CN108085305A - The 5-ALA mutant synthase albumen of people a kind of and its application - Google Patents
The 5-ALA mutant synthase albumen of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to the 5 aminolevulinic acid synthase mutains of people a kind of and its applications, using a large amount of porphyrias and leukaemic as research case, genetic test is carried out to case and is analyzed, determine the mutain of 5 aminolevulinic acid synthases of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the 5 aminolevulinic acid synthase mutains of the people, for the impulse that the gene diagnosis of human health status provides, the early diagnosis, early discovery and associated treatment of porphyria and leukaemia are realized.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to a kind of people 5-ALA mutant synthase albumen and
It is applied.
Background technology
5-ALA synthase is the rate-limiting step for the biosynthesis for being catalyzed ferroheme (iron-protoporphyrin).5- amino ketones penta
Acid synthase is house keeping enzyme；One individual encoding gene is specific to the form of erythrocytic tissue's enzyme.The protein of maturation coding
Level is adjusted by ferroheme：The ferroheme of high-content lowers maturase in mitochondria and low hemoglobin levels raise.The gene
Pseudogene be located at No. 12 chromosomes, alternative splice variant generates multiple variants coding different subtypes.With the 5- amino ketones penta
The relevant disease of synthase proteins includes porphyria and leukaemia.Therefore, to the inspection of the 5-ALA mutant synthase of people
Survey has certain impulse to the health status for judging human body.
The content of the invention
Present invention aims at the 5-ALA mutant synthase albumen and its application for providing a kind of people.
Technical solution of the present invention includes：
In a first aspect, the 5-ALA mutant synthase albumen of people a kind of is provided, amino acid sequence such as SEQ ID
NO:Shown in 1.
Second aspect provides a kind of encoding gene of the 5-ALA mutant synthase albumen of people, nucleotide sequence
Such as SEQ ID NO:Shown in 2.
The third aspect provides a kind of genetic chip, including：Solid phase carrier and fixed nucleotide probe on this carrier；
The nucleotide probe is according to the nucleotide sequence of the 5-ALA mutant synthase albumen of the people, the 5- amino ketones with people
The comparison result of the nucleotide sequence of the normal albumen of valeric acid synthase determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of monoclonal antibody of the 5-ALA mutant synthase albumen of specific recognition people,
Its amino acid sequence encoded is SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA examinations of the antibody for the 5-ALA mutant synthase albumen for being used to detect people
Agent box, the ELISA kit include：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection object of said monoclonal antibody
5-ALA synthase protein, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and termination
Liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
6th aspect provides a kind of 5-ALA synthase based on any of the above-described ELISA kit detection people
The method of the antibody of mutain, including：
A, said monoclonal antibody is diluted using the coating buffer solution, and the monoclonal after dilution is resisted
Body is loaded into the hole of ELISA ELISA Plates, and is incubated at room temperature；
B, the 5-ALA synthase protein for detecting object is diluted to various concentration ladder using the sample diluting liquid
Degree, and the 5-ALA synthase protein of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and room temperature is incubated
It educates；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
D, the ELIAS secondary antibody is added in, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
F, the developing solution is added in, room temperature is protected from light incubation, adds in the terminate liquid and terminates reaction；
G, 450nm measures OD values in microplate reader.
Preferably, further comprise：Blank control is tested.
The present invention provides the 5-ALA mutant synthase albumen of people a kind of and its application, by a large amount of porphyrias and
Leukaemic carries out case genetic test and analyzes, determine that the 5-ALA of people closes as research case
The mutain of enzyme prepares genetic chip, monoclonal antibody and ELISA according to the 5-ALA mutant synthase albumen of the people
Kit is the impulse that the gene diagnosis of human health status provides, and realizes that porphyria and the early of leukaemia diagnose, are early
It was found that and associated treatment.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate attached drawing be described in detail as after.
