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Ciliary neurotrophic factor mutants, modified mutants and uses thereof

A ciliary neurotrophic and mutant technology, applied in the direction of growth factors/inducing factors, applications, genetic engineering, etc., can solve the problems of unstable modification sites, difficulty in obtaining modified products, etc., and achieve inhibition of weight growth and high solubility , high biological activity

Active Publication Date: 2018-11-06
NAT VACCINE & SERUM INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in practice, it is found that the modification sites of CNTF or CNTF mutants are unstable by using PEG-based polymers, and as a result, it is difficult to obtain modified products with uniform properties after modification.

Method used

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  • Ciliary neurotrophic factor mutants, modified mutants and uses thereof
  • Ciliary neurotrophic factor mutants, modified mutants and uses thereof
  • Ciliary neurotrophic factor mutants, modified mutants and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0074] According to ciliary neurotrophic factor (CNTF) DNA sequence (SEQ ID NO: 2) and ciliary neurotrophic factor (CNTF) amino acid sequence (SEQ ID NO: 1), the DNA sequence of recombinant ciliary neurotrophic factor was synthesized from the whole gene sequence SEQ ID NO: 4, the encoded amino acid sequence is SEQ ID NO: 3. Among them, the recombinant ciliary neurotrophic factor retains the 17th Cys of the original CNTF amino acid sequence, and the 63rd glutamine in the ciliary neurotrophic factor is mutated into arginine, and the 15 amino acids at the C-terminal are removed to form a new CNTF mutants. The whole gene sequence synthesis of CNTF mutants can be completed by Shanghai Jierui Bioengineering Co., Ltd.

Embodiment 2

[0075] Example 2 Construction of CNTF Mutant Protein CNTFnew / CC22 Expression Vector and Identification of Engineering Bacteria

[0076] (1) Construction of CNTF mutant expression vector

[0077] After the whole gene sequence was synthesized, 5 μL (20 ng / μL) of the synthetic fragment was taken, and 1 μL (10 U / μL) of restriction enzymes NedI (10 U / μL) and 1 μL (15 U / μL) of EcorI were added (the above 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 (100 ng / μL) of the pET24a+ plasmid (purchased from Novagen), and add the same restriction enzymes NedI1 μL (10U / μL) and EcorI1 μL (15U / μL) (restriction enzymes purchased from TAKARA company) , add 11 μL of double distilled water to form a 20 μL system. In a water bath at 37°C, digest for 1 hour to digest the vector pET24a+.

[0078] After digestion...

Embodiment 3

[0092] Example 3 Expression of target protein CNTFnew / CC22

[0093] 1) Prepare LB agar plate: tryptone 10g, yeast extract 5g, NaCl 5g, agar 10g, add water to 1L, sterilize under high temperature and high pressure, add kanamycin when the temperature drops to about 50°C, and the final concentration is 50μg / ml, take 20ml and pour it into a petri dish, and after solidification, it will become an LB agar plate.

[0094] 2) Cultivate CNTFnew / CC22 engineered bacteria: Streak inoculate CNTFnew / CC22 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 h. Then transfer to the Erlenmeyer flask containing 200ml LB liquid culture solution (tryptone 10g, yeast extract 5g, NaCl 5g, add water to 1L, autoclave, divide into Erlenmeyer flasks, each bottle 200ml), and cultivate overnight at 37°C Prepare the seed solut...

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Abstract

The invention provides a ciliary neurotrophic factor mutant, and a modified mutant and application thereof. The ciliary neurotrophic factor mutant is obtained by retaining the seventeenth cysteine on the basis of an amino acid sequence of a ciliary neurotrophic factor, mutating the sixty-third glutamine into arginine and removing fifteen amino acids at the C-terminal. By virtue of the ciliary neurotrophic factor mutant, a modified ciliary neurotrophic factor with high modification specificity can be obtained, so that a series of problems of inhomogeneous modified products, instable result and the like caused by modification are solved, and the structure of a recombinant CNTF protein is more stable. Compared with a natural protein, the recombinant CNTF protein has higher biological activity and solubility. Compared with an unmodified ciliary neurotrophic factor, the modified ciliary neurotrophic factor has a long-acting effect.

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 Uses and pharmaceutical compositions of trophic factor mutants and modified ciliary neurotrophic factor mutants. Background technique [0002] Ciliary neurotrophic factor (CNTF for short) is a small molecular protein with a full length of 200 amino acids and a molecular weight of about 23KD. CNTF is stored in the form of two dimers in the body, and after being enzymatically hydrolyzed into monomers, it will undergo biological effects. CNTF can promote the growth and differentiation of various nerve cells and glial cells including motor neurons, sensory neurons, sympathetic neurons, and hippocampal neurons. [0003] CNTF got its name because it was originally extracted from ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/475C12N15/12C12N15/63C12N1/20C07K1/113A61K38/18A61P3/04A61P3/10
CPCA61K38/00C07K14/475
Inventor 王健刘君李莉莉郑秀玉张振龙
Owner NAT VACCINE & SERUM INST
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