CN105925584A - Novel mutant pathogenic gene PLEKHM1 of osteopetrosis, and encoded protein and application thereof - Google Patents
Novel mutant pathogenic gene PLEKHM1 of osteopetrosis, and encoded protein and application thereof Download PDFInfo
- Publication number
- CN105925584A CN105925584A CN201610463212.2A CN201610463212A CN105925584A CN 105925584 A CN105925584 A CN 105925584A CN 201610463212 A CN201610463212 A CN 201610463212A CN 105925584 A CN105925584 A CN 105925584A
- Authority
- CN
- China
- Prior art keywords
- plekhm1
- osteopetrosis
- gene
- sequence
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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
The present invention relates to the new sudden change Disease-causing gene PLEKHM1 of a kind of osteopetrosis and encoding proteins thereof and answer
With, belong to field of medical molecular biology.
Background technology
Osteopetrosis, is also called " Osteopetrosis ", is a kind of the rarest, owing to osteoclast function damages
Cause the general heredopathia (1-5) that bone sclerosis, bone density increase.The clinical characters of osteopetrosis predominantly tool
Have heterogeneous widely: some patients can show mortality Clinical symptoms, such as anemia, pancytopenia, pus
Toxemia, Secondary cases hepatosplenomegaly etc.；Some patients also shows as asymptomatic or light symptoms, is only capable of passing through
Skeletal image checks and could find.Skeletal image display basis cranii, pelvis, the vertebral bone of typical case's osteopetrosis
Density increases, and vertebral body presents " sandwich " sample and changes, likely the most broadening with bones of limbs metaphysis, presents
" triangular flask " sample changes.Visible " os in os " phenomenon at long bone, phalanx, pelvis.Bone fragility increases, bone
The wind from the northwest being apt to impair the small intestine danger increases (6,7).
Osteopetrosis clinical classification traditionally is divided into autosomal dominant inheritance, AD and autosomal recessive inheritance, AR
(7-9).Autosomal dominant inheritance, AD clinical manifestation is slightly light, only can show that bone density increases by bone imaging examination
Add.Autosomal recessive inheritance, AR type is more serious (10), is generally just found period at infant, and it is main
Reason is that bone resorption disorder causes excessive mineralising, is generally attended by anemia, haemolysis and hepatosplenomegaly, and prognosis is relatively
Difference, essential therapeutic arsenals relies on bone marrow transplantation (7).
In recent years, the research for osteopetrosis related genes is the most deep, examines the gene of osteopetrosis
It is broken into the Main Basis for the classification of this disease.Identify at present the Disease-causing gene obtained have LRP5, CLCN7, OSTM,
TNFSF11, TCIRG etc. (5,11), the osteopetrosis wherein caused by TCIRG gene unconventionality probably accounts for 50%,
But it is the most not comprehensive to work osteopetrosis Disease-causing gene and Mutation Screening at present, only has the osteosclerosis of 70% at present
Disease case can identify Disease-causing gene (12,13).Therefore, identify that new Disease-causing gene, site are to understand to cause
Pathogenesis system, the key of offer therapeutic scheme.
List of references involved by the application is as follows,
1.Tolar J,Teitelbaum SL,Orchard PJ.Osteopetrosis.N Engl J Med.2004；351(27):2839-49.Epub
2004/12/31.
2.Helfrich MH.Osteoclast diseases.Microsc Res Tech.2003；61(6):514-32.Epub 2003/07/25.
3.Askmyr M,Flores C,Fasth A,Richter J.Prospects for gene therapy of osteopetrosis.Curr
Gene Ther.2009；9(3):150-9.Epub 2009/06/13.
4.Aggarwal S.Skeletal dysplasias with increased bone density:evolution of molecular
pathogenesis in the last century.Gene.2013；528(1):41-5.Epub 2013/05/10.
5.Bonafe L,Cormier-Daire V,Hall C,Lachman R,Mortier G,Mundlos S,et al.Nosology and
classification of genetic skeletal disorders:2015revision.Am J Med Genet A.
2015；167A(12):2869-92.Epub 2015/09/24.
6.Balemans W,Van Wesenbeeck L,Van Hul W.A clinical and molecular overview of the human
osteopetroses.Calcif Tissue Int.2005；77(5):263-74.Epub 2005/11/25.