Description of the drawings
Fig. 1 is a kind of comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 2 is a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 3 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 4 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 5 is the standard curve that the offer of the embodiment of the present invention five is protein content；
Fig. 6 is the Western blot testing result schematic diagrames that the embodiment of the present invention five provides；
Fig. 7 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Fig. 8 is the Western blot testing result schematic diagrames that the embodiment of the present invention six provides.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment one, genetic chip
It is possible, firstly, to it is a kind of " detection side of mutain according to Application No. " 201710429915.8 ", patent name
Detection method described in the Chinese invention patent of method and device " determines the 5-ALA mutant synthase albumen of people
Encoding gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the 5- amino of people is determined according to the encoding gene
Ketone valeric acid mutant synthase albumen, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the 5-ALA mutant synthase albumen and its encoding gene of people, base is realized as follows
Because of the preparation of chip.
1st, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the nucleosides of the 5-ALA mutant synthase albumen of people
Acid sequence determines with the comparison result of the nucleotide sequence of the normal albumen of 5-ALA synthase of people, and according to as follows
The design principle of probe designs the specific nucleotide probe of the 5-ALA mutant synthase albumen for people.
Wherein, the principle of nucleotide probe design is as follows：
1. nucleotide probe Tm values should be close to the average Tm values of whole gene group, 5 DEG C of fluctuation up and down；
2. the single base that nucleotide probe intramolecular repeats continuously is no more than 5；
3. G+C contents are 40%-60%, the specificity that non-specific hybridization ensures hybridization is reduced；
4. nucleotide probe intramolecule stablizes secondary structure pairing bases longs less than 4bp, can so ensure will not
Hybridization efficiency is influenced due to the secondary structure of nucleotide probe internal stability；
5. the similitude through Homology search and other sequences is less than 40%；
6. continuously it is no more than 20 bases with the homologous fragment of non-probe alternative sequence by contrast.
Wherein, the corresponding amino acid sequence of 5-ALA mutant synthase albumen of people and the 5-ALA of people close
The comparison result of the corresponding amino acid sequence of the normal albumen of enzyme please refers to Fig.1, wherein, the Query sequences in Fig. 1 are the 5- ammonia of people
The corresponding amino acid sequence of base ketone valeric acid mutant synthase albumen, Sbjct sequences are the normal albumen of 5-ALA synthase of people
Corresponding amino acid sequence, as can be seen from FIG. 1, the 5-ALA mutant synthase albumen of people compared with people 5- amino ketones penta
The normal albumen of acid synthase is lacked at a position, according to SEQ ID NO:Nucleotide sequence and Fig. 1 shown in 2
Comparison result, whether the 5-ALA synthase in order to specific recognition object to be detected be mutated, then
When choosing nucleotide probe, when choosing nucleotide probe, nucleotide probe can be designed as follows：
Several nucleotide sequences are selected before deletion sites, with selecting several nucleotides sequences after deletion sites
Row generate nucleotide probe.
In the present embodiment, according to SEQ ID NO:It nucleotide sequence shown in 2 and is set according to above-mentioned nucleotide probe
Count principle, design one kind preferably nucleotide probe such as SEQ ID NO:Shown in 3, which is：ggccaagata ac
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2nd, the preparation for the genetic chip whether the 5-ALA synthase of detection people undergos mutation
In order to ensure to detect the quality of subject sample, blank control, positive control and the moon need to be also designed on genetic chip
Property control.Wherein, the layout of the genetic chip at least can be a kind of layout as shown in Figure 2.In fig. 2, is blank pair
According to, zero is negative control,For positive control,For experimental group.
Wherein, the position of experimental group is the point sample position of nucleotide probe.
For point sample needed for the negative internal reference Quality Control probe and positive control of point sample needed for blank control, negative control
Positive internal control Quality Control probe design it is as follows：
Blank control：The blank sampling liquid for being free from any genetic fragment refers to as the contamination monitoring in chip fabrication process
Mark.
