CN108424902A - The uroporphyrinogen decarboxylase mutain of people and its application - Google Patents
The uroporphyrinogen decarboxylase mutain of people and its application Download PDFInfo
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Abstract
The present invention relates to the uroporphyrinogen decarboxylase mutain of people a kind of and its applications, using several leukaemics as research case, genetic test is carried out to case and is analyzed, determine the mutain of the uroporphyrinogen decarboxylase of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the uroporphyrinogen decarboxylase mutain of the people, impulse is provided for the gene diagnosis of leukaemia, realizes the diagnosing and treating of leukaemia.
Description
Technical field
The present invention relates to the uroporphyrinogen decarboxylase mutain of a kind of genetic engineering field more particularly to a kind of people and its
Using.
Background technology
Uroporphyrinogen decarboxylase (uroporphyrinogen decarboxylase, UROD) is biological porphyrins
The key enzyme of branch's synthesis.The gene mutation of coding UROD can cause human body and generate metabolic disease, for example, Delayed onset skin
Skin porphyria (porphyria cutanea tarda, PCT).Wherein, porphyria cutanea tarda is that a kind of porphyrin metabolism is abnormal
Dermatoses can be divided into acquired (PCT I types) and genotype (PCT II types), be apt to occur in adult's exposure position.
Recent studies have found that the gene of the generation of leukaemia and coding UROD mutate there is also certain relationship, because
This, to the detection of the uroporphyrinogen decarboxylase of people to judging whether leukaemia has certain impulse to human body.
Invention content
Present invention aims at the uroporphyrinogen decarboxylase mutain of people of offer a kind of and its applications.
Technical solution of the present invention includes：
In a first aspect, a kind of uroporphyrinogen decarboxylase mutain of people is provided, amino acid sequence such as SEQ ID NO:1
It is shown.
Second aspect provides a kind of encoding gene of the mutain of the uroporphyrinogen decarboxylase 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 uroporphyrinogen decarboxylase mutain of people described above, the uroporphyrin with people
The comparison result of the nucleotide sequence of the former normal albumen of decarboxylase determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of monoclonal antibody of the uroporphyrinogen decarboxylase mutain of specific recognition people,
It can be with SEQ ID NO:Amino acid sequencespecific shown in 1 combines.
5th aspect provides a kind of ELISA reagents for detecting the antibody of the uroporphyrinogen decarboxylase mutain of people
Box, the ELISA kit include：It is coated with the ELISA ELISA Plates of said monoclonal antibody, ELIAS secondary antibody, detects object
Uroporphyrinogen decarboxylase albumen, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and terminate liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
The present invention provides the uroporphyrinogen decarboxylase mutain of people a kind of and its applications, and several leukaemics are made
To study case, genetic test is carried out to case and is analyzed, the mutain of the uroporphyrinogen decarboxylase of people is determined, according to this
The uroporphyrinogen decarboxylase mutain of people prepares genetic chip, monoclonal antibody and ELISA kit, is the gene of leukaemia
Diagnosis provides impulse, realizes the diagnosing and treating of leukaemia.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after coordinating attached drawing to be described in detail such as.
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.
Specific implementation mode
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 be a kind of " detection side of mutain according to application No. is " 201710429915.8 ", patent name
Detection method described in the Chinese invention patent of method and device " determines the volume of the uroporphyrinogen decarboxylase mutain of people
Code gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, the uroporphyrinogen of people is determined according to the encoding gene
Decarboxylase mutain, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the uroporphyrinogen decarboxylase mutain and its encoding gene of people, gene is realized as follows
The preparation of chip.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the nucleotide of the uroporphyrinogen decarboxylase mutain of people
Sequence determines with the comparison result of the nucleotide sequence of the normal albumen of uroporphyrinogen decarboxylase of people, and according to following probe
Design principle, design for people uroporphyrinogen decarboxylase mutain specificity nucleotide probe.
