CN106841593A - Application of the LACC1 and FHL2 genes in scoliosis detection product is prepared - Google Patents
Application of the LACC1 and FHL2 genes in scoliosis detection product is prepared Download PDFInfo
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Abstract
The invention discloses application of the LACC1 and FHL2 genes in scoliosis detection product is prepared.The invention also discloses a kind of biological reagent for detecting scoliosis.The present invention further discloses a kind of ELISA kit for detecting scoliosis.Early diagnosis be can not only fast and effectively accomplish using LACC1 and FHL2 genetic tests scoliosis, and for predictive diagnosis AIS develops, even therapy target and important evidence provided to treat AIS in biology level from now on.
Description
Technical field
The present invention relates to biomedicine field, and in particular to LACC1 and FHL2 genes are preparing scoliosis detection product
In application.
Background technology
Scoliosis (Scoliosis), is that a class is bent and with Vertebral rotation with backbone side also known as scoliosis
Complex three-dimensional deformity of spine.According to pathogenic factor, 3 major classes can be classified as：Congenital scoliosis, syndrome scoliosis
And idiopathic scoliosis.Congenital scoliosis is by caused by vertebral column development deformity；Syndrome scoliosis is by god
It is caused through muscle disease, skeletal diseases, connective tissue disease and neurofibromatosis etc.；The definite cause of disease of idiopathic scoliosis
It is unclear, correlative study think gene genetic factor, grow with hormone, connective tissue lesion, muscle systems it is abnormal,
Central nervous system exception, effect of epiphysin etc. all may be relevant with the morbidity of AIS.Wherein, adolescent idiopathic backbone side
Curved (Adolescent Idiopathic Scoliosis, AIS) is most commonly seen, accounts for the 80% of whole scoliosis.Teenager is special
The incidence of disease of the hair property scoliosis in -16 years old 10 years old teenagers is higher, is after eyesight abnormality, obesity, phimosis and social mentality
The fifth-largest common disease after obstacle.Although AIS does not generally cause serious pathological state in addition to deformity is showed, severe chest is curved
The heart, PFT may be caused to go down, cause deformity on obvious build, or even influence life-span.
Current treatment method is rescued primarily directed to deformity, including clinical observation, Brace Treatment and lateral bending is exceeded
45 ° of persons carry out operative treatment, and treatment is complicated, has often left deformity of spine and backbone moving obstacle, and effect is undesirable.Meanwhile, AIS
If patient does not carry out examination, the curved average angle of master is 38 ° when first medical, already close to the upper limit for carrying out Brace Treatment.
Therefore, early screening and diagnosis to AIS can strive for the chance of more expectant treatments for patient, reduce operation probability.At present
With examination mainly or by the physical examination to patient's outward appearance symmetry and imageological examination, this can make much for the diagnosis of AIS
The teenager of low-risk receives unnecessary radiological examination, and this examination means specificity is poor.With medical genetics
Learn and Medical Molecular Biology research deepening continuously and improves, the research of the Medical Molecular Biology mechanism of AIS also successively by
Report.Some relative biomarkers are found that during molecular biology research is carried out to AIS, such as calcium adjusts egg
In vain, leptin, Serum osteopontin, solubility CD44, SH3GL1 etc. in peripheral blood, but effect and conclusion still need to further checking.
Therefore, get on to study the AIS causes of disease from gene expression dose, continually look for AIS early screenings new, that specificity is good and diagnosis
Label is significant.
The content of the invention
In order to realize the early detection of Adolescent idiopathic scoliosis, early treatment, an object of the present invention is
Application of the LACC1 and FHL2 genes in scoliosis detection product is prepared is provided.
The second object of the present invention is to provide a kind of biological reagent for detecting scoliosis.
The third object of the present invention is to provide a kind of ELISA kit for detecting scoliosis.
To achieve the above object, present invention firstly provides LACC1 and FHL2 genes in scoliosis detection product is prepared
Application.
Preferably, the scoliosis is Adolescent idiopathic scoliosis.
