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Novel mutant pathogenic gene PLEKHM1 of osteopetrosis, and encoded protein and application thereof

A technology of PLEKHM1 and osteopetrosis, applied in the field of medical molecular biology, can solve incomplete problems and achieve the effect of enriching the resource base and effective molecular diagnosis basis

Active Publication Date: 2016-09-07
SHANDONG PROVINCIAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The currently identified pathogenic genes include LRP5, CLCN7, OSTM, TNFSF11, TCIRG, etc. (5,11), among which the abnormality of TCIRG gene accounts for about 50% of osteosclerosis, but the current research on the pathogenic genes of osteosclerosis and Mutation screening remains incomplete, with causative genes identified in only 70% of osteopetrosis cases (12,13)

Method used

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  • Novel mutant pathogenic gene PLEKHM1 of osteopetrosis, and encoded protein and application thereof
  • Novel mutant pathogenic gene PLEKHM1 of osteopetrosis, and encoded protein and application thereof
  • Novel mutant pathogenic gene PLEKHM1 of osteopetrosis, and encoded protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Obtaining and Identification of Mutant Gene PLEKHM1

[0053] 1. Sample Collection

[0054] The present inventor firstly carried out interrogation, general laboratory examination and bone imaging examination to the patient with osteosclerosis, and found that the patient had symptoms such as easy fracture, tooth loss, anemia, hepatosplenomegaly, and the bone X-ray photograph showed skull, Vertebrae, long bones, etc. have extensive bone density increase. Vertebral bone biopsy pathological smears show bone marrow cavity narrowing and hematopoietic tissue decrease, which are generally consistent with the pathological features of osteosclerosis (see appendix figure 1 ,2,3).

[0055] 2. DNA extraction

[0056] The peripheral blood of the proband and his family members was collected, and the whole genome DNA was extracted from the peripheral blood using a kit (TIANGEN BIOTECH, Beijing, China, DP304-03). The DNA concentration was measured by ultraviolet spectrophotom...

Embodiment 2

[0066] Construction of embodiment 2 expression vector

[0067] The wild-type PLEKHM1 protein expression vector (FLAG-PLEKHM1-WT) was provided by Professor Tamotsu Yoshimori of Osaka University in Japan. The inventor applied the overlap extension PCR method to construct the mutant PLEKHM1 protein expression vector (FLAG-PLEKHM1-MU). The vector plasmid backbone used For FLAG-HA-pcDNA3.1, the details are as Figure 5 shown. First, using the FLAG-PLEKHM1-WT plasmid as a template, the full-length sequence of PLEKHM1 was amplified by overlapping extension PCR technology, and the mutation was introduced into the target fragment, and then the target fragment with the mutation was combined with the vector, and the target gene was introduced into the recipient cells. Escherichia coli DH5α was used for screening and cultivation, and the target gene was obtained from the flora. It is a common technique in this field.

[0068] After identification, the mutant PLEKHM1 protein expression ...

Embodiment 3

[0069] Embodiment 3 Mutant protein expression and identification of function

[0070] PLEKHM1 protein can be combined with Rab7 protein, the wild-type and mutant proteins were expressed by HEK293 cells, and then immunofluorescence was used to detect related proteins, the results Figure 7 shown. Green shows Rab7 protein, red shows PLEKHM1 protein, when the two are fused, they show yellow. The wild-type PLEKHM1 protein is on the left, and the mutant PLEKHM1 protein is on the right. By comparison, it can be found that the expression of PLEKHM1 protein is reduced after mutation, and the combination with Rab7 protein is reduced.

[0071] Co-immunoprecipitation technique was used to detect the combination of wild-type and mutant PLEKHM1 protein and Rab7 protein, such as Figure 8 shown. The Rab7 protein and the wild-type or mutant PLEKHM1 protein were transfected into HEK293 cells, and the cells were lysed after 24 hours. First, immunoprecipitation was performed with FLAG-speci...

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Abstract

The invention relates to a novel mutant pathogenic gene PLEKHM1 of osteopetrosis, and encoded protein and application of the novel mutant pathogenic gene PLEKHM1. The novel mutant pathogenic gene PLEKHM1 is obtained through authentication, and compared with a wild type PLEKHM1 sequence, two bases of the 11th exon in a sequence of the novel mutant pathogenic gene PLEKHM1 are deleted. Compared with wild type PLEKHM1 protein, mutant PLEKHM1 encoded protein is in frameshift mutation starting from the 1018th amino acid. According to the novel mutant pathogenic gene PLEKHM1, the encoded protein and the application, disclosed by the invention, a new site can be provided for gene diagnosis of the osteopetrosis, a resource library of mutant sites related to the osteopetrosis is enriched, and more bases are provided for diagnosis of a patient in the later. The osteopetrosis can be diagnosed and typed through detecting a PLEKHM1 gene sequence of the patient suffering from the osteopetrosis, reasonable prenatal diagnosis can be carried out on puerperae and fetuses, and prenatal and postnatal care screening is achieved.

Description

technical field [0001] The invention relates to a new mutant pathogenic gene PLEKHM1 of osteosclerosis, its encoded protein and its application, and belongs to the field of medical molecular biology. Background technique [0002] Osteosclerosis, also known as "stone bone disease", is a relatively rare systemic genetic disease characterized by bone sclerosis and increased bone density due to impairment of osteoclast function (1-5). The clinical features of osteosclerosis are mainly characterized by extensive heterogeneity: some patients may present fatal clinical features, such as anemia, pancytopenia, sepsis, secondary hepatosplenomegaly, etc.; some patients may also present Asymptomatic or mildly symptomatic, only detectable by skeletal imaging. Skeletal imaging of typical osteopetrosis shows increased bone density in the skull base, pelvis, and vertebral body, and the vertebral body presents a "sandwich"-like change. It may also be accompanied by widening of the metaphysi...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/47C12Q1/68C07K16/18G01N33/68
CPCC07K14/47C07K16/18C12Q1/6883C12Q2600/112C12Q2600/156G01N33/6854G01N33/6893G01N2333/47G01N2800/10
Inventor 徐潮薄涛闫芳赵家军
Owner SHANDONG PROVINCIAL HOSPITAL
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