Recombined rockfish adenylate cyclase activated polypeptide gene and its expression system, expression product, producing method and use

A grouper adenylate cyclase and gene technology, applied in the field of genetic engineering, to achieve the effects of convenient production, high expression level, and easy purification

Inactive Publication Date: 2004-02-11
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is still no regulator that can effectively regulate...

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  • Recombined rockfish adenylate cyclase activated polypeptide gene and its expression system, expression product, producing method and use
  • Recombined rockfish adenylate cyclase activated polypeptide gene and its expression system, expression product, producing method and use
  • Recombined rockfish adenylate cyclase activated polypeptide gene and its expression system, expression product, producing method and use

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specific Embodiment Embodiment 1

[0026] Specific Examples Example 1 Synthesis of the cDNA of the grouper adenylate cyclase-activating polypeptide

[0027] According to the known mammalian and grouper grouper adenylyl cyclase-activating polypeptide cDNA sequences, three primers were designed: senseP1: 5'GCA(GT)G(AG)C(AC)A(AGT)( AG)TCTA GTA(AG)

[0028] AG C(GT) ACT 3'senseP2: 5'GA(AG)A(AG)GA(AG)C(GC)GAAA(GC)GCATGCAG 3'antisenseP3: 5'TA(AGC)(AGC)C(ACT) A(AG)(GCT)CG(AGC)CGTCCTTTG3'

[0029] senseP1 and antisenseP3 are primers for synthesizing PACAP 524 bp, and senseP2 and antisenseP3 are primers for synthesizing 288 bp cDNA of PACAP coding region. Using the reverse transcription product cDNA of brain total RNA as a template, the gene that synthesized PACAP 524bp and synthesized PACAP coding region 288bp was amplified by PCR. The PCR reaction conditions are: denaturation at 94°C for 3 minutes; 94°C for 45 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute and 3...

Embodiment 2

[0032]The PCR product containing the double restriction site and the PACAP sequence was digested with restriction enzymes KpnI and NotI and then subjected to gel electrophoresis to separate and purify the about 140bp grouper adenylate cyclase-activating polypeptide fragment; E. coli expression plasmid pTRX was digested with restriction enzymes KpnI and NotI, separated and purified the 5.8kb large fragment, mixed with the 140bp grouper adenylate cyclase-activating polypeptide fragment at a ratio of 1:3, connected with T4 ligase for 16 hours, and then transformed into In competent Escherichia coli BL21, select ampicillin-resistant transformants, use the Omega company plasmid extraction kit to screen recombinant plasmids with a size of about 6 kb, and digest the recombinant plasmids with restriction enzymes KpnI and NotI to obtain 140 bp and 5.8 kb The two fragments of the grouper adenylate cyclase-activating polypeptide and the expression vector pTRX are the same in size respecti...

Embodiment 3

[0033] Transform pTRX-PACAP into Escherichia coli BL21 by CaCl2 method, screen transformants on LB plates containing ampicillin, and obtain recombinant transformant pTRX-PACAP-BL21 containing pTRX-PACAP through plasmid detection and restriction analysis.

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Abstract

The recombinant rockfish adenylate cyclase qctivated polypeptide gene is obtained with rockfish brain tissue general RNA as template and through RT-PCR and RACE process and is the gene segment with whole-length cDNA sequence of rockfish adenylate cyclase qctivated polypeptide gene. The present invention also constructs carrier containing the gene and recombinant engineering strain. The present invention also relates to rockfish adenylate cyclase qctivated polypeptide recombined protein encoded with the gene and expressed with the recombined engineering strain, and the production process of the recombined protein. The present invention makes it possible to obtain rockfish adenylate cyclase qctivated polypeptide recombined protein with stable soruce and low cost for preparing growth and propagation regulator suitable for marine fishes.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a grouper adenylate cyclase-activating polypeptide gene, a carrier containing the gene, a recombinant engineering strain, and a growth product encoded and expressed by the gene for use in preparing seawater fish. and reproductive regulator applications. Background technique [0002] Pituitary adenylate cyclase activating polypeptide (pituitary adenylate cyclase activating polypeptide, referred to as: PACAP), is a peptide substance composed of 38 amino acids. PACAP was originally discovered from the extract of sheep hypothalamus, and it belongs to the gene family consisting of vasoactive intestinal peptide (VIP), glucagon, growth hormone releasing factor and secretin. At present, PACAP has been found in fish to regulate the secretion of pituitary hormones in carps, and it has been proved that PACAP produced by hypothalamus can promote the relea...

Claims

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Application Information

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IPC IPC(8): A23K10/16A23K20/147A23K50/80C07H21/00C12N1/21C12N15/12C12N15/63C12N15/70C12P21/02C12Q1/68
Inventor 李文笙江涌林浩然
Owner SUN YAT SEN UNIV
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