Negative internal reference Quality Control probe：Be one section does not have other genetic fragments of homology with detection gene, as hybridizing
The monitor control index of non-specific hybridization in journey, the nucleotide digit that negative internal reference Quality Control probe includes can be with nucleotide probes
Digit is identical, can not also be same.In the embodiment of the present invention, the negative internal reference probe sequence needed for genetic chip can be：
tgatgctgat aattgcatag。
Positive internal control Quality Control probe：It is one section of other genetic fragment for having homology with detection gene, in the 5- amino of people
In ketone valeric acid mutant synthase albumen, select sequence corresponding from nucleotide probe different, the core that positive internal control Quality Control probe includes
Thuja acid digit and the digit of nucleotide probe may be the same or different.Preferably, nucleotide digit is chosen to visit with nucleotide
The identical positive internal control Quality Control probe of the digit of pin.In the embodiment of the present invention, the positive internal control probe sequence needed for genetic chip
Can be：agtgatgatc ag.
It should be noted that in the deposition process of genetic chip, click and enter negative internal reference according to the layout of genetic chip and visit
Pin and positive internal control probe solution；The spotting buffer (10% aqueous trehalose) that reagent used in blank control is 1 times.
The application of embodiment two, genetic chip
1st, sample treatment
(1) the blood 1-3mL of acquisition testing object.
(2) the 1.5mLEP pipes that DEPC is handled are taken, to be detected sample processing tube, are added in sample processing tube is detected
The 300 μ L of blood of object are detected, add 700 μ L of Trizol, abundant mixing is placed at room temperature for 10Min.
(3) chloroform of 200 μ L is added in, centrifuge tube lid is covered tightly, firmly shakes centrifuge tube, be placed at room temperature for 3-5min, 12000r/
Min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(4) isometric isopropanol is added in, the abundant mixing liquid of centrifuge tube is gently overturned, is placed at room temperature for 10min, 12000r/
Min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(5) one time, 7500r/min, 4 DEG C centrifugation 15min is washed with 1mL75% ethyl alcohol, all supernatants is carefully sucked, ultra-clean
Dry 15min in platform adds in 10 μ L DEPC processing water dissolutions.
(6) products therefrom is RNA, if the purity of total serum IgE is not high, can influence the labeling effciency of probe and chip hybridization knot
Fruit.So use QIAGENPurify total serum IgE.
2nd, the first chains of cDNA and the second chain one-step synthesis method
(1) 2 μ g RNA is taken to configure following reaction solution in 1.5mL centrifuge tubes：
10 minutes ice baths of (2) 65 DEG C of heat preservations 5 minutes, in advance 5 × First Strand B μ ffer in 65 DEG C of 5 points of preheatings
Clock.
(3) following cDNA synthetic systems are configured：
| 5×First Strand Buffer | 4μL |
| 0.1M DTT | 2μL |
| 10mM dNTP mix | 1μL |
| MMLV RT | 1μL |
| RNase OUT | 0.5μL |
| Total volume | 8.5μL |
(4) above-mentioned 8.5 μ L mix are added in after being denatured in the RNA of ice bath.
(5) centrifuged with after pipette tips mixing.
(6) 40 DEG C of reaction 2h.
3rd, aaUTP marks cRNA synthesis
The configuration of NTP：
| 100mM ATP | 250μL |
| 100mM GTP | 250μL |
| 100mM CTP | 250μL |
| 100mM UTP | 187.5μL |
| RNase free H2O | 62.5μL |
| Total volume | 1000μL |
It is spare to be distributed into 10 pipes, notes：40 DEG C of heat preservation 1min before 50%PEG uses.Simultaneously by following operative configuration
Transcription mix；
(1) Transcription mix are configured
(2) 60 μ l Transcription mix and mixing are added in
(3) hot 60 DEG C of lid in PCR instrument, 40 DEG C of reaction 2h.
4th, cRNA is purified
QIAGEN RNeasy Mini kit purify cRNA, and specific method can be found in what QIAGEN companies provided with kit
Operation manual.
(1) 20 μ LRNase free water are added in, add in 350 μ L Buffer RLT and abundant mixing.
(2) 250 μ L absolute ethyl alcohols, Tip abundant mixing are added in.