7.Del Fattore A,Cappariello A,Teti A.Genetics,pathogenesis and complications of
osteopetrosis.Bone.2008；42(1):19-29.Epub 2007/10/16.
8.Aker M,Rouvinski A,Hashavia S,Ta-Shma A,Shaag A,Zenvirt S,et al.An SNX10mutation
causes malignant osteopetrosis of infancy.J Med Genet.2012；49(4):221-6.Epub 2012/04/14.
9.Bollerslev J,Mosekilde L.Autosomal dominant osteopetrosis.Clin Orthop Relat Res.
1993(294):45-51.Epub 1993/09/01.
10.Al-Tamimi YZ,Tyagi AK,Chumas PD,Crimmins DW.Patients with autosomal-recessive
osteopetrosis presenting with hydrocephalus and hindbrain posterior fossa crowding.J
Neurosurg Pediatr.2008；1(1):103-6.Epub 2008/03/21.
11.Warman ML,Cormier-Daire V,Hall C,Krakow D,Lachman R,LeMerrer M,et al.Nosology and
classification of genetic skeletal disorders:2010revision.Am J Med Genet A.
2011；155A(5):943-68.Epub 2011/03/26.
12.Frattini A,Orchard PJ,Sobacchi C,Giliani S,Abinun M,Mattsson JP,et al.Defects in TCIRG1
subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive
osteopetrosis.Nat Genet.2000；25(3):343-6.Epub 2000/07/11.
13.Kornak U,Schulz A,Friedrich W,Uhlhaas S,Kremens B,Voit T,et al.Mutations in the a3
subunit of the vacuolar H(+)-ATPase cause infantile malignant osteopetrosis.Hum Mol Genet.
2000；9(13):2059-63.Epub 2000/08/15.
Summary of the invention
For the deficiencies in the prior art, present invention is primarily aimed at, by the only one China bone accepted for medical treatment a few days ago
The gene screening of sclerosis patients and family thereof and following up a case by regular visits to: 1. identify the Disease-causing gene of osteopetrosis, orient this
The novel pathogenic sites of disease and catastrophe.2. this Disease-causing gene and coded protein thereof are provided, comprise this
The expression vector of mutant gene, expression host cell.3. exploitation is for the quick base of osteopetrosis of this mutant gene
Because of diagnostic kit, the test kit such as including mutant gene diagnostic primers, mutain diagnosis antibody.4. by right
The Clinical Follow-up of this patient, it is provided that disease risks evaluation, provides valuable data for diagnosing this disease further.
The present invention can be that the mechanism of causing a disease illustrating osteopetrosis is provided fundamental basis, and the treatment for osteopetrosis provides new
Drug target.
For achieving the above object, the present inventor only one China osteopetrosis patient to accepting for medical treatment a few days ago and family thereof are entered
Having gone gene screening and followed up a case by regular visits to, having identified and obtain a novel pathogenic sites, this site is spontaneous mutation
PLEKHM1 gene, compared with wild type PLEKHM1 sequence, the 11st exon in this mutant nucleotide sequence
G.59527_59528delCA, two bases there occurs disappearance (c.3051_3052CA).It may cause
The protein product generation frameshift mutation of PLEKHM1 gene code, mutain can result in osteopetrosis and is correlated with
Symptom.According to this mutant gene sequence, the external structure completing the artificial expression vector of this mutant gene, obtain
The expression product of this mutant gene.The PLEKHM1 albumen undergone mutation is the volume of said mutation sequence gene
Code product.Compared with wild type PLEKHM1 albumen, this mutain starts to move from 1018 amino acids
Code sudden change, causes its aminoacid sequence encoded to begin and wild type PLEKHM1 gal4 amino acid at 1018
Sequence is different, and termination codon position there occurs change simultaneously, causes mutain aminoacid sequence to extend 4
Individual residue.
The present inventor has carried out polymorphism analysis for the mutational site of said mutation sequence, normal right at 100
According to crowd does not find this sudden change, it was demonstrated that this mutational site does not exist in non-bone sclerosis patients, is can
The pathogenic mutation site of energy.