Preferably, the product can be by detecting LACC1 and FHL2 genes in biological sample or its expression product
Expression carrys out diagnosis of vertebral lateral bending.
Preferably, LACC1 the and FHL2 genes or its expression product express downward in scoliosis biological sample.
Preferably, described detection includes the detection of gene level and the detection of protein level, the gene level detection
Including real-time quantitative PCR method, gene chips and high-flux sequence method；The detection of the protein level includes SABC, glue
Body gold method, ELISA method and Western blot methods.
Preferably, the product includes chip, kit or biological reagent.
Preferably, the kit can be the kit for detecting LACC1 and FHL2 gene transcription levels, such as QPCR reagents
The kit of the expression of box, or detection LACC1 and FHL2 albumen, such as ELISA kit；The chip can be
Genetic chip or protein chip, its expression that can detect LACC1 and FHL2 genes；The biological reagent includes
The specific antibody of the specific primer of LACC1 and FHL2 and anti-LACC1 and FHL2 albumen.
Further, the invention provides a kind of biological reagent for detecting scoliosis, the biological reagent includes being used for
Two groups of primer pairs of scoliosis are detected, wherein, one group of primer pair has SEQ IDNO:1 and SEQ ID NO:Nucleosides shown in 2
Acid sequence；Another group of primer pair has SEQ IDNO:3 and SEQ ID NO:Nucleotide sequence shown in 4, two groups of primer pairs
The expression of LACC1 and FHL2 genes in body is detected by transcript mediated amplification technology, to judge that scoliosis is sent out
Raw risk.
Further, the invention provides a kind of ELISA kit for detecting scoliosis, the kit includes anti-
The specific antibody of LACC1 and FHL2 albumen.
Preferably, LACC1 the and FHL2 protein antibodies are monoclonal antibody or polyclonal antibody.
Preferably, the antibody can be obtained from commercial channels, it is also possible to be come using a series of methods known in the art
Prepare.For example, people LACC1 and the FHL2 albumen or its antigen fragment of purifying are injected into animal body to produce Anti-TNF-α
Body.Equally, the cell of expression people LACC1 and FHL2 albumen or its antigen fragment may also be used for causing animal immune and producing
Antibody.Monoclonal antibody can be prepared with hybridoma technology.
Preferably, LACC1 the and FHL2 protein antibodies are monoclonal antibody, LACC1 the and FHL2 protein antibodies can
It is mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, preferably mouse source antibody.Anti-human LACC1 and FHL2 that the present invention is used
Albumen mouse resource monoclonal antibody is standby by abcam company systems.
Preferably, LACC1 the and FHL2 protein antibodies are the antibody of horseradish peroxidase-labeled.
Preferably, the kit also includes the anti-coated ELISA Plate of LACC1 rabbit polyclonal antibodies, anti-FHL2 rabbit polyclonals
The coated ELISA Plate of antibody, protein standard substance LACC1 and FHL2, tmb substrate solution, dilution, lavation buffer solution, termination are molten
Liquid.
Preferably, stop bath composition is 2M H2SO4。
Beneficial effects of the present invention are as follows：The invention discloses a kind of the gene LACC1 and FHL2 related to scoliosis,
And LACC1 the and FHL2 genes or its expressing protein are further characterized by Adolescent idiopathic scoliosis patient derived biological sample
Middle expression is lowered.The generaI investigation of scoliosis is carried out in teenager using LACC1 and FHL2 genes, is solved and is examined in the prior art
The problem of disconnected measure Sensitivity and Specificity difference, decreases the infringement of x-ray radiation, can fast and effectively accomplish the morning of AIS
Phase diagnoses, and for predictive diagnosis AIS develops, even provides treatment to treat AIS in biology level from now on
Target spot and important evidence.
Brief description of the drawings
Scoliosis patient's LACC1 and FHL2 down regulation of gene expression in embodiment 2 in Fig. 1 present invention.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
In the conventional meanses that are well known to those skilled in the art of technological means used.