(3) 700 solution of the μ L containing total serum IgE altogether are transferred in the RNeasy pillars being sleeved in 2mL centrifuge tubes >=8000g from
Heart 15-30sec, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=15-30sec of 8000g centrifuge washings abandons
Filtered solution is gone to discard the casing of filtered solution and 2mL in >=8000g centrifuge washings 2min with 500 μ L Buffer RPE again will
RNeasy mini pillars are transferred in a new 1.5mL Eppendorf pipes.
(5) water for drawing 30 μ L RNase free stands 1min, >=8000g centrifugation elutions 1min.
(6) step (5) is repeated once.
5th, cRNA concentration mensurations
With spectrophotometric analysis RNA concentration.Need 260 and 280nm measure absorbance come determine the concentration of sample and
Purity A260/A280 should be purer RNA (ratio 1.9-2.1 also can) close to 2.0.
6th, cRNA fluorescents mark；
(1) above-mentioned cRNA4 μ g are taken and are concentrated into 6.6 μ L.
(2) 10 μ L DMSO mixings are added.
(3) 0.3M sodium acid carbonates (NaHCO3) pH9.0 and mixing of 3.4 μ L is added.
(4) above-mentioned 20 μ L cRNA mixtures are added in fluorescent dye Cy3 simultaneously mixing.25 DEG C of heat preservation 1h.
(5) plus 9 μ L 4M Hydroxylamine mixings after 25 DEG C heat preservation 15min.
7th, fluorescent marker cRNA Sample Purification on Single
The process that specific steps are purified with cRNA in the step 4 of the present embodiment, this step are not repeating.
8th, cRNA sample fragments and chip hybridization 4x44K microarrays
(1) according to the form below prepares fragmentation mixed liquor and then carries out fragmentation in 60 DEG C of warm bath 30min.
| Cy3cRNA green fluorescences | 875ng |
| 10XBlocking Agent | 11μL |
| 25X Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ L 2X GEx Hybridization Buffer are added in.
(3) 100 μ L hybridization solutions is taken to be added drop-wise on chip ware, at the same be added dropwise respectively as shown in Figure 2 by chip layout blank,
On negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C roll hybridization 16h.
9th, chip washs
(1 liter) configuration of washing lotion 1：
| DEPC-H2O | 700m L |
| 20*SSPE | 300m L |
| 20%N-Lauroylsarcosine | 0.25m L |
(1 liter) configuration of washing lotion 2：
| DEPC-H2O | 997m L |
| 20*SSPE | 3.0m L |
| 20%N-Lauroylsarcosine | 0.25m L |
Washing lotion 3：Stabilization and Drying Solution
(1) chip is taken out to wash 1 minute in washing lotion 1；
(2) chip is put into washing lotion 2 again and washs 1 minute (37 DEG C)；
(3) finally chip is washed 30 seconds in washing lotion 3.
10th, chip scanning
Chip in scanner is scanned, the 5-ALA synthase egg of detection object is determined according to scanning result
Whether it is mutated in vain.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control is
Green fluorescence, showing the sample quality of acquisition testing object has no problem, and experimental group is green fluorescence, then shows to examine
The 5-ALA synthase protein for surveying object is mutated.In result shown in Fig. 4, negative control is colorless fluorescent, sun
Property control for green fluorescence, showing the sample quality of acquisition testing object has no problem, and experimental group redgreen fluorescence, that
The 5-ALA synthase protein for showing to detect object is not undergone mutation.
Embodiment three, the preparation 1 of monoclonal antibody of 5-ALA mutant synthase albumen of specific recognition people, root
According to base sequence (such as SEQ ID NO of the 5-ALA mutant synthase albumen of people:Shown in 2) design sense primer such as SEQ
ID NO:Shown in 4 and, anti-sense primer such as SEQ ID NO:Shown in 5：
Sense primer (P)：agtgatgatc agccagagaa
Anti-sense primer (F)：attataagtt atcttggc
2nd, detect the DNA of object and carry out PCR amplification for template
| 10×Buffer | 5uL |
| dNTP | 2uL |
| Ex Taq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (P) | 3uL |
| Primer (F) | 3uL |
| Total system | 20uL |
PCR amplification is carried out as template using the DNA for detecting object, obtains the 5-ALA mutant synthase protein gene of people
Complete segment, and pMD19-T Vector (Takara companies) are connected, it is sequenced.Then prepared by special biotech firm anti-
Body is a kind of humanization or Chimeric antibodies.The antibody of preparation is measured into content using ELISA method.
