Fc fusion protein of long-acting recombinant human growth hormone

A fusion protein and biological activity technology, applied in the fields of molecular biology and medicine, can solve the problems of difficult construction of hGH-L-vFc fusion protein, no half-life GH derivatives, etc.

Active Publication Date: 2013-01-16
PHARMAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Due to the inherent difficulties in the construction of hGH-L-vFc fusion prot...

Method used

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  • Fc fusion protein of long-acting recombinant human growth hormone
  • Fc fusion protein of long-acting recombinant human growth hormone
  • Fc fusion protein of long-acting recombinant human growth hormone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Construction of encoding hGH-L-vFc γ2 gene encoding the fusion protein

[0082] 1. Preparation of human-growth hormone-containing gene sequence plasmid

[0083] hGH-L-vFc γ2 The fusion protein coding sequence is composed of several DNA fragments. The gene encoding the leader peptide and mature protein containing human GH is prepared by artificial synthesis method according to the human-growth hormone gene sequence contained in NCBI reference number NM_000515.3. The synthesis method is to advance along the double-stranded DNA sequence of the hGH gene from the 5' terminal to the 3' terminal, and prepare four oligonucleotide fragments with alternate upper and lower chains and a length of about 180 bases. The end of each fragment contains about 20 base complementary overlapping sequences with the end of the next complementary chain fragment respectively. Afterwards, the four oligonucleotide fragments were combined into a nucleotide fragment with a length of ab...

Embodiment 2

[0096] Example 2 Construction of encoding hGH-L-vFc γ4 gene encoding the fusion protein

[0097] Due to dissociation of the inter-heavy chain disulfide bonds in the hinge region, a portion of human IgG4 dissociates to form what is considered a half-antibody molecule. This situation does not normally occur in molecules of the other three human IgG isotypes. The literature shows that the 228 site in IgG1 and IgG2 is Pro, while the site is Ser228 in IgG4. Substituting the Ser228 residue of IgG4 with Pro as a single amino acid can keep IgG4 an intact antibody molecule (see Angal et al., Molec. Immunol., 30:105-108, 1993; Owens et al., Immunotechnology, 3:107-116, 1997; US Patent No. 6,204,007). Additionally, the Fc γ4 Carry out Leu235Ala mutation, can make this Fc γ4 The variants have reduced binding to FcyRs. This mutation, together with the aforementioned Ser228Pro mutation, will allow the fusion protein to obtain a more uniform and complete preparation during purification...

Embodiment 3

[0102] Example 3 Construction of encoding hGH-L-vFc γ1 gene encoding the fusion protein

[0103]The hinge region of the human IgGl heavy chain contains 15 amino acid residues including 3 cysteines (GluProLysSerCysAspLysThrHisThrCysProProCysPro). Among these 3 cysteine ​​residues, the 2nd and 3rd are involved in the formation of disulfide bonds between the two heavy chains. The first cysteine ​​residue is involved in disulfide bonding to the IgG light chain. Since there is no light chain in the Fc fusion protein molecule, this cysteine ​​residue may pair with other cysteine ​​residues, resulting in non-specific disulfide bonding. To prevent this non-specific disulfide bonding, the Fc γ1 The hinge region of was shortened to eliminate the first cysteine ​​residue (AspLysThrHisThrCysProProCysPro). Using RNA prepared from human leukocytes and appropriate 5' primers (SEQ ID NO: 13) and 3' primers (SEQ ID NO: 4), Fc-containing γ1 Region-encoding genes. The resulting Fc γ1 DNA ...

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Abstract

The invention discloses a Fc fusion protein of long-acting recombinant human growth hormone. The Fc fusion protein (hGH-L-vFc fusion protein) disclosed herein contains human growth hormone, flexible peptide linker of about 2-20 amino acids, and human IgG Fc mutant. The Fc mutant is not lytic and has tiny side effect of adverse Fc-mediator. The invention further discloses a method for preparing or generating the fusion protein with high expression level. The hGH-L-vFc fusion protein disclosed herein has prolonged serum half-life period and increased biological activity, so as to improve the pharmacokinetics and drug efficacy, and needs few times of injection required in treatment.

Description

technical field [0001] The present invention relates to the fields of molecular biology and medicine. More specifically, the present invention relates to a long-acting recombinant human growth hormone Fc fusion protein and its preparation method and use. Background technique [0002] Human growth hormone (hGH) is a peptide hormone with a full length of 191 amino acids and a molecular weight of 22 kDa, which is produced and released by the pituitary gland. It participates in the regulation of normal growth and development of most people, is the main hormone that stimulates body growth, and exhibits a variety of biological effects, including linear growth, physique formation, lactation, macrophage activation, and insulin-like effects. In addition, growth hormone can also stimulate the metabolism of bones, cartilage and muscles. [0003] Growth hormone and its specific growth hormone receptor (hGHR) bind to each other on the surface of target cells, mediate a biochemical casc...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/85C12P21/02
Inventor 金宜慧刘瑞贤周若芸严孝强王宇鹏李强孙乃超
Owner PHARMAB
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