The experimental technique of unreceipted actual conditions in embodiment, usually this area conventional method, such as according to normal condition
Such as Sambrook et al., molecular cloning, the condition in laboratory manual (third edition) (Science Press, 2002), or according to
Condition proposed by reagent manufacturing firm.
Technical scheme is specifically included：25 Adolescent idiopathic scoliosis clinical samples and 15 are compareed
Sample carries out high-flux sequence, and genescreen is carried out with reference to bioinformatics method, picks out candidate gene LACC1 and FHL2,
Do not have LACC1, FHL2 report related to Adolescent idiopathic scoliosis in existing research, further, inventor is carried out
Molecular biology method checking, it was confirmed that LACC1 and FHL2 expresses downward, its phase in scoliosis patient derived biological sample
Preparation is closed to can be used to diagnose Adolescent idiopathic scoliosis.
LACC1 and FHL2 genes of the invention are knowns before making the present invention, and its essential information is as follows：
Genbank accession number：LACC1GeneID:144811, FHL2GeneID:2274 derive from human genome.
LACC1 (laccase domain containing 1, laccase domain 1) is an albumen on No. 13 chromosomes
Encoding gene.Include rheumatoid arthritis and systemic onset juvenile leprosy with LACC1 diseases.
FHL2 (four and a halfLIM domains 2) also known as DRAL be FHL (four-and-half LIM,
FHL) a member in family.FHL families have 5 members, i.e. FHL1, FHL2, FHL3, FHL4 and FHL5/ACT, comprise only LIM
The activity of domain, mediating proteins-protein-interacting, activation or suppression transcription factor, and then influence gene expression；FHL2 is adjusted
The propagation of ganglion cell；Participate in the formation of cytoskeleton.
The experimental technique specifically studied mainly includes following components：
1. using high-flux sequence method to 25 levels of blood sample LACC1 and the FHL2 gene of scoliosis patient
Comparison in difference is carried out with 15 check samples.
2. the expression using RT-PCR method detection LACC1 and FHL2 genes in scoliosis patient and normal population,
And the gene is demonstrated for scoliosis expresses down-regulated gene.
3. the ELISA detection kit of scoliosis is assembled and used
(1) preparation of conventional reagent during ELISA is tested
(2) prepared by polyclonal antibody
(3) coating of ELISA Plate
(4) preparation of enzyme labelled antibody
(5) assembling of kit
The clinical practice of 4.ELISA kits
The ELISA kit prepared using the present inventor is detected scoliosis patient to be made a definite diagnosis and is detected with actual clinical
Compare that the validity of ELISA kit is determined.Specifically include LACC1 and FHL2 albumen in measure subject's blood sample
Expression quantity, and be compared with LACC1 and FHL2 expressing quantities in normal blood sample, it is that clinician quick and precisely grasps
The morbid state and coincident with severity degree of condition of patient, take the control prece of more personalized to provide support in time.
The nucleotides full length sequence or its fragment of LACC1 and FHL2 genes of the invention can generally use PCR TRAPs, weight
Group method or artificial synthesized method are obtained.Once obtain relevant sequence, it is possible to had in large quantity with recombination method
Close sequence.
At present, it is already possible to encode the DNA sequence dna of albumen of the invention (or its fragment) by chemical synthesis completely.So
The DNA sequence dna can be introduced into afterwards in the various DNA moleculars in this area (such as carrier) and cell.The fragment of albumen of the present invention except
Outside being produced with recombination method, also can use solid phase technique that (Stewart et al., (1969) are produced by being directly synthesized peptide
Soliod-Phase Peptide Synthesis, WH Freeman Co., San Francisco；Merrifield J.
(1963)J.Am Chem.Soc 85:2149-2154).Synthetic protein can be carried out by hand or automatically in vitro.For example,
Peptide can be automatically synthesized with the 431A types peptide synthesizer (Foster City, CA) of Applied Biosystems.Can be respectively
Each fragment of chemical synthesis albumen of the present invention, is then chemically connected to produce the molecule of total length.
When " scoliosis " used herein is not explained, Adolescent idiopathic scoliosis are typically refered in particular to.
