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
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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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