Preparation and purification method of recombinant proserum/growth hormone fusion protein for treating children dwarfism

A technology of human serum albumin and fusion protein, applied in peptide preparation methods, microbial-based methods, biochemical equipment and methods, etc., can solve problems such as low patient compliance, high treatment costs, and limited drug recipients , to save costs

Active Publication Date: 2019-06-07
TIANJIN LINDA SINOBIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although recombinant human growth hormone achieves the purpose of clinical treatment, because the frequency of administration is once a day, and the medication time for children is at least 1-3 years, the patient's compliance is extremely low, and the treatment cost is huge. It is difficult for ordinary families to cooperate with the doctor's requirements. treatment, which greatly limits the population receiving the drug

Method used

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  • Preparation and purification method of recombinant proserum/growth hormone fusion protein for treating children dwarfism
  • Preparation and purification method of recombinant proserum/growth hormone fusion protein for treating children dwarfism
  • Preparation and purification method of recombinant proserum/growth hormone fusion protein for treating children dwarfism

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Experimental program
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Effect test

Embodiment 1

[0076] Embodiment 1: Human serum albumin (HSA) gene expression and the construction of carrier plasmid

[0077] Using the total RNA extracted from human fetal liver as a template, the HSA gene was synthesized and amplified by reverse transcription polymerase chain polymerization. Under the action of reverse transcriptase (purchased from GIBCO / BRL Company), 5 micrograms of total RNA synthesized the corresponding DNA chain, and the oligonucleotide primer was 18 T+lN (N is a random nucleotide). Reaction condition is 45 ℃, 20 minutes, then be warming up to 55 ℃, continue to incubate for 40 minutes.

[0078] The oligonucleotide primer sequences are: 5'-GAATTCATGAAGTGGGTAACCTTTATTTCC-3' and 5'-GAATTCTTATAAGCCTAAGGCAGCTTGACTTGC-3'

[0079] two EcoRI recognition sites were added to the two ends of the HSA gene and cloned into an expression vector. The PCR reaction conditions were 94°C for 4 minutes, and the DNA encoding HSA was further amplified for 35 cycles: 94°C for 30 seconds;...

Embodiment 2

[0084] Example 2: Expression of recombinant human serum albumin / growth hormone fusion gene (rHSA / GH) and construction of vector plasmid

[0085] The nucleotide sequence of GH mature peptide (GeneBank registration number: NP_000506) was artificially synthesized by DNA full-sequence synthesis method (synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.). respectively add at its N-terminus Bsu36 Ⅰ Restriction site, add at its C-terminus xho Ⅰ Restriction site, use these two enzymes to respectively digest the synthesized target gene GH and pYZ-HSA recombinant plasmid; the connection of the target gene GH and the pYZ-HSA recombinant plasmid: specific steps: the target gene GH after digestion and the pYZ-HSA recombinant plasmid The pYZ-HSA recombinant plasmid was mixed according to a certain ratio, and ligation buffer and ligase were added, and ligated for 1 hour at 22°C; the ligation product was transformed into DH5α competent cells, and the specific steps were: add the l...

Embodiment 3

[0094] Embodiment 3: Transformation and preparation of Pichia pastoris expression engineered bacteria

[0095] Pichia pastoris strain X33 colony was inoculated in a 50ml centrifuge tube containing 5ml YPD culture medium, and cultured overnight at 30°C at a speed of 250 rpm. The next day, take 0.2ml of the overnight culture and transfer it into 500ml of YPD culture solution, and place it in a 2-liter Erlenmeyer flask. Incubate with rotation at 30°C for 2-3 hours to bring the cell density to OD 600 =1.3-1.5. The yeasts were collected by centrifugation, resuspended in 500ml ice-cold sterile water and washed twice. Then the yeast was suspended in 20ml of ice-cold 1M Sortbitol solution and washed once.

[0096] The pYZ-HSA / GH plasmid DNA constructed in Example 2 was subjected to Pme After I restriction endonuclease treatment, a linear plasmid molecule is formed. 5 μg of linearized plasmid DNA was mixed with 80 μl of treated yeast and placed in a 0.2 cm thick electrode cup and...

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Abstract

The invention discloses recombinant proserum/growth hormone fusion protein, a preparation and purification method of the recombinant fusion protein, and the use of the recombinant fusion protein to preparation of medicines for treating children dwarfism. The amino acid sequence of the recombinant proserum/growth hormone fusion protein is SEQID NO.1, and the nucleotide sequence of the recombinant proserum/growth hormone fusion protein is SEQID NO.2. According to a preparation technology of the recombinant proserum/growth hormone fusion protein disclosed by the invention, yeast engineering bacteria are constructed and expressed, so that high-density expression recombinant fusion protein is obtained; and through a purification technology, the recombinant proserum/growth hormone fusion proteinwhich can be used clinically is obtained. The recombinant proserum/growth hormone fusion protein obtained by the preparation and purification method adopts a creative medicine structure for treatingthe children dwarfism, has long residual action that administration can be performed once every two weeks, is more suitable for children medication demands, and has more excellent treatment effects, less administration frequency and lower production cost.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically to a recombinant human serum albumin / growth hormone fusion protein for treating dwarfism in children, its preparation and purification method and its use in the preparation of medicines for treating dwarfism in children. Background technique [0002] Human serum albumin (HSA) is a soluble monomeric protein that constitutes half of the total protein in blood. As a basic carrier, albumin carries molecules such as fatty acids, steroids and hormones, and its stable and inert nature is an important factor in maintaining blood pressure. Serum albumin is a globular non-glycosylated serum protein with a molecular weight of 65 kilodaltons and 585 amino acids. This protein (albumin precursor) is then processed by the Golgi apparatus to remove the leading polypeptide and secreted out of the cell. Serum albumin has 35 cysteines, and in blood, albumin is a monomer with 17 disulfide bonds (see ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N1/19C07K1/22C07K1/20C07K1/18C07K1/16A61K38/27A61K47/42A61P5/06C12R1/84
Inventor 侯琼杨小楠陈颖富岩于在林
Owner TIANJIN LINDA SINOBIOTECH CO LTD
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