Terms used herein " biological sample " includes but is not limited to the samples such as blood, serum, saliva, urine, synovia, cartilage
Product.Any tissue sample (such as interverbebral disc, articular process, spinal cord, the vertebra of any subject are derived from for biological sample of the invention
Other flesh or blood sample) or cell sample (such as Gegenbaur's cell, cartilage cell or blood cell samples) can be made according to the inventive method
With.These samples can therefrom be obtained and nothing is included but is not limited to using subject's example of these samples according to the inventive method
The subject of symptom subject, performance or more scoliosis symptom, clinical diagnosis are the subject with scoliosis, are susceptible to suffer from
Scoliosis subject (if any the subject of scoliosis family history, have scoliosis genetic predisposition subject and
Life style makes the subject that scoliosis possibility is suffered from its susceptible scoliosis or raising), suspect and being received with scoliosis
Examination person, just receiving scoliosis treatment subject, with scoliosis and it is non-receive scoliosis treatment subject, opening
Doctor (such as doctor) is defined as health or the subject's (i.e. normal) without scoliosis, has cured the subject, just of scoliosis
Control the subject of its scoliosis and have not been diagnosed as the subject of scoliosis.
The high-flux sequence of embodiment 1 screens difference expression gene
1st, sample
The scoliosis patient gone to a doctor in BJ Union Hospital's orthopaedics during choosing in October, 2012 in December, 2015, disease
Example group collects 25 altogether, and all patients have typical clinical manifestation, are made a definite diagnosis through x-ray inspection.Control derives from same time orthopaedics
Other diseases patient in hospital, collects 15 altogether.Patient is the teenager of -18 years old 10 years old.Gather the blood of all research objects
Liquid sample, numbers rearmounted -80 DEG C of low temperature refrigerators and preserves.All clinical samples of this research, to patient know the inside story and inform simultaneously
Pass through through this Hospital Ethical Committee.
2nd, Total RNAs extraction is carried out to blood sample
UsingLS(Invitrogen:RNA extractions 10296-010) are carried out to the blood sample for gathering, is tested
Operation is carried out by product description, and the concrete operations of every group of experiment are as follows：
The new blood being collected into is taken, 3 times of volume erythrocyte cracked liquids are added, room temperature placement 10 minutes after mixing, 10,
000rpm is centrifuged 1 minute.Thoroughly inhale and abandon supernatant, collect leukocyte cell pellet.
(1) 1mLTrizol, room temperature preservation 5 minutes are entered；
(2) the imitative 0.2mL of chlorination, uses forced oscillation centrifuge tube, fully mixes, and room temperature is placed 3-5 minutes；
(3) 12000rpm high speed centrifugations draw upper strata aqueous phase (inhale 70%) in another new centrifuge tube pipe after 15 minutes, note
Meaning should not be drawn onto the interface between two-layer water phase.New pipe is moved into, -20 DEG C of isometric pre- cold isopropanols are added, it is fully reverse mixed
It is even, it is placed in 10 minutes on ice；
(4) 12000rpm carefully discarded supernatant after 15 minutes at a high speed, and 75% is added in the ratio of 1mL/mL Trizol
DEPC ethanol washes paint precipitation (4 DEG C of preservations), washes paint sediment, and vibration mixes, and 8000rpm is centrifuged 2 minutes at 4 DEG C.Discard ethanol
Liquid, places 2 minutes fully to dry precipitation at room temperature, adds the water dissolves precipitation that DEPC is treated；
(5) RNA purity and concentration are measured with Nanodrop2000 ultraviolet specrophotometers, is frozen in -80 DEG C.RNA mass
Criterion：The OD260/OD280 values of RNA samples are between 1.7-2.2；Total serum IgE electrophoresis pattern has clearly 28S, 18S bar
Band；70 DEG C of water-baths be incubated 1 hour after electrophoresis pattern and water-bath insulation before collection of illustrative plates no significant difference.
