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Ciliary neurotrophic factor mutant and its modified mutant and application

A ciliary neurotrophic and mutant technology, applied in the field of biomedicine, can solve problems such as the inability to provide modification sites, achieve the effects of treating or preventing obesity and related diseases, stable structure, and high solubility

Active Publication Date: 2020-09-08
BEIJING BIOLOGICAL PROD INST CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, neither the natural full-length CNTF nor the CNTF mutants developed by the above-mentioned patent applications can provide unique modification sites

Method used

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  • Ciliary neurotrophic factor mutant and its modified mutant and application
  • Ciliary neurotrophic factor mutant and its modified mutant and application
  • Ciliary neurotrophic factor mutant and its modified mutant and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The whole gene sequence synthesis of embodiment 1.CNTF mutant

[0074] According to the natural ciliary neurotrophic factor (CNTF) DNA sequence (SEQ ID NO: 1) and natural ciliary neurotrophic factor (CNTF) amino acid sequence (SEQ ID NO: 2), the whole gene sequence was used to synthesize recombinant ciliary neurotrophic factor ( The DNA sequence of CNTF mutant) is SEQ ID NO: 3 (completed by Shanghai Jierui Biotechnology Co., Ltd.), and the encoded amino acid sequence is SEQ ID NO: 4.

[0075] Among them, CNTF mutants are lysine at position 26 of natural ciliary neurotrophic factor mutated to leucine, lysine at position 46 to arginine, lysine at position 160 to gluten Aminoamide, the 17th cysteine ​​is mutated to alanine, the 63rd glutamine is mutated to arginine, and the CNTF mutant formed by removing 15 amino acids at the C-terminal - CNTFnew / Clys3 mutation body.

Embodiment 2

[0076] Example 2. Construction of CNTF Mutant CNTFnew / Clys3 Expression Vector and Identification of Engineering Bacteria

[0077] (1) Construction of CNTF mutant CNTFnew / Clys3 expression vector

[0078] After the whole gene sequence was synthesized, 5 μL of the synthesized fragment was taken, and 1 μL of restriction enzyme NedI and 1 μL of EcorI were added (restriction enzymes were purchased from TAKARA Company). Add 2 μL of restriction endonuclease buffer and 11 μL of double distilled water to form a 20 μL system. 37°C water bath, enzyme digestion for 1 hour. At the same time, take 5 μL of the pET24a+ plasmid (purchased from Novagen), add 1 μL of the same restriction enzymes NedI and EcorI (restriction enzymes purchased from TAKARA), and add 11 μL of double distilled water to form a 20 μL system. In a water bath at 37°C, enzyme digestion was performed for 1 hour, and the vector pET24a+ was digested.

[0079] After digestion, 1% agarose gel electrophoresis was performed. T...

Embodiment 3

[0095] Example 3. Expression of the target protein CNTFnew / Clys3.

[0096] 1) Prepare LB agar plate: tryptone 10g, yeast extract 5g, NaCl 5g, agar 10g, add water to 1L (pH 7.2±0.1), high temperature and high pressure sterilization, when the temperature drops to about 50 ℃, add card Namycin, to a final concentration of 50 μg / ml, take 20 ml and pour it into a petri dish, and form an LB agar plate after solidification.

[0097] 2) Cultivate CNTFnew / Clys3 engineered bacteria: Streak inoculate CNTFnew / Clys3 engineered bacteria on LB agar plate, culture overnight at 37°C. Pick a single colony in good growth state from the LB plate with the culture medium overnight, inoculate it into a test tube containing LB, and incubate at 37°C for 10 hours. Then inoculate to contain 200ml LB liquid culture medium (tryptone 10g, yeast extract 5g, NaCl 5g, add water and dilute to 1L (pH is 7.2 ± 0.1), autoclave, pack in Erlenmeyer flask, every bottle 200ml) Cultivate overnight at 37°C in a Erlenm...

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Abstract

The present invention relates to a CNTF mutant, a modified CNTF mutant, and applications of the CNTF mutant and the modified CNTF mutant. According to the present invention, mutation occurs on the basis of the amino acid sequence of natural CNTF to obtain the CNTF mutant, and comprises the mutation of 26 site lysine into isoleucine or leucine, the mutation of 46 site lysine into arginine, and the mutation of 160 site lysine into glutamine or methionine, and the CNTF mutant retains the 40 site lysine of the natural CNTF; and through the CNTF mutant, the modified CNTF mutant having high modification specificity can be obtained so as to overcome the problems of non-uniform modified product, unstable results and the like caused by the modification, the structure of the recombinant CNTF protein is stable, the recombinant CNTF protein has high biological activity and high solubility compared to the natural protein, and the modified CNTF has a long-acting effect compared to the unmodified CNTF.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a modified ciliary neurotrophic factor mutant, DNA, a recombinant expression vector, a genetically engineered host cell, a modified ciliary neurotrophic factor mutant, the ciliary neurotrophic factor Application and pharmaceutical composition of trophic factor mutant and modified ciliary neurotrophic factor mutant. Background technique [0002] Ciliary neurotrophic factor (CNTF) got its name because it was originally extracted from chicken ciliary body and can maintain the survival of chicken parasympathetic ganglion. The full length of CNTF consists of 200 amino acids, with Cys at the 17th position, no intramolecular disulfide bond, and no glycosylation site. The full-length molecular weight of CNTF is about 23KD. It is stored in the form of two dimers in the body and undergoes biological effects after enzymatic hydrolysis into monomers. CNTF can promote the growth and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/475C12N15/12C12N15/70C12N1/21A61K38/18A61P3/04
CPCA61K38/00C07K14/475
Inventor 王健柳森李素贞郑秀玉蔡觅张夕燕晁华于世荣
Owner BEIJING BIOLOGICAL PROD INST CO LTD
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