GLP-1 fusion protein and its preparation method and medicinal uses
A fusion protein, GLP-1 technology, applied in pharmaceutical formulations, peptide/protein components, chemical instruments and methods, etc., can solve problems such as limiting clinical applications
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Embodiment 1
[0093] Construction of Fusion Protein Vector
[0094] The GLP-1 part of the fusion protein can be obtained by PCR amplification using any cDNA expressing human GLP-1, such as pancreas, small intestine, and brain tissue cDNA as a template. Mouse and human have the same GLP-1 amino acid sequence, so it can replace human cDNA.
[0095] The DNA encoding the GLP-1 of the present invention can be prepared by various methods including the above-mentioned cloning method and the method of chemically synthesizing DNA.
[0096] The glucagon receptor part of the fusion protein can be obtained by PCR amplification using human liver tissue cDNA as a template, and primers 5'-ATGCCCCCCTGCCAGCCACAG-3' and 5'-TCAGAAGGGGCTCTCAGCCAATC-3'. The amplified glucagon receptor is 1431bp, which is cloned into TOPO TA vector (Invitrogen), and the recombinant vector is identified by EcoRI digestion and sequence analysis. The resulting plasmid is referred to herein as TOPO TA-glucagon receptor.
[0097] ...
Embodiment 1a
[0109] Construction of fusion protein (G in position 2 replacing A) GLP-1-middle linker-glucagon receptor N-terminal extramembrane domain-human immune protein G1Fc recombinant vector:
[0110] Using mouse brain tissue cDNA as a template, the following primers were used:
[0111] (a) 5'-CCCAAGCTT ATGAAGACCATTTACTTTGT-3'
[0112] (b) 5'-GGTCCCTTCAGCATGCTGCCAGCTGCCTTGCAC-3'
[0113] (c) 5'-CATGCTGAAGGGACCTTTAC-3'
[0114] (d) 5'-AAAAGGCGCGCCTTCCTCGGCCTTTCACCAGCC-3'
[0115] PCR amplification can obtain proglucagon signal peptide and GLP-1 coding sequence respectively. The 3' end of the obtained signal peptide was inserted into the 15 bp GLP-1 5' end sequence through primers, and then the signal peptide and GLP-1 were fused together by overlapping PCR with primers (a) and (d). In primers (b) and (c), Ala was changed to Gly (GAA was changed to GCT), and the 5' and 3' ends of the obtained fragments were respectively inserted into HindIII and AscI restriction sites for cloning. ...
Embodiment 1b
[0131] Construction of recombinant vector of fusion protein (G in position 2 replacing A) GLP-1-middle linker-glucagon receptor functional region-1-middle linker-human immune protein G1Fc
[0132] The preparation of signal peptide-(G at position 2 replacing A) GLP-1 fragment was the same as in method 1a.
[0133] Glucagon receptor functional domain-1 can be obtained by PCR amplification using the following primers:
[0134] The primers for amplifying the glucagon receptor functional domain-1 are:
[0135]5'-CCGGCGCGCCTGTCATTGATGGGCTGCTCAG-3' (with Asc I restriction site) and 5'-AAAAGCCCGGGCCAGCCACCGCTCCATCACT-3' (with Srf I restriction site).
[0136] The fragment obtained by the above PCR was digested with HindIII and SrfI and then cloned into the Fc of the vector pV05-human immunoglobulin G1 that had been digested with HindIII and SrfI. The inserted sequence of the recombinant plasmid was identified by nucleotide sequencing and its sequence was completely correct.
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