Therefore, applicant has carried out the Clinical Follow-up of 4 years to this patient, this patient's self diagnosis be osteopetrosis it
Day plays (in June, 2011), follows the doctor's advice and takes calcitriol (Rocaltrol, 0.5 μ g/day), by serum first shape
Parathyrine (PTH) raises symptom and has alleviated, but anemia and hepatosplenomegaly symptom are without alleviating.This patient is every
Within six months, carrying out blood routine examination, hemoglobin level is close to 70g/L.This patient took in June, 2013
Adrenocortical hormone (prednisone, 30mg/day), but Anemia is still without taking a turn for the better.To 2015 9
Month, patient's liver function and serum PTH levels are stable, are engaged in the work of light work amount, there is no fracture and occur.Logical
Cross the Clinical Follow-up to this patient, it is possible to provide disease risks evaluation to the type osteopetrosis, for examining further
This disease disconnected provides valuable data.
It is further directed to identify that this mutational site of PLEKHM1 obtained, design provide a kind of hard for this bone
Changing the rapid gene diagnostic kit of disease pathogenic sites, described test kit comprises can specific amplification PLEKHM1
The primer of gene purpose section, and can the antibody of PLEKHM1 mutain described in specific detection.Institute
It can expand the fragment (g.59229_59655) of PLEKHM1 gene 427bp length, this fragment to state primer
Contain the mutational site of described PLEKHM1 mutant gene.Described antibody and described PLEKHM1 mutain
Can be specific binding, and not with the protein bound of wild type PLEKHM1 gene code.
Therefore, the present invention
On the one hand, it is provided that the sudden change Disease-causing gene PLEKHM1 that osteopetrosis is new, sequence such as SEQ ID NO.1
Shown in, the PLEKHM1 gene of this sudden change and wild type PLEKHM1 (sequence is as shown in SEQ ID NO.2)
Comparing, two bases CA of its 11st exon there occurs disappearance.The present invention studies this sudden change energy of discovery
Enough cause osteopetrosis related symptoms.
On the other hand, it is provided that the protein coded by this sudden change Disease-causing gene PLEKHM1, sequence such as SEQ ID
Shown in NO.3, this protein and the protein sequence (sequence such as SEQ ID NO.4) of wild type PLEKHM1
Compare, from mutational site after aminoacid sequence there occurs frameshift mutation: i.e..This mutain is from 1018
Amino acids starts frameshift mutation, causes its aminoacid sequence encoded to begin and wild type at 1018
PLEKHM1 protein amino acid sequence is different, and termination codon position there occurs change simultaneously, causes sudden change
Protein amino acid sequence extends 4 residues.
Another further aspect, it is provided that containing the carrier of described sudden change Disease-causing gene PLEKHM1.The expression of described carrier
Product is the albumen of described mutant gene PLEKHM1 coding.
On the other hand, it is provided that described mutant gene PLEKHM1 is at the drug target as treatment osteopetrosis
Application.
Another further aspect, it is provided that the gene PLEKHM1 of described sudden change as osteopetrosis molecular diagnosis and
Application in prenatal diagnosis, i.e. as the application in osteopetrosis diagnostic kit.
Another further aspect, it is provided that a kind of diagnostic kit for diagnosing osteopetrosis, described test kit includes energy
The primer of specific amplification described gene PLEKHM1, described primer sequence such as SEQ ID NO.5 and SEQ ID
Shown in NO.6.Described primer can expand the fragment of PLEKHM1 gene 427bp length, and this fragment contains
The mutational site of described PLEKHM1 mutant gene.
Another further aspect, it is provided that a kind of antibody, described antibody and the protein coded by mutant gene PLEKHM1
(SEQ ID NO.3) specific bond, and do not act on wild type PLEKHM1 coding protein (sequence is such as
SEQ ID NO.4)。
Another further aspect, it is provided that a kind of diagnostic kit for diagnosing osteopetrosis, described test kit includes
Stating can the antibody of specific detection PLEKHM1 mutain.