3rd, the quality analysis of RNA sample
RNA extract after agarose gel electrophoresis, from electrophoresis result can with preliminary judgement extract RNA sample it is up-to-standard with
It is no, if to can be used for further transcriptome analysis.And then RNA sample is detected by NanoDrop1000 spectrophotometers
Extraction situation, the sample requirement of RNA-seq sequencings：OD260/OD280 is 1.8-2.2.
4th, high-flux sequence
Microarray dataset is the high-flux sequence platforms of HiSeq 2500 of Illumina companies, carries out high flux transcript profile depth
Sequencing, we use Fast-QC (http after sequencing://www.bioinformatics.babraham.ac.uk/projects/
Fastqc/) software carries out total evaluation to the quality of sequencing data, including base quality Distribution value, the position point of mass value
Cloth, G/C content, PCR duplication contents, frequency of kmer etc..In differential genes expression analysis, according to obtaining
FPKM values, differential screening is carried out using internationally recognized algorithm EBSeq.Wherein, during screening, LOG2FC>1 or<- 1, FDR<
0.05.In order to be better understood from the function of difference expression gene, we difference expression gene has been carried out Gene Ontology and
Signal path is analyzed, and functional annotation and protein interaction network analysis are carried out to difference expression gene, in view of above number
According to the result of analysis, with reference to document, we have screened difference expression gene LACC1 and FHL2, LACC1 and FHL2 genes in backbone
Expressed in lateral bending blood samples of patients sample and lowered.
The qRT-PCR of embodiment 2 verifies the expression of scoliosis patient's LACC1 and FHL2 gene
1st, material
The scoliosis patient 10 gone to a doctor in BJ Union Hospital's orthopaedics during choosing in October, 2012 in December, 2015
Example, all patients have typical clinical manifestation, are made a definite diagnosis through x-ray inspection.Other diseases that control is in hospital from same time orthopaedics
Patient, collects 8 altogether.Patient is the teenager of -18 years old 10 years old.The blood sample of all research objects is gathered, after numbering
- 80 DEG C of low temperature refrigerators are put to preserve.All clinical samples of this research, to patient know the inside story and inform and entrusted through this hospital ethics
Member can pass through.
2nd, method
2.1 pairs of blood samples carry out Total RNAs extraction, with the extracting method of embodiment 1.
2.2 reverse transcriptions synthesize cDNA
UsingRT reagent kit (TaKaRa, article No. DRR037A) carry out cDNA reverse transcriptions, real
Test operation is carried out by product description, and concrete operations are as follows：
Using Reverse Transcriptase kit, converse record synthesis cDNA is carried out to 0.3 μ g total serum IgEs with RT Buffer.Using 10 μ
L reaction systems：5xPrimerScript Buffer 2μL、PrimeScript RT Enzyme Mix I 0.5μL、OligodT
The μ L of Primer 0.5, Random 6mers 0.5 μ L, RNA templates are 0.3 μ g and RNase Free dH2O is mended to 10 μ L.Obtain
CDNA preserve that to put -20 DEG C of refrigerators standby.
2.3 Real-Time PCR
Using online primer-design software, LACC1 gene orders are with reference to NCBI:NM_001128303.1, FHL2 gene sequence
Row are with reference to NCBI:NM_001039492.2, interior participation in the election GAPDH, is synthesized after design of primers by invitrogen companies.Specific primer
As shown in table 1：
The primer sequence table of table 1
WithPremix Ex TaqTMII (TaKaRa, article No. DRR081A) is expanded, and experimental implementation presses product
Specification is carried out.The μ L reaction systems of quantitative fluorescent PCR 20 are as follows：Premix Ex TaqTMII：10 μ L, primer (10
μM)：Forward and reverse each 0.8 μ L, ROX Reference DyeII (50 ×) of primer：0.4 μ L, dH2O：6 μ L, template cDNA：2μL.
Response procedures are carried out on ABI7500, and amplification program is：95 DEG C of 30sec, (95 DEG C of 5sec, 60 DEG C of 34sec) × 40 circulations.
