Method for preparing salmon calcitonin

A salmon calcitonin and segment technology, applied in the biological field, can solve the problems of high price, many chemical synthesis steps, low yield and the like

Active Publication Date: 2017-05-31
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

[0081] Example 1. Expression of salmon calcitonin by transgenic rapeseed oil body expression system

[0082] 1. Construction of recombinant plasmid pBO-sCT2301

[0083] 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 BamHI, the 7th to 21st are the recognition sequences of enterokinase, and the 22nd to 120th are salmon calcitonin The coding gene of the precursor of sCT (hereinafter referred to as sCT gene), the 121st to 126th positions are two stop codons, and the 127th to 132nd positions are the recognition sequence of the restriction endonuclease XhoI.

[0084] 2. Ligate the double-stranded DNA molecule synthesized in step 1 to the vector pUC57 to obtain the recombinant plasmid psCT.

[0085] 3. Extract the total RNA of the seeds of Yuzhi No. 4 5 weeks after flowering, and then u...

Embodiment 2

[0210] Example 2. Expression of salmon calcitonin by transgenic rapeseed oil body expression system

[0211] According to the method of step 1 to step 3 of Example 1, the recombinant plasmid pBO-sCT2301 in step 1 was replaced with recombinant plasmid pBO-sCT2301-DZ, and other steps were all unchanged to obtain salmon calcitonin solution; 0.16 mg salmon calcitonin (at 100% purity).

[0212] The construction method of the recombinant plasmid pBO-sCT2301-DZ is as follows: according to the method of step 1 of Example 1, replace the "synthetic double-stranded DNA molecule shown in sequence 1 in the sequence listing" in step 1 with "artificially synthesized sequence in the sequence listing The double-stranded DNA molecule shown in 7", and the other steps were kept unchanged, and the recombinant plasmid pBO-sCT2301-DZ was obtained. The recombinant plasmid pBO-sCT2301-DZ also expresses fusion protein A shown in sequence 5 in the sequence listing.

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Abstract

The invention discloses a method for preparing salmon calcitonin. The method provided by the invention comprises the following steps: (1) importing a recombinant vector containing an expression cassette into a receiver plant, so as to obtain a transgenic plant, wherein the expression cassette comprises the following elements from top to bottom in sequence: a promoter of a plant oil body protein gene, a fusion gene and a termination sequence; the fusion gene comprises a segment III used for coding a protein tag, a segment I used for coding a plant oil body protein, a digestion identification sequence of protease and a nucleic acid molecule of a precursor used for coding salmon calcitonin; (2) taking seeds of the transgenic plant for extraction of oil bodies; (3) purifying a fusion protein from the oil bodies by utilizing the protein tag; (4) digesting the fusion protein with protease; (5) separating salmon calcitonin; (6) acetifying; (7) performing desalting. Salmon calcitonin with comparatively high biological activity can be obtained by utilizing the method provided by the invention. 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): C12N15/84C12N15/62C07K14/585
CPCC07K14/585C12N15/8205
Inventor 刘昱辉谭杰李梅贾士荣王志兴
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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