At present, the gene diagnosis to osteopetrosis patient is carried out the most comprehensively, and one of reason is the bone of only 70%
Sclerosis case can identify Disease-causing gene.Also the exploitation of osteopetrosis related gene diagnostic kit is had no,
Osteopetrosis Disease-causing gene and Mutation Screening are worked the most comprehensive, therefore, identify new Disease-causing gene, position
Point is to understand mechanism of causing a disease, the key of offer therapeutic scheme.
The present invention is by having carried out gene screening to the osteopetrosis patient accepted for medical treatment and its family a few days ago, first
The secondary new mutation site being found that an osteopetrosis Disease-causing gene, and research and develop osteopetrosis for this site
Rapid gene diagnostic kit.
There is advantages that
(1) a new site can be provided for the gene diagnosis of osteopetrosis, enrich this disease related mutation
The resources bank in site, provides more foundation for patient diagnosis afterwards.Can be by detection osteopetrosis patient
Its disease is diagnosed and typing by PLEKHM1 gene order, it is also possible to carry out reasonably for puerpera fetus
Prenatal diagnosis, reaches prenatal and postnatal care examination.
(2) acquisition and vivo and vitro by the osteopetrosis mutain above-mentioned to the present invention are studied, energy
The most deep enough molecular mechanism disclosing osteopetrosis morbidity, can be that osteopetrosis mechanism of causing a disease research provides
Preferably object of study, thinking and theoretical basis.
(3) by the Clinical Follow-up for many years that this patient is carried out, it is possible to obtain what sudden change of the present invention caused
Osteopetrosis develops, disease risk and the related data of prognosis, provides new for treatment osteopetrosis
Path.
(4) present invention can be that treatment osteopetrosis provides a new possible drug target.
(5) test kits based on mutational site of the present invention research and development can be osteopetrosis fast and effectively
Patient provides molecular diagnosis foundation, and can consider to extend to other known or unknown osteopetrosises and cause a disease position
Point.
Accompanying drawing explanation
Fig. 1 is osteopetrosis patient's pedigree chart that the present invention identifies.Wherein square represents male, and circle represents
Women, solid for diseased individuals, hollow for normal individual.
Fig. 2 is the skeletal image figure of the osteopetrosis patient that the present invention identifies.(A) skull X-ray photographs shows
Basion hardening symptom；(B-E) the hardening pathomorphism of lumbar X ray display skeleton；(F) thigh bone X-ray
Photo shows that the long bone including femur, tibia shows bone density widely and increases symptom.
Fig. 3 is osteopetrosis patient's vertebral bone biopsy pathology smear immunostaining figure that the present invention identifies.(A) bone
Occurring unabsorbed calcified cartilage in pulp cavity, bone trabecula substantially increases slightly, and medullary cavity narrows, and hemopoietic tissue is inconspicuous.
(B) calcium deposition seen from cartilaginous tissue and a small amount of osteoclast.(C) medullary cavity Serious Stenosis, part is closed
Plug, hemopoietic tissue significantly reduces, rarely seen a small amount of hematopoietic cell.(D) the rarely seen a small amount of medullary cell of hemopoietic tissue
And hematopoietic cell.
Fig. 4 is the osteopetrosis patient that identifies of the present invention and its lineal relative PLEKHM1 order-checking peak figure.On
Part is PLEKHM1 protein structure domain schematic diagram and PLEKHM1 exons structure schematic diagram；Lower part is
The osteopetrosis patient of present invention qualification and its lineal relative PLEKHM1 order-checking peak figure.Black arrow represents
The site undergone mutation.
Fig. 5 is that the present invention builds saltant type PLEKHM1 protein expression vector (FLAG-PLEKHM1-MU), institute
Scheme with vector plasmid skeleton FLAG-HA-pcDNA3.1.
Fig. 6 is saltant type PLEKHM1 protein expression vector (FLAG-PLEKHM1-MU) constructed by the present invention
Qualification result figure.From left to right: empty plasmid, insertion plasmid, mutant plasmid, marker, double digestion are (empty
Charge material grain, insert plasmid, mutant plasmid), hind3 single endonuclease digestion (empty plasmid, insert plasmid, mutant plasmid),
Kpn1 single endonuclease digestion (empty plasmid, insertion plasmid, mutant plasmid): 5.4kb, Insert Fragment 3171kb.
Fig. 7 is the fluoroscopic examination result of wild type and saltant type PLEKHM1 albumen.