3rd, statistical analysis
Real-time quantitative PCR amplification curve flex point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is flat and nothing raises up now, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve is all unimodal, illustrates that amplified production only has one, is specific amplification；Relative quantification formula according to qRT-PCR：2-ΔΔCt× 100%, compare expression of the LACC1 and FHL2 genes in scoliosis blood samples of patients and in control group blood.
Result shows：The expression water of qRT-PCR stable amplification results, wherein LACC1 and FHL2 genes in scoliosis blood samples of patients
In flat respectively control group blood 40% and 35%, as shown in Figure 1.Result above demonstrates high flux transcript profile expression data
Confluence analysis LACC1 and FHL2 gene the result of downward is expressed in scoliosis patient.
The ELISA kit of the detection scoliosis of embodiment 3 is assembled and used
Conventional reagent in 1.ELISA experiments：
Coating buffer solution (carbonate buffer solution of pH9.6)：Na2CO31.59g, NaHCO32.93g, plus distilled water is to 1L.
Lavation buffer solution (pH7.4)：8.0g NaCl；0.2g KH2PO4；2.9g Na2HPO4·12H2O；0.2g KCl；
The Tween-20s of 0.5mL 0.05%, plus ddH2O to 1L.
Dilution:Bovine serum albumin(BSA) (BSA) 0.1g adds lavation buffer solution to 100mL；
The anti-human LACC1 and FHL2 albumen mouse resource monoclonal antibody that the present invention is used is standby by abcam company systems.
Protein standard substance LACC1 and FHL2 are purchased from Beijing Aureal Dongyuan County limited public affairs of biotechnology for people source recombinant protein
Department.
2. prepared by polyclonal antibody：
LACC1 and FHL2 albumen is prepared using solid-phase synthesis；LACC1 and FHL2 will be synthesized using carbodiimide coupling method
Albumen and carrier protein BSA (bovine serum albumin(BSA)) coupling preparation immunogenes LACC1-BSA and FHL2-BSA, combined U V are (ultraviolet
Scanning) and SDS-PAGE (polyacrylamide gel electrophoresis) monitoring conjugates LACC1-BSA and FHL2-BSA；The idol that will be prepared
Connection thing LACC1-BSA and FHL2-BSA is immunized the big ear of New Zealand as immunogene with reference to Freund's complete adjuvant and Freund's incomplete adjuvant
White rabbit, each conjugate antigen immune dosage is 1mg, and immunization wayses are the subcutaneous multi-point injection of nape part；Detected after being immunized through 3 times
Immune intravenous rabbit drop of blood degree, reaches 1:After more than 10000, arteria carotis is carried out to immune rabbit and takes the immune whole blood of blood collection, centrifugation point
From rear collection rabbit immune serum；Carried out using the rabbit immune serum of Protein A affinity columns confrontation LACC1 and FHL2 albumen
Purifying, prepares the polyclonal antibody of anti-LACC1 and FHL2 albumen, detects titre, specificity, sensitivity of polyclonal antibody etc.,
Obtain antiserum LACC1 and FHL2 protein antibodies.
3. the coating of ELISA Plate：
The described anti-coated ELISA Plate of LACC1 and FHL2 rabbit polyclonal antibodies is prepared via a method which：With pH9.6's
Carbonate coating buffer solution will be diluted to the μ g/mL of purpose concentration 0.63 by anti-LACC1 and FHL2 rabbit polyclonal antibodies after purification；Will be dilute
The antibody-solutions released are added in micropore after mixing, 100 μ L/ holes, and 4 DEG C overnight；Board-washing 3 times, 200 μ L/ holes；Add 3%BSA envelopes
Close liquid, 300 μ L/ holes, 4 DEG C are overnight；Board-washing 3 times, 200 μ L/ holes；- 20 DEG C of preservations.
4. the preparation of enzyme labelled antibody：
Anti- LACC1 and FHL2 albumen mouse resource monoclonal antibody is taken, is coupled with HRP respectively, obtain enzymic-labelled antibody.Take
A certain amount of enzymic-labelled antibody is added in dilution, is fully mixed, and makes its final concentration of 2 μ g/mL (can be according to specific condition
Depending on), 2-8 DEG C keeps in dark place.
