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Surface expression vector for fusion protein of Myo-2 peptide multimer and myostatin, and microorganism transformed by thereof

A myostatin and fusion protein technology, applied in the direction of immunoglobulin, animal/human protein, peptide, etc., can solve the problem that the surface anchoring motif has not been developed

Active Publication Date: 2010-09-29
BIOLEADERS CORP +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, surface anchoring motifs satisfying all the above requirements remain unexplored

Method used

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  • Surface expression vector for fusion protein of Myo-2 peptide multimer and myostatin, and microorganism transformed by thereof
  • Surface expression vector for fusion protein of Myo-2 peptide multimer and myostatin, and microorganism transformed by thereof
  • Surface expression vector for fusion protein of Myo-2 peptide multimer and myostatin, and microorganism transformed by thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Determination of the antigenic site of the myostatin peptide

[0081] To enhance the induction of immunity against myostatin, two antigenic sites (Myo-1 and Myo-2) of myostatin were identified on the basis of the predicted 3D structure of myostatin and myostatin The mature protein was purified and administered transdermally to broiler breeder hens. Chicks were hatched from the eggs of the immunized hens, and the enhanced growth rate was compared between the chicks.

[0082] like figure 1 As shown, Myo-1 (SEQ ID NO: 2) and Myo-2 (SEQ ID NO: 1) were identified as antigenic sites on the peptide based on the predicted 3D structure of the mature myostatin protein. In this experiment, three experimental groups: purified myostatin mature protein administration group (mAV group), Myo-1-administration group and Myo-2-administration group, and control group were used, and each group included 5 breeding birds (4 females and 1 male).

[0083] For initial transdermal ...

Embodiment 2

[0085] Example 2: Construction of 4Myo-2 peptide multimer (4xMyo-2) and surface expression vector (pHCE2LB: pgsA-4xMyo-2-CMyom)

[0086] Multimers whose repeat number is a multiple of 4 showed the highest body weight gain among chickens hatched from breeder hens dosed with myostatin-derived peptide and myostatin mature protein (4xMyo-2) The gene encoding Myo-2 peptide in chicken was constructed, and its C-terminus was fused with the gene encoding chicken myostatin mature protein (CMyom), thereby constructing the surface expression vector pHCE2LB: pgsA-4xMyo-2-CMyom.

[0087] In order to synthesize the subunits used to construct the 4Myo-2 peptide multimer, the primers having the nucleotide sequence sequence 5 and sequence 6 were diluted to a concentration of 10 pM / μL respectively, mixed in equal volumes, and reacted at 37°C for 1 hour , followed by image 3 pRSET ( A): Myo-2.

[0088] Sequence 5: 5'-gatccgtttttcttcaaaagtatccacatacacatcttgttcatcaagctggtggttcaagatctggtac-3' ...

Embodiment 3

[0097] Example 3: Cell surface expression of pgsA:4xMyo-2-CMyom and pgsA:CMyom

[0098] It was examined whether a fusion protein containing a 4xMyo-2 peptide multimer fused to a mature protein was fused to pgsA and a mature protein fused to pgsA (pgsA:CMyom) was used with a protein containing a fusion protein encoding myostatin. The surface expression vector pHCE2LB of the gene: pgsA-4xMyo-2-CMyom transformed lactic acid bacteria and the surface expression vector pHCE2LB containing the gene encoding myostatin mature protein constructed in Example 2: pgsA-4xMyo-2-CMyom transformation expressed in lactic acid bacteria.

[0099] For the expression of 4xMyo-2-CMyom protein and CMyom protein respectively fused to the C-terminus of the pgsA gene involved in poly-γ-glutamic acid synthesis, cheese milk transformed with pHCE2LB:pgsA-4xMyo-2-CMyom will be used The Lactobacillus casei strain and the Lactobacillus casei strain transformed with pHCE2LB:pgsA-CMyom were cultured statically ...

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Abstract

The present invention relates to a fusion protein in which a myostatin mature protein is fused to a multimer of myostatin-derived antigenic peptide Myo-2, a surface expression vector containing a polynucleotide encoding the fusion protein, a recombinant microorganism transformed with the vector, and a feedstuff additive or a pharmaceutical composition containing the microorganism as an effective ingredient. The feedstuff additive or pharmaceutical composition according to the present invention can be used for muscle development and regulation of muscle growth in livestock and poultry, as wellas for preventing and treating muscle- wasting diseases and degenerative diseases such as muscular dystrophy, muscular atrophy and the like. In addition, the transformed strain shows the same effect even if the strain itself after culture thereof is directly used, and thus it is very economical.

Description

technical field [0001] The present invention relates to a fusion protein wherein a myostatin mature protein is fused to a multimer derived from myostatin antigen peptide Myo-2, a surface expression vector containing a polynucleotide encoding the fusion protein, using the A recombinant microorganism transformed with a carrier and a feed additive or a pharmaceutical composition containing the microorganism as an active ingredient. Background technique [0002] GDF-8 (growth differentiation factor-8), also known as myostatin as a growth control factor, selectively negatively regulates skeletal muscle growth and was discovered in 1997 (McPherron et al., Nature, 387:83, 1997). The research group that discovered myostatin announced that two breeds of cows, Belgianblue and Piedmontese, which are of high quality due to their high muscle mass and tender meat quality, have mutations in the gene encoding myostatin, which lead to muscle development ( McPherron and Lee, Proc.Natl.Acad....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/02
CPCC07K14/475A61P21/00A61P21/04A61P3/00A61P43/00C07K16/00C07K19/00
Inventor 成文喜金哲仲夫夏玲金智渊金英淑许龙春
Owner BIOLEADERS CORP
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