Method for preparing salmon calcitonin

A salmon calcitonin and segment technology, applied in the direction of calcitonin, chemical instruments and methods, hormone peptides, etc., can solve the problems of high price, many chemical synthesis steps, low yield and the like

Active Publication Date: 2018-02-16
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The genetic engineering method is to express salmon calcitonin in Escherichia coli or yeast, but since the 32nd amino acid at the C-terminal of salmon calcitonin cannot be amidated, the biological activity of the expressed salmon calcitonin is only 100-200U / mg
So far, salmon calcitonin sold commercially is produced by chemical synthesis, which has many steps, low yield and high price. Salmon calcitonin in injection is 12,000 yuan / mg, and the price of the original drug is Also as high as 18000-20000 yuan / g

Method used

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  • Method for preparing salmon calcitonin
  • Method for preparing salmon calcitonin
  • Method for preparing salmon calcitonin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Expression of salmon calcitonin by transgenic rapeseed expression system

[0076] 1. Construction of recombinant plasmid pPha(p / gc / t)2300

[0077] 1. Artificially synthesize the double-stranded DNA molecule shown in sequence 1 in the sequence listing. In Sequence 1 in the sequence listing, the 1st to 6th positions from the 5' end are the recognition sequence of the restriction endonuclease SacI, the 7th to 1625th are the nucleotide sequence of the promoter of phaseolin storage protein, and the 1626th to 1631st The bit is the recognition sequence of the restriction endonuclease KpnI.

[0078] 2. Digest the double-stranded DNA molecule synthesized in step 1 with restriction endonuclease SacI and KpnI, and recover 1631bp DNA fragment A.

[0079] 3. Digest the vector pCAMBIA2301 with restriction endonucleases SacI and KpnI to recover the vector skeleton A of about 11.6bp.

[0080] 4. Linking the DNA fragment A and the vector skeleton A to obtain the intermedia...

Embodiment 2

[0201] Example 2. Expression of salmon calcitonin by transgenic rapeseed expression system

[0202] According to the method of step 1 to step 3 of Example 1, the recombinant plasmid pPha(p / gc / t)2300 in step 1 was replaced with recombinant plasmid pPha(p / gc / t)2300-DZ, and other steps were all unchanged, and obtained Salmon calcitonin solution; yields 0.15 mg salmon calcitonin (at 100% purity) per 10 g rapeseed.

[0203] The construction method of the recombinant plasmid pPha(p / gc / t)2300-DZ is as follows: according to the method of step 1 of Example 1, in step 13, "artificially synthesized double-stranded DNA molecule shown in sequence 4 in the sequence listing" is replaced with " Artificially synthesize the double-stranded DNA molecule shown in Sequence 8 in the Sequence Listing", and keep other steps unchanged to obtain the recombinant plasmid pPha(p / gc / t)2300-DZ. The recombinant plasmid pPha(p / gc / t)2300-DZ also expresses the fusion protein A shown in sequence 6 in the sequen...

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Abstract

The invention discloses a method for preparing salmon calcitonin. The method provided by the invention sequentially comprises the following steps: (1) introducing a recombinant vector containing an expression cassette into a receptor plant, thus obtaining a transgenic plant, wherein the expression cassette sequentially comprises the following elements from the upstream to the downstream: a promoter of a plant seed specific expression protein gene, a fusion gene and a termination sequence; the fusion gene comprises a section c for coding a protein label, an identification sequence of formic acid, and nucleic acid molecules encoding the precursor of the salmon calcitonin; (2) taking the seeds of the transgenic plant, and extracting crude protein after degreasing; (3) purifying the fusion protein from the oil body by utilizing the protein label; (4) carrying out cutting by using formic acid; (5) separating salmon calcitonin; (6) carrying out acetification; and (7) carrying out desalting.The experiment proves that with the method provided by the invention, salmon calcitonin with high biological activity is obtained. Therefore, the method provided by the invention has an important application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing salmon calcitonin. Background technique [0002] Salmon calcitonin is a single-chain polypeptide composed of 32 amino acids secreted by the hindbranch body of salmon. Salmon calcitonin, as a preventive drug for the treatment of metabolic bone diseases such as osteoporosis, has been included in the pharmacopoeia of many countries and has been marketed in many countries for nearly 30 years. Salmon calcitonin has an important biological function of regulating calcium and phosphorus metabolism and bone metabolism in the body, can reduce blood calcium level, and also has antihistamine, anticholine, and inhibitory effects on gastric acid and pancreatic secretion. Clinically, it is mainly used for the treatment of osteoporosis. The production methods of salmon calcitonin mainly include extraction method, chemical synthesis method and genetic engineering method. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/62C07K14/585
CPCC07K14/585
Inventor 刘昱辉李梅贾士荣王志兴
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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