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FKBP5 gene fragment containing 163G>C mutation, coded protein fragment and application thereof

A fragment and protein technology, applied in the field of biomedicine, can solve the problems of lack of immunosuppressive drug binding ability and PPIase activity, and achieve the effect of increasing osteoclast ability, strong clinical guiding significance, and increasing phosphorylation level.

Active Publication Date: 2017-09-05
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, coded protein fragment and application thereof
  • FKBP5 gene fragment containing 163G>C mutation, coded protein fragment and application thereof
  • FKBP5 gene fragment containing 163G>C mutation, coded protein fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Obtaining the mutation site of human FKBP5 gene

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

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

[0049] Primers were designed for the known paget susceptibility genes, including SQSTM1, TNFRSF11A, TNFRSF11B, OPTN, VCP, CSF1, and DCSTAMP, and sanger sequencing was used to an...

Embodiment 2

[0064] Example 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 sequences containing the mutation site in the FKBP5 gene exon 4 of all family members and 200 normal controls were analyzed. Perform sequencing analysis.

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

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

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

[0069] The PCR amplification system is as follows:

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

[0071] 10×Ex Taq buffer 2μL

[0072] 2μL of dNTP mix (2.5mM each)

[0073] DNA 100ng

[0074] Primers (upstream and downstrea...

Embodiment 3

[0110] Example 3: Effects 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 CDS sequence of the FKBP5 gene, 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 this primer, the target fragment was amplified with the CDS sequence of the wild-type or mutant FKBP5 gene as a template. PCR products were recovered and purified using a universal DNA purification and recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

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

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Abstract

The invention belongs to the field of biomedicine and provides FKBP5 genes containing novel single base variation and a coded sequence thereof. The gene is mutated from G to C (163G>C) at the 163rd nucleotide corresponding to SEQ ID NO.1, so that an amino acid coding the genes is mutated from valine to leucine, namely the 55th leucine corresponding to SEQ ID NO.4. The mutated FKBP5 protein has the effects of increasing the Akt Ser473 phosphorylation level, increasing the osteoclast ability and causing formation of Paget bone disease, so that the FKBP5 protein provides a novel marker for early diagnosis of the Paget bone disease and 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 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 epidemiological characteristics of Paget bone disease have significant regional differences. It has a high incidence in some regions such as the United Kingdom, Australia and North America. It is the second largest bone disease after osteoporosis. However, it is extremely high in Africa and Asia. Rare (2). [0003] The etiology of Paget 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, SQSTM1 gene mutation is considered to be the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C07K14/705
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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