Fig. 8 is wild type and saltant type PLEKHM1 protein immunization co-precipitation testing result.
Detailed description of the invention
Below in conjunction with concrete test method and accompanying drawing to technical scheme and produced technique effect thereof
Being further elaborated, the description below is merely to explain the present invention, but adds the present invention never in any form
To limit, based on present invention teach that any conversion or replacement made, belong to protection scope of the present invention.
Method used in the present invention if no special instructions, is this area conventional method.In following embodiment
Used test material, reagent etc., if no special instructions, the most commercially obtain.
The present invention has carried out the order-checking examination of a large amount of related genes first against the osteopetrosis patient accepted for medical treatment,
1st step. obtain the novel mutation site of a Disease-causing gene PLEKHM1.For this mutational site, applicant
Carry out following work: 2. external structure PLEKHM1 gene and the expression vector of said mutation gene.
3. establish the vitro expression systems of above-mentioned expression vector, it is thus achieved that PLEKHM1 gene expression product albumen with
And mutant protein.4. designed and synthesized and can expand the 427bp nucleotide sequence that comprises this site
Primer, product can be used for later stage order-checking and identifies catastrophe.Primer sequence is such as SEQ ID NO.5, shown in 6.5.
Devise the monoclonal antibody for mutain specific amino acid, it is possible to specific combination
The albumen of this site mutation of PLEKHM1, does not combines with wild type PLEKHM1 simultaneously.
The acquisition of embodiment 1 mutant gene PLEKHM1 and qualification
1. sample collection
First the present inventor carries out inquiring, common lab inspection and skeleton shadow to the osteopetrosis patient accepted for medical treatment
Check as learning, find that patient has the symptoms such as easy fracture, loss of tooth, anemia, hepatosplenomegaly, skeleton X
Actinogram display skull, vertebra, long bone etc. have the bone density of popularity to increase, vertebral bone biopsy pathology smear
Display medullary cavity is narrow, and hemopoietic tissue reduces, and totally meets osteopetrosis pathological characters (see accompanying drawing 1,2,3).
2.DNA extracts
Collection proband and the peripheral blood of family member thereof, employing test kit (TIANGEN BIOTECH, Beijing,
China, DP304-03) from peripheral blood, extract complete genome DNA, ultraviolet spectrophotometer method measures DNA
Concentration, adjusts DNA concentration to 50ng/ul, and-20 DEG C save backup.
3.PCR
Design of primers: PLEKHM1 gene order comes from Gene ID:9842, NCBI ReferenceSequence:
NC_000017.10, mRNA:NM_014798.2, devise 12 pairs of primers altogether for expanding PLEKHM1's
12 exons and the intron sequences of flank thereof.This research the primer is all by Shanghai biotechnology
Services Co., Ltd synthesizes.The equal dissolved dilution of primer is the working solution of 10UM, and-20 DEG C save backup.
Primer sequence is as shown in the table:
| Exon | Forward (Forward) 5'-3' | Reversely (Reverse) 5'-3' | Annealing temperature |
| 2 | tgggaatggactctggctgg | acagtgcccaaggaagtgagc | 60℃ |
| 3 | ttgcacacgtatttggcacc | tctgttggcttcctgaacagc | 60.5℃ |
| 4 | tagttattcctggttcctgctaacca | agtaacagacaatcacatgc | 60℃ |
| 5 | cagcagttttgcattcttcc | ctcacatgccaagaaacagc | 59℃ |
| 6 | cagctactcaggagactggg | atgatggcattctggaatgagg | 60℃ |
| 7-1 | atgcactgcagggcatcagg | acatttcagagcctccaagg | 61℃ |
| 7-2 | agggcacacatttgactgg | cagaagctgtagggcaagcc | 61℃ |
| 8 | agctcccaagttactaggagc | agctccccaacatcacagtg | 62℃ |
| 9 | taagtgagcaggtcatgtgg | tgcatgtgcacgagtgcctgc | 61℃ |
| 10 | aggccgaattacactcctag | tgcagaaaagctacagactg | 62℃ |
| 11 | gccatggcacctcagtgcagc | atgggctgccccaacaactgc | 59℃ |
| 12 | gtgtgacgtatgggtaagc | atggcaaacccagccgggatg | 61℃ |
Archaeal dna polymerase used by PCR be high-fidelity PrimeSTAR Max Premix (Code No.:R045A,
Takara), the PCR reaction system of 25ul is separately added into 2.0ul dNTP, 2.5ul 10 × PCR buffer,
0.15ul Taq enzyme, 2ul genomic DNA and the upstream and downstream primer of each 0.5ul 10um, 17.35ul ddH2O。
PCR reaction condition is: 94 DEG C of denaturations 3min on grads PCR instrument；94 DEG C of degeneration 30s subsequently, anneal 30s
(annealing temperature sees the above table), 72 DEG C extend 30s, totally 40 circulations；Last 72 DEG C extend 10min.