Example IV, the ELISA kit for detecting the antibody of the 5-ALA mutant synthase albumen of people
In the present embodiment, the composition of ELISA kit is：It is coated with the ELISA enzymes of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, the 5-ALA synthase protein for detecting object, sample diluting liquid, coating buffer solution, ELISA enzyme marks
Plate cleaning solution, developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition at least can be a kind of following parameter：
ELISA ELISA Plates can be the ELISA enzyme marks in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) marks；Wherein, dilution times
Number can be 8000 times.
It can be 1 × PBS, pH to be coated with buffer solution:7.4；
ELISA ELISA Plates cleaning solution can be 1 × PBS solution containing 0.05%Tween-20；
Developing solution can be 3,3 ', 5,5 '-tetramethyl benzidine；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
In embodiments of the present invention, using porphyrin patient as detection object, and the porphyrin is detected using ELISA kit
Whether the 5-ALA synthase protein of patient is mutated, and this method can at least include following a kind of：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, such as be diluted to
2.0ug/ml, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h；
B, the 5-ALA synthase protein of porphyrin patient is diluted to various concentration gradient using sample diluting liquid,
And the 5-ALA synthase protein of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and be incubated at room temperature
1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
D, the ELIAS secondary antibody is added in, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions is added in, washs and dry；
F, the developing solution is added in, room temperature, which is protected from light, is incubated 10-20min, adds in the terminate liquid and terminates reaction；
G, 450nm measures OD values in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the OD values that standard items measure are ordinate, make standard
Curve；Calculate standard curve regression coefficient R2, work as R2This measures effective during ＞ 0.99；Gone out according to ELISA Calc Software on Drawing
Standard curve can draw the R according to the ELISA Calc softwares2=0.99944, therefore, this measures effective.It will detect
To OD450 values bring the concentration that standard curve can acquire the 5-ALA synthase protein of porphyrin patient into.Using building
The content of 5-ALA synthase protein in vertical ELISA method detection porphyria patient serum sample.And use Western
Blot is identified.Fig. 5 is refer to, is the standard curve of OD values.Wherein, X-axis is OD values, and Y-axis is corresponding concentration.
I, Western blot are identified
1st, glue
(1) clean glass plate and dry；
(2) reference molecule cloning process prepares SDS-PAGE glue：
A. lower floor's separation gel prepares system (Total Volum：15mL)
| ddH2O | 5.9mL |
| 30% acrylamide mixed liquor | 5.9mL |
| 1.5mol/L Tris(PH 8.8) | 3.8mL |
| 10%SDS | 0.15mL |
| 10% ammonium persulfate | 0.15mL |
| TEMED | 0.006mL |
B. upper strata spacer gel concentration preparation system (Total Volum：8mL)
| ddH2O | 5.5mL |
| 30% acrylamide mixed liquor | 1.3mL |
| 1.0mol/L Tris(PH 6.8) | 1.0mL |
| 10%SDS | 0.08mL |
| 10% ammonium persulfate | 0.08ml |
| TEMED | 0.008mL |
(3) lower floor's separation gel of respective volume is added in each offset plate, adds the sterile ddH of 1mL2O flattens separation
Glue, after gelling to be separated is solid, remaining moisture in plastic plate is blotted with filter paper, upper strata concentration glue is then added in, after being inserted into comb
Wait upper strata concentration gelling solid.
2nd, albumen loading electrophoresis：
(1) comb is pulled up, duct mark is carried out, adds in 5 × SDS electrophoretic buffers, then to being added in protein sample
SDS-PAGE albumen sample-loading buffer (5 ×), boiling water bath heating 3-5min, loading；
(2) electrophoresis first is carried out with 80V voltages, after treating that band ran concentration glue, uses 100V voltages instead and run about 100min.
