Fusion protein possessing growth facilitation action and its coding gene and uses

A technology of fusion protein and action, applied in the direction of peptide/protein components, medical preparations containing active ingredients, hybrid peptides, etc., can solve the problems of long treatment cycle, short half-life of GHRH, unbearable for patients, etc., and achieve broad application prospects , high expression, easy to purify

Inactive Publication Date: 2007-05-02
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the half-life of GHRH is short, and only frequent administration can obtain satisfactory curative effect. For example, in clinical research, once-a-day injection is often used.
However, such frequent medication combined with a long treatment cycle makes it difficult for patients to bear

Method used

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  • Fusion protein possessing growth facilitation action and its coding gene and uses
  • Fusion protein possessing growth facilitation action and its coding gene and uses
  • Fusion protein possessing growth facilitation action and its coding gene and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, the acquisition of fusion protein gene HSA-GHRH

[0064] Primers were designed according to the nucleotide sequences of the human serum albumin gene (HSA) and the GHRH protein gene (GHRH), and the human serum albumin gene and the GHRH protein gene were respectively amplified by PCR. The primer sequences were as follows:

[0065] Primer 1: 5'-GGGAATTCCTATTACAATCTAGCTCTAGCACCTCT-3';

[0066] Primer 2: 5'-GTGGTGGTGGTTCCGGTGGTGGTGGTGGTTCTTACGCTGACGCTATCTTCACTAAC-3';

[0067] Primer 3: 5'-ACGCTGACGCTATCTTCACTAACTCCTACCGTAAGGTTTTGGGTCAATTGTCCGCTAGAAAGTTGTTGCAAGACATCATG-3';

[0068] Primer 4: 5'-CAATCTAGCTCTAGCACCTCTCTCTTGGTTGGACTCACCTTGTTGTCTGGACATGATGTCTTGCAACAACTTTCTAGGGGA-3'.

[0069] 1. Amplification of the GHRH gene

[0070] Using primers 3 and 4 as templates and primers, the full-length sequence of the GHRH gene was assembled by PCR. The 50 μl PCR reaction system was: 5 μl of 10x PCR buffer, 1.5 μl of dNTP, 0.5 μl of Pfu enzyme, primers 3 and 4 2 μl ea...

Embodiment 2

[0076] Example 2, Expression of Human Serum Albumin and Human Growth Hormone Releasing Hormone Fusion Protein Gene HSA-GHRH in Pichia pastoris and Purification of Expression Products

[0077] 1. Expression of human serum albumin and human growth hormone releasing hormone fusion protein gene HSA-GHRH in Pichia pastoris

[0078] 1. Screening of high expression transformants

[0079] The recombinant yeast expression vector HSA-GHRH / pPIC9 of HSA-GHRH constructed in Example 1 was transformed into Pichia pastoris GS115 his4(Mut+his-)NRRLY-15851 by the yeast protoplast transformation method, and the specific method was: linear Add about 10 μg of HSA-GHRH / pPIC9 plasmid DNA into 100 μl Pichia pastoris GS115 protoplasts, and let stand at room temperature for ten minutes. Add 1.0 mL of fresh 40% PEG / CaT solution, mix gently and incubate at room temperature for 10 minutes. After centrifugation at 750g for 10 minutes, carefully aspirate the PEG / CaT solution, and resuspend the cells with ...

Embodiment 3

[0084] Embodiment 3, animal experiments

[0085] 1. In vivo activity detection of fusion protein HSA-GHRH in mice

[0086] Twenty 3-week-old weaned male Kunming mice were randomly divided into 4 groups, 5 mice in each group, one group was the control group, and the other 3 groups were the treatment groups. In the treatment group, 0.2 mL of the HSA-GHRH fusion protein obtained in step 2 was subcutaneously injected per day, and the injection volumes in each group were 20, 2, and 0.2 μg; the control group was subcutaneously injected with 0.2 mL of PBS per day. The body weight was recorded daily, and the administration was continued for two weeks. The experimental results are shown in Fig. 10, the group whose daily dosage is 0.2 μg has the most obvious weight gain effect, and the average daily weight gain percentage is 46% higher than that of the control group.

[0087] 2. In vivo long-term detection of fusion protein HSA-GHRH in mice

[0088] Twenty 3-week-old weaned male Kunm...

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Abstract

The invention discloses fusion protein with the action of promoting body accretion, its encoding gene and use in preparing medicament for treating children's and adults' growth hormone deficiency. The fusion protein is protein having one of the following amino acid residue sequences: (1) SEQ ID No.1 in the sequence table 1, (2) protein related to tumor and obtained through substitution and / or deletion and / or addition of one to ten amino acid residuals of SEQ ID No.1 in the sequence table.

Description

technical field [0001] The present invention relates to a fusion protein and its encoding gene and application, in particular to a fusion protein with the function of promoting body growth, its encoding gene and its application in preparing children and adults with growth hormone deficiency therapeutic medicine. Background technique [0002] Human growth hormone (hGH) is a polypeptide containing 191 amino acid residues, which is secreted by the anterior pituitary gland cells. It has the effect of promoting body growth, maintaining muscle matrix and length, and has the effect of resisting insulin and promoting its anabolism. The secretion of growth hormone (hGH) from the pituitary gland is mainly regulated by two peptide hormones secreted by the hypothalamus: growth hormone releasing hormone (growth hormone releasing hormone, GHRH or GRH), which induces hGH release, and growth hormone inhibitory hormone (somatostatin, SS), which inhibits the release of hGH, that is, the form...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/27A61P5/00
Inventor 刘志敏王楠赵洪亮薛冲张伟熊向华杨秉芬姚学勤
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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