Agonist peptide for adiponectin receptor
一种序列、氨基酸的技术,应用在激素肽、肽、特定肽等方向,能够解决损伤或破坏、注射的不便或反感大等问题
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Embodiment 1
[0033] Example 1: Synthesis and separation and purification of ADIPOR1 recombinant protein
[0034] ADIPOR1 protein sequence Using the same N-terminal (N-terminal) excluding 88 amino acid sequence used in ADIPOR1 structural analysis, a gene (IDT) that can produce the protein sequence was synthesized through codon optimization (codon optimization) process. At this point, for good cloning and purification of expressed proteins, a BamHI restriction enzyme site, a flag tag (Flag tag) and a TEV protease recognition sequence (ENLYFQG) were inserted at the 5' end (end), 3 ' end into an EcoRI restriction enzyme site. The obtained gene was put into the pFASTbacl vector (pFASTbacl vector) required for the production of baculovirus (baculovirus) to perform cloning. The cloned vector was injected into DH10bac (Invitrogen) and recombined to obtain a bacmid containing the ADIPOR1 gene. In order to make baculovirus, the bacmid obtained by transfection (Transfectine) (Qiagen) was used to tr...
Embodiment 2
[0035] Example 2: Preserving cells
[0036] C2C12 and HepG2 cells were purchased from ATCC, 10% fetal bovine serum (FBS) (Hyclone) and 1% penicillin / streptomycin were added to DMEM (Hyclone) medium, and the cells were incubated at 37°C and 5% CO 2 The environment is cultivated in an incubator for use.
Embodiment 3
[0037] Example 3: C2C12 Myotube Differentiation
[0038] After C2C12 cells were seeded in 12 wells (well), when there was more than 80% confluency, they were washed once with phosphate buffered saline (PBS), and then 2.5% horse serum (Horse serum) and 1 % penicillin / streptomycin differentiation medium, after that, replace the medium with a new differentiation medium every day, and use it in the experiment after 5-7 days of differentiation.
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