3rd, transferring film and incubation：
(1) first pvdf membrane is activated in methyl alcohol about 30s.Respectively by gel, foam-rubber cushion and pvdf membrane in advance in electrotransfer
30min is balanced in buffer solution.It is clipped by the order of filter paper-glue-film-filter paper, transferring film device is put into the principle of black flour according to black flour
In, refrigerator (ice bag) is added in, about 120min is run with the electric current of 150mA in ice；
(2) take the film out after the completion of, be put at once in 5% skim milk, gently room temperature close membrane 1h on shaking table；
(3) film that PBST cleanings have been closed, is cleaned 3 times, each 10min；
(4) primary antibody for being diluted to corresponding multiple is added in, 4 DEG C of incubator overnights are incubated；
(5) primary antibody is recycled, PBST cleans film three times, each 10min；
(6) secondary antibody is (with 3% milk with 1:5000-10000 dilutes) incubation at room temperature film 2h；
(7) film is cleaned three times with PBST, each 10min；
(8) after the isometric mixing of Pierce ECL Western Blotting Substrate kit A, B liquid, dropwise
It is added dropwise on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment are analyzed with Image J.
Wherein, primary antibody is the 5-ALA mutant synthase protein monoclonal antibody of the standby people of company system, and secondary antibody is peppery
The Goat anti-Human IgG of root peroxidase labelling.
J, Western blot Analysis of test results：It is shown according to Western blot experimental procedures as a result, such as Fig. 6 institutes
Show, wherein, the Marker in Fig. 6 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 24KD
There is apparent band, wherein, the molecular weight of 5-ALA mutant synthase albumen is about 24KD, shows porphyrin patient
5-ALA synthase protein be implicitly present in mutation.
Embodiment six
In embodiments of the present invention, using leukaemic as detection object, and the white blood is detected using ELISA kit
Whether the 5-ALA synthase protein of patient is mutated, and this method can at least include following a kind of：
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer solution, for example, being diluted to
2.0ug/ml, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plates, it is incubated at room temperature 1-2h；
B, the 5-ALA synthase protein of leukaemic is diluted to various concentration gradient using sample diluting liquid,
And the 5-ALA synthase protein of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and be incubated at room temperature
1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；
D, the ELIAS secondary antibody is added in, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions is added in, washs and dry；
F, the developing solution is added in, room temperature, which is protected from light, is incubated 10-20min, adds in the terminate liquid and terminates reaction；
G, 450nm measures OD values in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the OD values that standard items measure are ordinate, make standard
Curve；Calculate standard curve regression coefficient R2, work as R2This measures effective during ＞ 0.99；Gone out according to ELISA Calc Software on Drawing
Standard curve can draw the R according to the ELISA Calc softwares2=0.99878, therefore, this measures effective.It will detect
To OD450 values bring the concentration that standard curve can acquire the 5-ALA synthase protein of leukaemic in sample into.
Utilize the content of 5-ALA synthase protein in the ELISA method detection serum of leukaemia sample of foundation.It is used in combination
Western blot are identified.Fig. 7 is refer to, is the standard curve of OD values.Wherein, X-axis is OD values, and Y-axis is corresponding dense
Degree.
I, Western blot are identified
J, Western blot Analysis of test results
Wherein, step i and step j are identical in embodiment five, and details are not described herein, refer to Fig. 8, in Fig. 8
Marker is used to characterize the size of labelled protein.The experimental display result of the present embodiment, compared with blank control, only in experimental group
Nearby there is apparent band in 24KD, wherein, the molecular weight of 5-ALA mutant synthase albumen is about 24KD, shows that this is white
The 5-ALA synthase protein of blood patient is implicitly present in mutation.
