Pituitary adenylate cyclase activated polypeptide derivatives and process for preparing the same

A technology of adenylate cyclase and peptide derivatives, applied in the field of genetic engineering, can solve the problems of low harvest rate and high cost

Inactive Publication Date: 2004-12-29
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Natural PACAP is composed of 38 amino acids, most of which are currently chemically synthesized, which is expensive and has a low yield

Method used

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  • Pituitary adenylate cyclase activated polypeptide derivatives and process for preparing the same
  • Pituitary adenylate cyclase activated polypeptide derivatives and process for preparing the same
  • Pituitary adenylate cyclase activated polypeptide derivatives and process for preparing the same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The amplification of embodiment 1PACAP gene and the construction of PTY-PDT plasmid

[0036] According to the amino acid sequence of PACAP mature peptide, its nucleotide sequence was designed according to the codon preference of Escherichia coli. Segmented synthetic primers, according to image 3 As shown, the PACAP gene was synthesized in vitro by PCR. Three primers for the synthesis of PACAP:

[0037] F1 5'ATG CAC TCT GAC GGC ATC TTC ACA GAC TCT TAT TCC CGCTAC CGA AAA CAA ATG3';

[0038]F2 5'TCC CGC TAC CGA AAA CAA ATG GCT GTC AAG AAA TAC TTGGCG GCC GTG CTA GGG AAA AGG3';

[0039] F3 5’TTA CTA TTT GTT TTT AAC CCT CTG TTT ATA CCT TTT CCC TAGCAC GGC CGC3’

[0040] For the construction of expression fusion protein plasmid, see Figure 8 . Primers were designed to introduce the Ndel recognition site upstream of the PACAP gene and the Sapl recognition site downstream; the PACAP gene was inserted into PTYB1 to obtain the expression vector PTY-PDT. For PCR identificati...

Embodiment 2

[0041] Embodiment 2 Expression of recombinant fusion protein and cleavage of target protein

[0042] Transform the expression plasmid into the expression host strain E.coli Strain ER2566, pick a single clone and culture it overnight, inoculate it at 1:20 in 1L LB medium containing 50 mg / L kanamycin, and shake it at 37°C until the OD is 0.5- 0.8, add IPTG to a final concentration of 0.3-0.5mmol / L, and induce at 30°C for 3h. The cells were collected by centrifugation, resuspended in BufferA (20mM Tris.HCl, 500mM NaCl, 1mM EDTA, pH7.5), ultrasonically disrupted at 4°C, and centrifuged to obtain the supernatant.

[0043] Take 6mL chitin beads slurry and equilibrate with 60mL Buffer A after loading into the column; load the broken supernatant of the bacteria at a flow rate of Figure 11 , westernblot identification results see Figure 12 , mass spectrometry results see Figure 13 .

Embodiment 3

[0044] Activity and stability comparison of embodiment 3PDT and PACAP38

[0045] After the S1990 cells in the logarithmic growth phase were digested and detached, the concentration of the cell suspension was adjusted to 1×10 7 each / mL, add the same concentration of PDT and PACAP38 respectively, and use the culture medium as the control group; after incubation for 5 minutes, take out 200 μL suspension from the experimental group and the control group, add trichloroacetic acid, shake and mix well, centrifuge, and take the upper Clear 0.5 mL, wash the supernatant with water-saturated ether, and evaporate to dryness. Cyclic AMP Enzyme Immunoassay Kit was used to determine cAMP concentration.

[0046] The activity data of PDT and PACAP38 promoting cAmp production in S1990 pancreas cells by the above method are shown in Table 1, and the graph is shown in Figure 14 , the results showed that the activities of PDT and PACAP38 were comparable.

[0047] Tabl...

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Abstract

The present invention belongs to the field of gene engineering technology, and relates to one kind of adenylate cyclase activating polypeptide derivative. The derivative has amino acid sequence as shown in the sequence list and features one added methionine molecule in the amino terminal and one beta-mercaptoethanol molecule connected via thioester bond to the carboxyl terminal. Compared with natural and chemically synthesized adenylate cyclase activating polypeptide, the adenylate cyclase activating polypeptide derivative and its serial hydrolytic products have the same activity but raised stability. The present invention also provides the production process of the derivative.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to an adenylate cyclase-activating polypeptide derivative and a production method of the derivative. Background technique [0002] Pituitary adenylate cyclase activating peptide (English name pituitary adenylate cyclase activating polypeptide, hereinafter referred to as PACAP) and its receptors are widely distributed in the central nervous system, peripheral nervous system and non-nervous tissues, such as brain, spinal cord, adrenal gland, testis, Ovary, liver, kidney, pancreas, pineal gland, heart, spine, ganglia, respiratory system and digestive system, etc. PACAP regulates the second signal history cAMP, IP3 and Ca in target cells by activating specific G protein-coupled receptors on the target cell membrane. 2+ Concentration, thereby exerting important biological functions. It has been confirmed that PACAP has the functions of neurotransmitter,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P9/04A61P9/10A61P9/12C07K14/435C12N15/12C12N15/63C12N15/70C12P21/00
Inventor 洪岸余榕捷
Owner JINAN UNIVERSITY
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