5. the assembling of kit：
Detecting the ELISA kit of scoliosis includes the component in table 2：
The ELISA detection kit of table 2
Use for convenience, the kit can also include positive control：Normal human AB serum or the μ L of hyclone 500 and feminine gender
Control：1%PBS 1mL.
The application method of the kit is as follows：
(1) LACC1 and FHL2 standard proteins concentration respectively 900pg/mL, 600pg/mL, 300pg/mL are taken respectively,
The μ L of 150pg/mL, 75pg/mL solution 50 add the ELISA Plate of coated antibody to draw standard curve；
(2) blank well (blank control wells are not added with sample and enzyme marking reagent, and remaining each step operation is identical) is set respectively, treat test sample
Sample wells, first adds the μ L of sample diluting liquid 40 in testing sample hole on ELISA Plate, and the μ L of testing sample 10 are then added again, and (sample is finally dilute
Degree of releasing is 5 times).Sample is added on ELISA Plate bottom hole portion by sample-adding, and hole wall is not touched as far as possible, gently rocks mixing；
(3) rearmounted 37 DEG C of shrouding film shrouding is used to incubate 30 minutes；
(4) carefully take shrouding film off, discard liquid, dry, cleaning solution is filled it up with per hole, discarded after standing 30 seconds, so weight
It is multiple 5 times, pat dry；
(5) the μ L of enzyme marking reagent 50 are added per hole；
(6) incubate, washing, step is ibid；
(7) the μ L of tmb substrate solution A 50 are first added per hole, add the μ L of tmb substrate solution B 50, gently concussion is mixed,
37 DEG C of lucifuges develop the color 15 minutes；
(8) the μ L of terminate liquid 50, terminating reaction are added per hole；
(9) returned to zero with blank well, 450nm wavelength sequentially measures the absorbance (OD values) in each hole, in each serum sample of calculating
Protein content.
The clinical detection of the ELISA kit of embodiment 4
1st, case
20 scoliosis patients for treating examination are chosen, is sampled during deriving from October, 2012 in December, 2015 in north
The medical patient of capital Concord Hospital orthopaedics.The blood sample of all research objects is gathered, rearmounted -80 DEG C of low temperature refrigerators is numbered and is protected
Deposit.All clinical samples of this research, to patient know the inside story and inform and pass through through this Hospital Ethical Committee.
2nd, method
Sample disposal serum separation gel vacuum test tube gathers venous blood 2mL, first room temperature 3000r/min and 10min is centrifuged,
Transfer upper serum, then at 4 DEG C, 16000 × g centrifugation 10min, draws the acellular serum packing in the upper strata often μ L of pipe 200, puts -70
DEG C preserve.Aforesaid operations are in completion in 2h after collection of specimens.Normal human AB serum uses implementation as positive control with kit
LACC1 and FHL2 expressing quantities change with respect to normal human AB serum in ELISA kit detection blood sample in example 3.
3rd, result
Result shows, have in 20 blood samples of preceding scoliosis patient for treating examination in 7 clinical samples LACC1 and
Expression quantity is without significant difference in FHL2 expressing quantities and normal human AB serum；Have in 13 blood samples of patients samples LACC1 and
FHL2 expressing quantities are less than 60% of expression quantity in normal human AB serum, wherein have in 9 blood samples of patients samples LACC1 and
The expression quantity of FHL2 albumen is less than the 40% of normal human AB serum's expression quantity.Through clinical further detection, 20 are treated examination
9 preceding scoliosis are made a definite diagnosis in patient, this 9 preceding scoliosis patients are consistent with kit testing result prepared by the present invention.