4. order-checking
Using shrimp alkaline phosphotase and excision enzyme I purified pcr product, the product of purification is through agarose gel electricity
After swimming quantitatively, application ABI Prism TM3730 automated DNA analyser (U.S. Applied Biosystems)
Carry out unidirectional or two-way order-checking.Autoassembler 2.0 software (U.S. Perkin Elmer) is used to check order
Result is analyzed with normal wild type PLEKHM1 genome sequence.For ensureing the reliable of sequencing result
Property and repeatability, all sites different from normal gene sequence all carry out positive and negative two-way order-checking.
5. experimental result
Passing through gene sequencing, it was found that a new mutational site, this site is positioned at the 11st exon, is scarce
Lose frameshift g.59527_59528delCA, c.3051_3052CA (as shown in Fig. 4 black arrow).Amplification
These 11 exon primers F: 5 '-TTGGGAGGAGACGGGAGCAG-3 '；R:
5’-GATGGGCATCAGGCGAAA-3’。
The structure of embodiment 2 expression vector
Wild type PLEKHM1 protein expression vector (FLAG-PLEKHM1-WT) is by Japan OsakaUniversity
Tamotsu professor Yoshimori provide, the present inventor applies Overlap extension PCR method to construct saltant type
PLEKHM1 protein expression vector (FLAG-PLEKHM1-MU), used carrier plasmid backbone is
FLAG-HA-pcDNA3.1, details are as shown in Figure 5.First with FLAG-PLEKHM1-WT plasmid as mould
Plate, application overlap extension pcr amplification PLEKHM1 full length sequence, and sudden change is introduced purpose fragment,
Then the purpose fragment with sudden change is combined with carrier, genes of interest is imported recipient cell, apply large intestine bar
Bacterium DH5 α carries out screening and cultivating, obtain genes of interest from flora.For technology commonly used in the art.
Through identifying, saltant type PLEKHM1 protein expression vector (FLAG-PLEKHM1-MU) constructed by the present invention
Being successful, qualification result is as shown in Figure 6.The a length of 5.4kb of used carrier plasmid, Insert Fragment length
For 3171b.First restricted enzyme (Hind III and Kpn I) enzyme action vector plasmid is applied, so during structure
After the purpose fragment with above-mentioned restriction enzyme site is attached with carrier.Therefore, with Hind III and Kpn I
When carrying out double digestion, the fragment (see 6-7 swimming lane) of available 5.4kb Yu 3.2kb size, and use respectively
When Hind III or Kpn I carries out single endonuclease digestion, the fragment of 8.6kb size can be obtained (see 9-10 and 12-13
Swimming lane).
Embodiment 3 mutain is expressed and the qualification of function
Wild type and mutein can be passed through HEK293 with Rab7 protein binding by PLEKHM1 albumen
Cell is expressed, then application Immunofluorescence test associated protein, shown in result Fig. 7.Green show
Rab7 albumen, redness is PLEKHM1 albumen, is shown as yellow after the two merges.Left side is wild type
PLEKHM1 albumen, right side is saltant type PLEKHM1 albumen.By contrast it is found that after Tu Bian
The expression of PLEKHM1 albumen reduces, and reduces with the combination of Rab7 albumen.