The above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Sequence table
<110>Tianjin lakeside Pan Gu's genetic science Development Co., Ltd
<120>The 5-ALA mutant synthase albumen of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 95
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
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1 5 10 15
Phe Glu Leu Asp Ser Cys Asn Gly Ser Gly Lys Val Cys Leu Val Tyr
20 25 30
Lys Ser Gly Lys Pro Ala Leu Ala Glu Asp Thr Glu Ile Trp Phe Leu
35 40 45
Asp Arg Ala Leu Tyr Trp His Phe Leu Thr Asp Thr Phe Thr Ala Tyr
50 55 60
Tyr Arg Leu Leu Ile Thr His Leu Gly Leu Pro Gln Trp Gln Tyr Ala
65 70 75 80
Phe Thr Ser Tyr Gly Ile Ser Pro Gln Ala Lys Ile Thr Tyr Asn
85 90 95
<210> 2
<211> 285
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<213>Species home sapiens (Homo sapiens)
<400> 2
agtgatgatc agccagagaa gcctcacttt gactctcgca gtgtgatatt tgagctggat 60
tcatgcaatg gcagtgggaa agtttgcctt gtctacaaaa gtgggaaacc agcattagca 120
gaagacactg agatctggtt cctggacaga gcgttatact ggcattttct cacagacacc 180
tttactgcct attaccgcct gctcatcacc cacctgggcc tgccccagtg gcaatatgcc 240
ttcaccagct atggcattag cccacaggcc aagataactt ataat 285
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<213>Artificial sequence (Artificial Sequence)
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ggccaagata ac 12
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<213>Artificial sequence (Artificial Sequence)
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agtgatgatc agccagagaa 20
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<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
attataagtt atcttggc 18
Claims (9)
- A kind of 1. 5-ALA mutant synthase albumen of people, which is characterized in that its amino acid sequence such as SEQ ID NO:1 institute Show.
- A kind of 2. encoding gene of the 5-ALA mutant synthase albumen of people, which is characterized in that its nucleotide sequence such as SEQ ID NO:Shown in 2.
- 3. a kind of genetic chip, which is characterized in that including：Solid phase carrier and fixed nucleotide probe on this carrier；It is described Nucleotide probe is according to the nucleotide sequence of the 5-ALA mutant synthase albumen of the people, the 5-ALA with people The comparison result of the nucleotide sequence of the normal albumen of synthase determines.
- 4. genetic chip according to claim 3, which is characterized in that the nucleotide probe is SEQ ID NO:Shown in 3 Base sequence.
- 5. the monoclonal antibody of the 5-ALA mutant synthase albumen of a kind of specific recognition people, which is characterized in that it is compiled The amino acid sequence of code is SEQ ID NO:Shown in 1.
- 6. a kind of ELISA kit of the antibody for the 5-ALA mutant synthase albumen for being used to detect people, which is characterized in that The ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection of monoclonal antibody described in claim 5 5-ALA synthase protein, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and the end of object Only liquid.
- 7. it is used to detect the ELISA reagents of the antibody of the 5-ALA mutant synthase albumen of people according to claim 6 Box, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
- 8. a kind of 5-ALA mutant synthase albumen based on the ELISA kit detection people of claim 6 or 7 is anti- The method of body, which is characterized in that including：A, monoclonal antibody described in claim 5 is diluted using the coating buffer solution, and by the list after dilution Clonal antibody is loaded into the hole of ELISA ELISA Plates, and is incubated at room temperature；B, the 5-ALA synthase protein for detecting object is diluted to various concentration gradient using the sample diluting liquid, and The 5-ALA synthase protein of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and is incubated at room temperature；C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；D, the ELIAS secondary antibody is added in, and is incubated at room temperature；E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added in, washs and dry；F, the developing solution is added in, room temperature is protected from light incubation, adds in the terminate liquid and terminates reaction；G, 450nm measures OD values in microplate reader.
- 9. the side of the antibody of the 5-ALA mutant synthase albumen of ELISA kit detection people according to claim 8 Method, which is characterized in that further comprise：Blank control is tested.
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2017
- 2017-11-29 CN CN201711221271.XA patent/CN108085305A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 于广军等: "《医疗大数据》", 31 January 2015, 上海科学出版社 * |
| 无: "XP_011524219.1", 《GENBANK》 * |
| 无: "XP_016861362.1", 《GENBANK》 * |
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