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Fkbp5 gene fragment containing 163g>c mutation, encoded protein fragment and application thereof

A gene and nucleic acid fragment technology, applied in the field of FKBP5 gene mutation, can solve the problems of lack of immunosuppressive drug binding ability and PPIase activity, and achieve the effect of increasing osteoclast ability, increasing phosphorylation level, and strong clinical guidance

Active Publication Date: 2020-05-26
SUZHOU BASECARE MEDICAL DEVICE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The structure of the FK2 domain is similar to that of FK1, but it does not have the ability to bind immunosuppressive drugs and PPIase activity

Method used

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  • Fkbp5 gene fragment containing 163g>c mutation, encoded protein fragment and application thereof
  • Fkbp5 gene fragment containing 163g>c mutation, encoded protein fragment and application thereof
  • Fkbp5 gene fragment containing 163g>c mutation, encoded protein fragment and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: the acquisition of human FKBP5 gene mutation site

[0047] A case of familial clustered Paget bone disease in the Chinese Han population was obtained from the Third People's Hospital of Jinan City, see the pedigree figure 1 , family members gave informed consent to this study. There were 3 patients in the family, namely Ⅱ3, Ⅱ6 and Ⅱ11. According to the imaging examination results of the skeletal system, serological examination results, clinical symptoms and signs, Paget's disease of bone can be clearly diagnosed. The peripheral blood whole blood of the patient and other family members was extracted, and the genomic DNA was extracted using the QIAGEN whole blood small amount DNA extraction kit for gene mutation detection and analysis.

[0048] 1. Sequencing analysis of Paget bone disease susceptibility genes

[0049] Primers were designed for known paget susceptibility genes, including SQSTM1, TNFRSF11A, TNFRSF11B, OPTN, VCP, CSF1, and DCSTAMP, and the e...

Embodiment 2

[0064] Embodiment 2: FKBP5 gene mutation verification

[0065] In order to verify the gene mutation in the family and confirm whether the mutation is an unreported SNP site in the Han population, the gene sequence of the exon 4 of the FKBP5 gene of all family members and 200 normal controls contains the mutation site Perform sequencing analysis.

[0066] 1. Fragment PCR containing mutation site 163G>C

[0067] Using the genomic DNA of all family members and 200 normal controls as templates, the gene fragment containing the FKBP5c.163G>C gene mutation site was amplified.

[0068] The amplification primer fragments are: upstream 5'-ATTAAACCAATGAACAGGAT-3' (SEQ ID NO.5); downstream 5'-CACAACTCAAAAAAAATACCC-3' (SEQ ID NO.6); product length: 446bp.

[0069] The PCR amplification system is as follows:

[0070] TaKaRa Ex Taq HS (5U / μL) 0.1μL

[0071] 10×Ex Taq buffer 2μL

[0072] dNTP mix (2.5mM each) 2μL

[0073] DNA 100ng

[0074] Primers (upstream and downstream primer mix,...

Embodiment 3

[0110] Example 3: Effect of FKBP5 gene mutation on NF-κB and Akt signaling pathways

[0111] 1. Construction of eukaryotic expression vectors expressing wild-type and mutant FKBP5 proteins

[0112] The pcDNA3.1 vector was purchased from Invitrogen (Carlsbad, CA, USA). By designing primers, a BamH I restriction site was introduced at the 5' end of the FKBP5 gene CDS sequence, and an EcoR I restriction site was introduced at the 3' end. The primer sequences are as follows: upstream 5'-GGATCCATGACTACTGATGAAGGTG-3'; downstream 5'-GAATTCTCATACGTGGCCCTCAGGTT-3'. Using the primers, the target fragment is amplified using the wild-type or mutant FKBP5 gene CDS sequence as a template. The PCR product was recovered and purified using a general-purpose DNA purification and recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0113] The purified PCR product was connected to the pMD18-T (Takara, Tokyo, Japan) connection vector, and the positive clones connected with the w...

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Abstract

The invention belongs to the field of biomedicine and provides a FKBP5 gene containing a new single base variation and its coding sequence, the gene is mutated from G to C at the 163rd nucleotide corresponding to SEQ ID NO.1 (163G>C ), thereby causing the amino acid encoded by it to be mutated from valine to leucine, which corresponds to the 55th leucine of SEQ ID NO.4. The mutated FKBP5 protein increases the phosphorylation level of Akt Ser473, increases the ability of osteoclasts, and leads to the formation of Paget's bone disease, thus providing a new marker for the early diagnosis of Paget's bone disease, which has strong clinical guiding significance.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to the mutation of FKBP5 gene. Background technique [0002] Paget's disease of bone is a chronic metabolic bone disease characterized by localized abnormal remodeling of bone tissue, mainly affecting the elderly older than 55 years (1). The prevalence of Paget's bone disease has significant regional differences. It has a relatively high incidence in some areas such as the United Kingdom, Australia, and North America. It is the second largest bone disease after osteoporosis. Uncommon (2). [0003] The etiology of Paget's bone disease is still unclear, and it is generally believed that genetic factors play an important role in its occurrence and development. Paget bone disease susceptibility genes mainly include: SQSTM1, TNFRSF11A, TNFRSF11B, OPTN, VCP, CSF1 and DCSTAMP, etc. (3-6). Among them, the SQSTM1 gene mutation is considered to be the causative gene mutation of Paget's bone disease...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/11C07K14/705
CPCC07K14/705C12Q1/6883C12Q2600/156
Inventor 赵跃然焦玉莲卢冰如崔彬陈子江
Owner SUZHOU BASECARE MEDICAL DEVICE CO LTD
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