Teduglutide tandem polypeptide and preparation method of teduglutide

A technology for teduglutide and polypeptide, applied in the field of preparation of teduglutide, can solve the problems of lack of teduglutide and the like

Active Publication Date: 2018-01-16
SHANGHAI DUOMIRUI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In summary, there is still a lack o...

Method used

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  • Teduglutide tandem polypeptide and preparation method of teduglutide
  • Teduglutide tandem polypeptide and preparation method of teduglutide
  • Teduglutide tandem polypeptide and preparation method of teduglutide

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preparation example Construction

[0090] The preparation method provided by the present invention comprises the following steps: (a) adding an aqueous solution containing (or not containing) Triton X-100 to the bacterium, and releasing the expression product by heating; (b) coarsely separating the expression product by means of precipitation , the crudely separated product is a precipitate; (c) the crudely separated product is acid-digested at high temperature first, the acid solution is removed, dissolved in an enzyme-cleavage reaction buffer, and then digested with enterokinase or digested with enterokinase first, and the enzyme-cleavage reaction buffer is removed After high temperature acid cutting, the crude product of teduglutide was obtained; (d) the crude product of teduglutide was loaded on the reverse phase chromatography medium, eluted with acetonitrile solution containing TFA, analyzed by chromatography, collected and combined eluent; (e) take the combined solution in the above steps, remove TFA and ...

specific Embodiment approach

[0112] This application describes preferred implementation modes and examples, and those skilled in the art can make appropriate changes to the implementation modes and examples described in this application on the basis of reading this application. This application therefore includes all equivalent modifications and variations of the subject matter recited in the claims of this application within the scope permitted by law.

Embodiment 1

[0135] Example 1: Plasmid construction of Ted2

[0136] In this example, an expression vector containing Ted2 was constructed. In this example, the Escherichia coli expression vector pET28a was used.

[0137] According to the amino acid sequence of Ted, the DNA sequence of Ted2 was designed, and the restriction enzyme cutting sites of Nco I and EcoR I were respectively contained at the 5' end and the 3' end. The DNA sequence of Ted2 was synthesized by Shanghai Jierui Bioengineering Co., Ltd., and connected to the pGHn plasmid (Shanghai Jierui Bioengineering Co., Ltd.), that is, pGH-Ted2. The DNA sequence encoding Ted2 was amplified with the following primers:

[0138] 5'-CACACAGGAAACAGCTATGACCATG-3'SEQ ID NO:6

[0139] 5'-CCCAGTCACGACGTTGTAAAACG-3'SEQ ID NO:7

[0140] A conventional PCR reaction was performed using EasyPfu DNA polymerase (product number: AP211-01) from Beijing Quanshijin Biotechnology Co., Ltd. The Ted2 fragment was introduced into the Escherichia coli ...

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Abstract

The invention provides a teduglutide tandem polypeptide, which contains two or more teduglutide polypeptides connected by joint peptide, and the joint includes acid digestion and enzyme digestion site. The invention also provides a plasmid and host cell containing the teduglutide tandem polypeptide sequence, and a method for preparation of teduglutide from the teduglutide tandem polypeptide.

Description

technical field [0001] The invention relates to a teduglutide tandem polypeptide, a plasmid and a host cell comprising the teduglutide tandem polypeptide sequence, and a method for preparing teduglutide. Background technique [0002] GLP-2 is composed of 33 amino acids and is a hormone obtained by enzymatic cleavage of proglucagon. It can reduce gastric emptying and secretion, and regulate the growth, proliferation and repair of small intestinal lining cells. It is used for the treatment of short bowel syndrome . However, the natural GLP-2 has a short half-life and is easily hydrolyzed by DPPIV. Teduglutide is the N-terminal Ala of GLP-2 2 is replaced by Gly 2 , after alanine is replaced, the half-life is prolonged. [0003] Chinese patent CN1244872A discloses a solid-phase synthesis preparation method for teduglutide and its analogues. For solid-phase synthesis of longer polypeptides, no matter through sequential coupling or fragment coupling methods, there are high sy...

Claims

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Application Information

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IPC IPC(8): C07K14/605C12N15/70C12N1/21A61K38/26A61P1/00
Inventor 许俊路建光黄宗庆姜旖旎郭新昆
Owner SHANGHAI DUOMIRUI BIOLOGICAL TECH CO LTD
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