention discloses a high-stability VPAC2 type receptor specific activator MHDBAY, a preparation method and application thereof. Compared with the traditional VPAC2 type recombinant receptor specific activator, the recombinant activator MHDBAY has higher stability and physiological activity, and through key amino acid mutation, the specific activation activity of the recombinant activator MHDBAY to the VPAC2 type receptor is obviously improved. The in vitro stability of the recombinant activator MHDBAY is about 25 times of that of BAY55-9837 and about 8 times of that of chemical synthesis MHDBAY polypeptide. Proved through experiments, the selective activation activity of the recombinant activator MHDBAY to the VPAC2 type receptor is obviously higher than that of BAY55-9837. The recombinant activator MHDBAY can be applied to preparing medicaments having the functions of treating diabetes mellitus and complications thereof, treating hyperglycemia, treating hypertension, treating thrombosis, treating adiposes, treating cardiovascular diseases, and the like.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a highly stable VPAC2-type receptor-specific agonist MHDBAY and a preparation method thereof, and its use in the preparation of drugs for treating VPAC2-type receptor-related diseases, such as: promoting glucose-dependent insulin secretion , improve the body's emergency response to high concentrations of blood sugar, effectively reduce blood sugar concentration, protect the function of pancreatic β cells, increase the number of β cells, and regulate the function of the immune system. 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12A61K38/17A61P3/10A61P7/02A61P9/12A61P3/04A61P9/00A61P25/00
Inventor 洪岸马义
Owner JINAN UNIVERSITY