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Mutated hFGF-21 protein mature peptide and mutated hFGF-21 protein mature peptide-polyethylene glycol cross-linking agent and applications thereof

A protein and protein technology, which is applied to the mature peptide of mutant hFGF-21 protein and its cross-linked product with polyethylene glycol and their application fields, which can solve the problems of prone to antigen-antibody reaction, short half-life in vivo, and influence on therapeutic effect, etc. problems, to achieve good hypoglycemic activity, long half-life, and low immunogenicity

Active Publication Date: 2013-07-10
TIANJIN TASLY PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the results of long-term research and clinical trials show that FGF-21, like other protein drugs, has poor stability, short half-life in vivo, and is prone to antigen-antibody reactions, which directly affects its clinical therapeutic effect.

Method used

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  • Mutated hFGF-21 protein mature peptide and mutated hFGF-21 protein mature peptide-polyethylene glycol cross-linking agent and applications thereof
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  • Mutated hFGF-21 protein mature peptide and mutated hFGF-21 protein mature peptide-polyethylene glycol cross-linking agent and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Preparation of FGF-21 mature peptide and mFGF-21 mature peptide

[0042] 1. Construction of recombinant plasmid pSUMO-FGF-21

[0043] 1. Synthesize the double-stranded DNA molecule shown in Sequence 2 of the Sequence Listing.

[0044] 2. Using the double-stranded DNA molecule obtained in step 1 as a template, using a primer pair consisting of P1 and P2, and using rTaq enzyme to perform PCR amplification to obtain a PCR amplification product.

[0045] P1: 5′- GGTCTC TAGGT CACCCCATCCCTGACTCCAGT-3';

[0046] P2: 5’-CGC GGATCC TTA GGAAGCGTAGCTGGGGCTTCGG-3.

[0047] PCR amplification program: 95°C pre-denaturation 5min; 95°C 30s, 56°C 30s, 72°C 45s, 25 cycles; 72°C extension 10min.

[0048] 3. Double-cut the PCR amplified product of step 2 with restriction enzymes BsaI and BamHI, and recover the digested product.

[0049] 4. Double digestion of the prokaryotic expression vector pSUMO with restriction enzymes BsaI and BamHI to recover a 5700bp vector backbone.

[0050] 5. Conn...

Embodiment 2

[0079] Example 2: Comparison of the activity of FGF-21 mature peptide and mFGF-21 mature peptide

[0080] 1. Cell viability test

[0081] The FGF-21 mature peptide prepared in Example 1 and the mFGF-21 mature peptide prepared in Example 1 were used as the test proteins to be identified as follows:

[0082] After starving HepG2 cells for 12 hours, use protein concentration of 10, 100 or 1000 nmol / L respectively (use cell culture medium as DMEM medium, and test protein is FGF-21 mature peptide solution prepared in Example 1 or mFGF-21 mature peptide The test protein was added in the form of a solution to stimulate the cells for 24 hours, and then use the glucose (Glu) determination kit (Beijing Jinhao Pharmaceutical) to use the GOD-POD method (document describing the "GOD-POD method": Yang Guizhi, Gao Xiaoping, Yan Jufang) , Et al. The establishment of the GOD-POD method for micro-determination and its application in the sugar uptake of 3T3-L1 adipocytes and HepG2 cells. Sichuan Journ...

Embodiment 3

[0095] Example 3. Establishment of related parameters for modification of mFGF-21 mature peptide

[0096] mPEG-SPA (Methoxy Polyethylene Glycol-Succinimidyl Propionate): 20KD, purchased from Beijing Kaizheng Biological Engineering Development Co., Ltd. mPEG-ALD (monomethoxy polyethylene glycol propionaldehyde): 20KD, purchased from Beijing Kaizheng Biological Engineering Development Co., Ltd.

[0097] 1. The choice of modifiers

[0098] Take the mFGF-21 mature peptide solution with a protein concentration of 2mg / mL, add the modifier (mPEG-ALD or mPEG-SPA) to make the mass ratio of mFGF-21 mature peptide to modifier 1:4 or 1:6, add cyanide Make it 10 times the mole number of mPEG-ALD with sodium borohydride, adjust the pH to 6.0, react at 4°C for 6 hours or 8 hours, and sample for SDS-PAGE analysis. See the result Image 6 . Lane 1 is the product of the mass ratio between mFGF-21 and PEG-ALD at 1:4 and reacted for 6 hours, Lane 2 is the product of the mass ratio between mFGF-21 and...

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Abstract

The invention discloses a mutated hFGF-21 protein mature peptide and coding gene and applications thereof, as well as polyethylene glycol-modified mutated hFGF-21 protein mature peptide and applications thereof. The mutated hFGF-21 protein mature peptide is protein shown in sequence 3 of a sequence table. The invention further discloses a conjugate of the protein and polyethylene glycol, namely the protein is modified by the polyethylene glycol. The drug for treating diabetics has a good hypoglycemic activity, especially the drug taking the conjugate as an active ingredient also has the advantages of being high in stability, long in half-life period, low in immunogenicity and the like.

Description

Technical field [0001] The invention relates to a mutant hFGF-21 protein mature peptide and its coding gene and application, as well as a polyethylene glycol modified mutant hFGF-21 protein mature peptide and its application. Background technique [0002] Diabetes is an absolute or relative lack of insulin in the blood, which leads to high blood sugar and diabetes, which can lead to disorders of fat and protein metabolism. Clinical manifestations such as polyuria, polydipsia, polydipsia, polyphagia and weight loss can occur. Acute complications such as ketoacidosis or chronic complications such as blood vessels and nerves. [0003] Diabetes is a common endocrine and metabolic disease. In 1980, the prevalence rate in my country was 0.67%. According to Shanghai statistics, the prevalence rate rose from 1.01% to 2.23% from 1978 to 1989. The increase was mainly type 2 patient. The above facts show that with the improvement of people's social and economic life, the rapid increase of di...

Claims

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Application Information

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IPC IPC(8): C07K14/50C12N15/12C07K1/113A61K38/19A61K47/48A61P3/10
Inventor 李德山叶贤龙任桂萍孙国鹏刘铭瑶
Owner TIANJIN TASLY PHARMA CO LTD
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