Use of protein in preparation of drugs for prevention and treatment of Alzheimer's disease
A technology for Alzheimer's disease, uses, application in the field of biomedicine
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Embodiment 1
[0045] Preparation of embodiment 1 sTREM2 protein
[0046] Construction of pFUSE-sTREM2-TEV-hIgG1-Fc1 expression plasmid: pFUSE-hIgG1-Fc1 (available for purchase) was selected as the eukaryotic expression vector, and EcoR Ⅰ and Xho Ⅰ restriction sites were selected as the cloning site to construct pFUSE-sTREM2-hIgG1- Fcl expression plasmid. The steps are as follows: Design upstream primer: 5'-CG GAATTC ATGGAGCCTCTCCGGCTGC-3'SEQ ID NO: 5 where the underline is the EcoR Ⅰ restriction site;
[0047] Downstream primer: 5'-CCG CTCGAG TT TGGGAAGGGGATTTCTC-3'SEQ ID NO: 6, where the underline is the Xho I restriction site; where the double underline corresponds to the coding sequence of the TEV restriction site: GAAAACCTGTATTTTTCAGGGA SEQ ID NO: 7, and the amino acid sequence of the TEV restriction site is: ENLYFQG SEQ ID NO: 8.
[0048] The nucleotide sequence of TREM2 is (purchased from Beijing Yiqiao Sino Biotechnology Co., Ltd., article number HG11084-M):
[0049] ATGGAGC...
Embodiment 2
[0053] Example 2 Preparation of sTREM2-Fc fusion protein
[0054] Construction of pFUSE-sTREM2-hIgG1-Fc1 expression plasmid: pFUSE-hIgG1-Fc1 was selected as the eukaryotic expression vector, and EcoR Ⅰ and Xho Ⅰ restriction sites were selected as the cloning site to construct the pFUSE-sTREM2-hIgG1-Fc1 expression plasmid. The steps are as follows: Design upstream primer: 5'-CG GAATTC ATGGAGCCTCTCCGGCTGC-3'SEQ ID NO: 5 where the underline is the EcoRI restriction site;
[0055] Downstream primer: 5'-CCG CTCGAG TTTGGGAAGGGGATTTCTC-3'SEQ ID NO: 9 where the underline is the XhoI restriction site;
[0056] Using cDNA (SEQ ID NO: 4) as a template, it can be artificially synthesized, and the sTREM2 expression gene can be cloned by PCR. Gel recovery and purification of the target product, and then restriction endonucleases EcoR Ⅰ and Xho Ⅰ digest pFUSE-hIgG1-Fc1 plasmid, and gel recovery of large fragments. The PCR product was digested with the same enzyme, and the digested prod...
Embodiment 3
[0058] Example 3 Identification of sTREM2 protein and sTREM2-Fc fusion protein
[0059] Identification of protein purity: The sTREM2-Fc fusion protein obtained in Example 2 and the sTREM2 protein obtained in Example 1 were subjected to SDS-PAGE electrophoresis, followed by silver staining to identify their purity. Identification results such as image 3 As shown, the sTREM2-Fc fusion protein and sTREM2 protein have single bands and high purity.
[0060] Identification of protein types: The sTREM2-Fc fusion protein obtained in Example 2 and the sTREM2 protein obtained in Example 1 were transferred to PVDF membrane after SDS-PAGE electrophoresis, and an antibody specifically recognizing the extracellular region of human TREM2 (R&Dsystems, AF1828) were identified by western blot. Identification results such as Figure 4 As shown, both sTREM2-Fc fusion protein and sTREM2 can be recognized by the antibody that recognizes human TREM2, indicating that the constructed protein is co...
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