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Fc fusion protein of long-acting recombinant human growth hormone

A fusion protein, hgh-l-vfc technology, applied in the fields of molecular biology and medicine, can solve the problems of no half-life GH derivatives, difficulties in the construction of hGH-L-vFc fusion protein and the like

Active Publication Date: 2014-06-11
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 protein, there is no satisfactory GH derivative with significantly prolonged half-life so far.

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 fusion protein

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

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

Embodiment 2

[0096] Example 2 Construction of encoding hGH-L-vFc γ4 gene encoding 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 a molecule considered to be a half-antibody. This situation does not normally occur in the other three human IgG isotype molecules. The literature shows that the 228 position in IgG1 and IgG2 is Pro, while in IgG4 this position is Ser228. A single amino acid substitution of the Ser228 residue of IgG4 with Pro allows IgG4 to remain intact as an 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). In addition, the Fc γ4 Mutation of Leu235Ala can make this Fc γ4 Variants have reduced binding to FcγRs. This mutation, together with the aforementioned Ser228Pro mutation, will allow the fusion protein to be purified in a more uniform and complete preparation.

[0098]...

Embodiment 3

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

[0103]The hinge region of a human IgG1 heavy chain contains 15 amino acid residues including 3 cysteines (GluProLysSerCysAspLysThrHisThrCysProProCysPro). Among these three cysteine ​​residues, the second and third are involved in the formation of disulfide bonds between the two heavy chains. The first cysteine ​​residue is involved in disulfide bonding with 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 nonspecific disulfide bonding. To prevent this non-specific disulfide bonding, the Fc γ1 The hinge region was shortened to eliminate the first cysteine ​​residue (AspLysThrHisThrCysProProCysPro). Fc-containing primers were obtained by reverse transcription and PCR using RNA prepared from human leukocytes and appropriate 5' primers (SEQ ID NO: 13) and 3' primers (SEQ ID NO: 4...

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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 191 amino acids and a molecular weight of 22 kDa, which is produced and released by the pituitary gland. It is involved in the regulation of normal growth and development in 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 stimulate the metabolism of bone, cartilage and muscle. [0003] Growth hormone and its specific growth hormone receptor (hGHR) bind to each other on the surface of target cells, mediating biochemical cascade reactio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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