Application Immunoprecipitation detects the combination of wild type and saltant type PLEKHM1 albumen and Rab7 albumen
Situation, as shown in Figure 8.Rab7 albumen is transfected respectively with wild type or saltant type PLEKHM1 albumen
HEK293 cell, cell lysis after 24 hours, first carry out immunoprecipitation by the specific antibody of FLAG,
Immunoblotting is carried out the most respectively by shown specific antibody.Result display saltant type PLEKHM1 albumen with
The binding capacity of Rab7 albumen relatively wild type significantly reduces.
Embodiment 4 one kinds is for diagnosing the diagnostic kit of osteopetrosis
Described test kit includes the primer of the specific amplification gene PLEKHM1 of energy, primer sequence F:5 '-TT
GGGAGGAGACGGGAGCAG-3’(SEQ ID NO.5)；R:5’-GATGGGCATCAGGCGAAA-3’(S
EQ ID NO.6)。
25ul reaction system:
2.0ul dNTP
2.5ul 10 × PCR buffer
0.15ul Taq enzyme (archaeal dna polymerase is high-fidelity PrimeSTAR Max Premix)
2ul genomic DNA
The forward primer of 0.5ul 10um
The downstream primer of 0.5ul 10um
17.35ul ddH2O。
PCR reaction condition is: 94 DEG C of denaturations 3min on grads PCR instrument；94 DEG C of degeneration 30s subsequently, 55 DEG C
Annealing 30s, 72 DEG C extend 30s, totally 40 circulations；Last 72 DEG C extend 10min.
Embodiment 5 one kinds is for diagnosing the diagnostic kit of osteopetrosis
Preparation can the monoclonal antibody of specific binding PLEKHM1 mutant protein, use prior art,
Process description is as follows: prepared by (1) antigen: genes of interest fragment is cloned；Expression vector establishment；Protein expression
Purification；(2) immunity and ELISA detection: 4 BALB/C female mices of antigen immune；Positive serum ELISA
Detection, titer > 10000；(3) merge and cell strain screens: the mice taking titer the highest is cooked fusion experiment；From
Screening positive clone in 500 monoclonal cell strain；(4) antibody purification: Protein A/G affinity purification.
Preparation comprises the test kit of above-mentioned antibody, for Testing and appraisal wild type and saltant type PLEKHM1 albumen.
Claims (6)
1. the sudden change Disease-causing gene PLEKHM1 that osteopetrosis is new, is characterized in that, sequence is as shown in SEQ ID NO.1.
2. the protein of mutant gene PLEKHM1 coding described in claim 1, is characterized in that, sequence such as SEQ ID NO.3 institute
Show.
3. a carrier, is characterized in that, requires sudden change Disease-causing gene PLEKHM1 described in 1 containing claim.
4. for diagnosing a diagnostic kit for osteopetrosis, it is characterized in that, described test kit includes can specific amplification power
Profit requires the primer of gene PLEKHM1 described in 1, and described primer sequence is as shown in SEQ ID NO.5 and SEQ ID NO.6.
5. an antibody, is characterized in that, protein coded by mutant gene PLEKHM1 described in described antibody and claim 2
Specific bond, and do not act on the protein of wild type PLEKHM1 coding.
