A kind of recombinant natriuretic peptide and its preparation method
A technology of natriuretic peptide and atrial natriuretic peptide, applied in botany equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problem of short half-life, weak physiological effect, and acute heart failure treatment effect Unsatisfactory problems, to achieve the effect of prolonging the half-life of the drug, simple and feasible preparation process, and preventing and treating heart failure
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[0052] The present invention also provides a method for preparing the above-mentioned genetically engineered novel recombinant natriuretic peptide, the method comprising the following steps:
[0053] S1. Respectively obtain the N-terminal gene fragment of the atrial natriuretic peptide gene and the C-terminal gene fragment of the snake natriuretic peptide gene. Preferably, the obtained atrial natriuretic peptide gene is human atrial natriuretic peptide gene.
[0054] S2. Synthesize a recombinant natriuretic peptide gene by PCR, the N-terminal of the recombinant natriuretic peptide gene is the N-terminal gene fragment of the atrial natriuretic peptide gene, and the C-terminal of the recombinant natriuretic peptide gene is the C-terminal gene fragment of snake natriuretic peptide gene;
[0055] S3, constructing an expression vector;
[0056] S4. Expression.
[0057] Preferably, the above step S1 further includes:
[0058] S101. Obtaining the full-length human atrial natriure...
Embodiment 1
[0085] The novel recombinant natriuretic peptide (ADNP) gene that was amplified by PCR splicing and sequencing was cloned into the prokaryotic system Escherichia coli expression vector pGEX-4T-1, and a prokaryotic expression plasmid containing the novel recombinant natriuretic peptide (ADNP) was constructed pGEX-ADNP. BL21 and Rosetta were used as prokaryotic expression host bacteria. Extract high-quality and high-concentration prokaryotic expression plasmid pGEX-ADNP, using cold CaCl 2 Prepare competent host bacteria by conventional methods, transform the host bacteria by conventional methods, transform the above plasmids into BL21 and Rosetta strains respectively, and culture them at 37°C for 12-18 hours; pick a single clone expression strain, and inoculate LB medium (adding ampicillin , with a final concentration of 100 μg / ml), shake (250-300 rpm) at 37°C for overnight culture, collect the bacterial liquid by centrifugation, extract protein after ultrasonic crushing, and p...
Embodiment 2
[0087] The novel recombinant natriuretic peptide (ADNP) gene amplified by PCR splicing and sequencing was cloned into the prokaryotic expression vector pCW, and the prokaryotic expression plasmid pCW-ADNP containing the novel recombinant natriuretic peptide (ADNP) was constructed. BL21 and Rosetta were used as prokaryotic expression host bacteria. Extract high-quality and high-concentration prokaryotic expression plasmid pCW-ADNP, using cold CaCl 2 Competent host bacteria were prepared by conventional methods, and the above plasmids were transformed into BL21 and Rosetta strains respectively by conventional methods, and cultured at 37°C for 12-18 hours; single-clonal expression strains were picked and inoculated into LB medium (adding ampicillin, The final concentration is 100 μg / ml), cultured overnight at 37°C with shaking (250-300rpm), centrifuged to collect the bacterial liquid, extracted the protein after ultrasonic crushing, and carried out protein electrophoresis detecti...
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