Ciliary neurotrophic factor mutant and modified mutant and application thereof

A technology of ciliary neurotrophy and mutant, applied in the field of biomedicine, can solve problems such as inability to provide modification sites, achieve treatment or prevention of obesity and related diseases, high biological activity and solubility, and high biological activity Effect

Active Publication Date: 2017-05-10
北京生物制品研究所有限责任公司 +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 modified mutant and application thereof
  • Ciliary neurotrophic factor mutant and modified mutant and application thereof
  • Ciliary neurotrophic factor mutant and modified mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] The whole gene sequence synthesis of embodiment 1.CNTF mutant

[0071] 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.

[0072] Among them, the CNTF mutant is the 40th lysine of natural ciliary neurotrophic factor is mutated into cysteine, the 17th cysteine ​​is mutated into alanine, and the 63rd glutamine is mutated into arginine, and remove the C-terminal 15 amino acids to form a CNTF mutant——CNTFnew / CC40 mutant.

Embodiment 2

[0073] Example 2. Construction of CNTF Mutant CNTFnew / CC40 Expression Vector and Identification of Engineering Bacteria

[0074] (1) Construction of CNTF mutant CNTFnew / CC40 expression vector

[0075] 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.

[0076] After digestion, 1% agarose gel electrophoresis was performed. The g...

Embodiment 3

[0092] Example 3. Expression of the target protein CNTFnew / CC40.

[0093] 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.

[0094] 2) Cultivate CNTFnew / CC40 engineered bacteria: Streak inoculate CNTFnew / CC40 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 Erlenmeye...

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Abstract

The invention relates to a CNTF (ciliary neurotrophic factor) mutant and a modified mutant and an application thereof. The CNTF mutant is prepared by mutating on the basis of an amino acid sequence of natural CNTF. The mutation comprises: lysine at the 40th locus is mutated into cysteine, and cysteine at the 17th locus is mutated into alanine. According to the CNTF mutant disclosed by the invention, the modified CNTF with high modification specificity can be obtained, so that the problems that the modified products are non-uniform and the result is unstable due to modification and the like are solved. Moreover, the recombinant CNTF protein structure is stable. Compared with natural proteins, the CNTF protein has high biological activity and solubility, and the modified CNTF has a long-acting effect compared with non-modified 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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IPC IPC(8): C07K14/475C12N15/12C12N15/70C12N1/21A61K38/18A61P3/04
CPCA61K38/00C07K14/475
Inventor 王健郑秀玉柳森张夕燕李莉莉蔡觅李素贞刘君
Owner 北京生物制品研究所有限责任公司
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