6. for diagnosing a diagnostic kit for osteopetrosis, it is characterized in that, described test kit includes described in claim 5 anti-
Body.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610463212.2A CN105925584B (en) | 2016-06-23 | 2016-06-23 | The new mutation Disease-causing gene PLEKHM1 of osteopetrosis and its coding albumen and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610463212.2A CN105925584B (en) | 2016-06-23 | 2016-06-23 | The new mutation Disease-causing gene PLEKHM1 of osteopetrosis and its coding albumen and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105925584A true CN105925584A (en) | 2016-09-07 |
| CN105925584B CN105925584B (en) | 2019-06-21 |
Family
ID=56831710
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610463212.2A Active CN105925584B (en) | 2016-06-23 | 2016-06-23 | The new mutation Disease-causing gene PLEKHM1 of osteopetrosis and its coding albumen and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105925584B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100034788A1 (en) * | 2004-07-28 | 2010-02-11 | Wim Camiel Augusta Van Hul | Method for diagnosing and treating bone-related diseases |
| CN105586389A (en) * | 2014-10-21 | 2016-05-18 | 天津华大基因科技有限公司 | Kit and application thereof in detection on hereditary bone disease genes |
-
2016
- 2016-06-23 CN CN201610463212.2A patent/CN105925584B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100034788A1 (en) * | 2004-07-28 | 2010-02-11 | Wim Camiel Augusta Van Hul | Method for diagnosing and treating bone-related diseases |
| CN105586389A (en) * | 2014-10-21 | 2016-05-18 | 天津华大基因科技有限公司 | Kit and application thereof in detection on hereditary bone disease genes |
Non-Patent Citations (2)
| Title |
|---|
| GENBANK: "NCBI Reference Sequence: NG_012932.1", 《GENBANK》 * |
| 庞倩倩等: "骨硬化症研究进展", 《中华骨质疏松和骨矿盐疾病杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105925584B (en) | 2019-06-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102622309B1 (en) | Detection of chromosomal interactions | |
| Perdikari et al. | BATLAS: deconvoluting brown adipose tissue | |
| US11197897B2 (en) | Methods and compositions for reducing vancomycin-resistant enterococci infection or colonization | |
| CN105829589A (en) | Cell-free nucleic acids for the analysis of the human microbiome and components thereof | |
| CN109266766B (en) | Application of intestinal microorganisms as bile duct cell cancer diagnosis marker | |
| Emerling | Genomic regression of claw keratin, taste receptor and light-associated genes provides insights into biology and evolutionary origins of snakes | |
| CN108531581A (en) | The mankind IBGC Disease-causing genes XPR1 and its detection method that 490th site mutates | |
| Schaffner et al. | Spondylodiscitis caused by Tropheryma whippelii | |
| Hsieh et al. | Molecular cloning and tissue distribution of ferritin in Pacific white shrimp (Litopenaeus vannamei) | |
| CN107419008B (en) | Method and kit for accurately diagnosing Parkinson's disease in early stage | |
| Izza et al. | Factors Affecting the Occurrence of Stunting in Indonesia. | |
| CN105925584A (en) | Novel mutant pathogenic gene PLEKHM1 of osteopetrosis, and encoded protein and application thereof | |
| EP4347895A1 (en) | Rna profiling of the microbiome and molecular inversion probes | |
| CN105733988B (en) | Composition and application | |
| Ardesjö et al. | Autoantibodies to glutathione S-transferase theta 1 in patients with primary sclerosing cholangitis and other autoimmune diseases | |
| CN112266955A (en) | Ankylosing spondylitis diagnosis marker and application thereof | |
| Teh et al. | P925 Deep functional assessment of the colonic mucosa-associated microbiota in Crohn’s disease and health identifies pro-and anti-inflammatory secretomes and associated bacterial consortia. The ENIGMA study | |
| CN111705134B (en) | Molecular marker of esophageal cancer and application of molecular marker in preparation of esophageal cancer detection kit | |
| Pranoto et al. | Vitamin D Receptor Gene Polymorphism Fok 1 and Vitamin D (25-OH) D Status in Type 2 Diabetes Mellitus Patients with Pulmonary Tuberculosis. | |
| Mahboobi et al. | Evolution of miR-155 and its probable targets: MST1R, Adam10, and CD9 genes expression levels in adults and children gastritis patients with H. pylori infection | |
| CN101104852A (en) | Human new gene LOC344967 correlated with nasopharyngeal carcinoma and coding protein product thereof | |
| CN116287341A (en) | Application of genetically engineered bacteria in preparation of preparation for treating multiple myeloma or relapsed refractory multiple myeloma | |
| Bittner et al. | OA30 Primary osteoarthritis chondrocyte map of chromatin conformation reveals novel effector genes | |
| Wang et al. | First isolation and Genetic characterization of G8P [8] rotavirus strains emerged recently from Guangzhou, China | |
| CN114231648A (en) | Fusobacterium periodontopaeum as esophageal squamous cell carcinoma biomarker and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20160907 Assignee: Beijing Fujun gene Biotechnology Co.,Ltd. Assignor: SHANDONG PROVINCIAL Hospital Contract record no.: X2021980001168 Denomination of invention: Plekhm1, a novel mutated pathogenic gene of osteosclerosis and its encoded protein and Application Granted publication date: 20190621 License type: Common License Record date: 20210209 |