Infer accordingly, diagnostic kit of scoliosis can clearly distinguish preceding scoliosis patient before this, and be carried as clinic
For diagnostic clue.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Beijing Sai Erda Bioisystech Co., Ltd
<120>Application of the LACC1 and FHL2 genes in scoliosis detection product is prepared
<130> p16zjcw18
<160> 6
<170> PatentIn version 3.5
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<212> DNA
<213>Artificial sequence
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<212> DNA
<213>Artificial sequence
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<212> DNA
<213>Artificial sequence
<400> 3
tgcctggctg agaactgtg 19
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<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
gtgccggtcc ttgtaagaca 20
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<212> DNA
<213>Artificial sequence
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ggagcgagat ccctccaaaa t 21
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<212> DNA
<213>Artificial sequence
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Claims (10)
- Application of the 1.LACC1 and FHL2 genes in scoliosis detection product is prepared.
- 2. application as claimed in claim 1, it is characterised in that the scoliosis is Adolescent idiopathic scoliosis.
- 3. application as claimed in claim 1, it is characterised in that the product can by detect in biological sample LACC1 and The expression of FHL2 genes or its expression product carrys out diagnosis of vertebral lateral bending.
- 4. application as claimed in claim 1, it is characterised in that LACC1 the and FHL2 genes or its expression product are in backbone side Expressed in curved biological sample and lowered.
- 5. applied as described in Claims 1 to 4 is any, it is characterised in that the product includes chip, kit or biological examination Agent.
- 6. a kind of biological reagent for detecting scoliosis, it is characterised in that the biological reagent is included for detecting scoliosis Two groups of primer pairs, wherein, one group of primer pair has SEQ IDNO:1 and SEQ ID NO:Nucleotide sequence shown in 2；It is another Group primer pair has SEQ IDNO:3 and SEQ ID NO:Nucleotide sequence shown in 4, two groups of primer pairs are situated between by transcribing Lead amplification technique to detect the expression of LACC1 and FHL2 genes in body, to judge the risk that scoliosis occurs.
- 7. it is a kind of detect scoliosis ELISA kit, it is characterised in that the kit include anti-LACC1 and FHL2 eggs White specific antibody.
- 8. kit as claimed in claim 7, it is characterised in that LACC1 the and FHL2 protein antibodies be monoclonal antibody or Polyclonal antibody.
- 9. kit as claimed in claim 8, it is characterised in that LACC1 the and FHL2 protein antibodies are horseradish peroxidase The antibody of enzyme mark.
- 10. kit as claimed in claim 7, it is characterised in that the kit also includes anti-LACC1 rabbit polyclonal antibodies bag The ELISA Plate of quilt, the anti-coated ELISA Plate of FHL2 rabbit polyclonal antibodies, protein standard substance LACC1 and FHL2, tmb substrate solution, Dilution, lavation buffer solution, stop bath.
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| CN107312846A (en) * | 2017-07-12 | 2017-11-03 | 北京赛尔维康生物医学科技有限公司 | Application of the CAPG and PTGIS genes in scoliosis detection kit is prepared |
| CN107326076A (en) * | 2017-07-12 | 2017-11-07 | 北京赛尔维康生物医学科技有限公司 | A kind of scoliosis early stage auxiliary detection kit and its application |
| CN108852286A (en) * | 2018-05-03 | 2018-11-23 | 腾讯科技（深圳）有限公司 | Show the method, apparatus and terminal of backbone measurement data |
| CN114622013A (en) * | 2022-05-17 | 2022-06-14 | 中南大学湘雅医院 | Juvenile idiopathic scoliosis detection product |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107312846A (en) * | 2017-07-12 | 2017-11-03 | 北京赛尔维康生物医学科技有限公司 | Application of the CAPG and PTGIS genes in scoliosis detection kit is prepared |
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| CN108852286A (en) * | 2018-05-03 | 2018-11-23 | 腾讯科技（深圳）有限公司 | Show the method, apparatus and terminal of backbone measurement data |
| CN114622013A (en) * | 2022-05-17 | 2022-06-14 | 中南大学湘雅医院 | Juvenile idiopathic scoliosis detection product |
| CN114622013B (en) * | 2022-05-17 | 2022-08-19 | 中南大学湘雅医院 | Juvenile idiopathic scoliosis detection product |
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