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 4；
3. G+C contents are 40%-60%, the specificity that non-specific hybridization ensures hybridization is reduced；
4. it should be less than 4bp with the bases longs of probe molecule internal stability secondary structure pairing in nucleotide probe sequences,
Can ensure that hybridization efficiency will not be influenced because of the secondary structure of nucleotide probe internal stability in this way；
5. the similitude through Homology search and other sequences is less than 40%；
6. being continuously no more than 20 bases with the homologous fragment of non-probe alternative sequence by contrast.
Wherein, the corresponding amino acid sequence of uroporphyrinogen decarboxylase mutain of people and the uroporphyrinogen decarboxylase of people be just
The comparison result of the corresponding amino acid sequence of normal albumen please refers to Fig.1, wherein the Query sequences in Fig. 1 are the uroporphyrinogens of people
The corresponding amino acid sequence of decarboxylase mutain, Sbjct sequences are the corresponding ammonia of the normal albumen of uroporphyrinogen decarboxylase of people
Base acid sequence, the sequence between Query sequences and Sbjct sequences are comparison result, and as can be seen from FIG. 1, the uroporphyrinogen of people is de-
Uroporphyrinogen decarboxylase normal albumen of the carboxylic acid mutain relative to people, is inserted at a position, according to SEQ
ID NO:The comparison result of nucleotide sequence and Fig. 1 shown in 2, in order to the urine porphin of specific recognition object to be detected
Whether quinoline original decarboxylase is mutated, then when choosing nucleotide probe, it can be according to any in following several modes
Kind mode designs nucleotide probe：
1. choosing the insertion nucleotides sequence column-generation nucleotide probe of insertion position；
2. before insertion position, and/or, after insertion position, several nucleotide sequences are respectively selected, by selection
Several consecutive nucleotides and the insertion nucleotide sequence of insertion position generate nucleotide probe jointly, wherein the nucleosides of generation
The sequencing of adjacent nucleotide and its sequence consensus in the corresponding nucleotide sequence of mutain in acid probe；
3. several nucleotide sequences are selected before insertion position, at the starting position of insertion position select it is several
A nucleotide sequence generates nucleotide probe；
4. several nucleotide sequences are selected after insertion position, at the end position of insertion position select it is several
A nucleotide sequence generates 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
Principle is counted, 3. designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, the nucleotide probe
For：tctgcaggtg ag.
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether uroporphyrinogen decarboxylase of detection people mutates
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.
Point sample needed for negative internal reference Quality Control probe and positive control for 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 number that negative internal reference Quality Control probe includes can be with nucleotide probes
Base number is identical, can also be different.In the embodiment of the present invention, the negative internal reference probe sequence needed for genetic chip can be：
tgatgctgat aattgcat。
Positive internal control Quality Control probe：It is one section of other genetic fragment for having homology with detection gene, in the uroporphyrin of people
In former decarboxylase mutain, select sequence corresponding from nucleotide probe different, and can be with of nucleotide probe base
Number is identical, and one section of nucleotide sequence that can also be different from the number of nucleotide probe base is as positive internal control Quality Control probe.
Preferably, nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen.The present invention is implemented
In example, the positive internal control probe sequence needed for genetic chip can be：gagaggaaac ag.
It should be noted that in the deposition process of genetic chip, clicks and enters negative internal reference according to the layout of genetic chip and visit
Needle 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
1, sample treatment
(1) the blood 1-3mL of acquisition testing object.
(2) take the 1.5mL EP pipes that DEPC is handled that detection object is added in pipe as detected sample processing tube
300 μ L of blood, add 700 μ L of Trizol, mix well, be placed at room temperature for 10min.
(3) chloroform of 140 μ L is added, covers tightly pipe lid, firmly shakes, be placed at room temperature for 3-5min, be placed in a centrifuge,
12000r/min, 4 DEG C of centrifugation 15min, carefully draws supernatant in centrifuge tube, the supernatant of absorption is transferred to clean centrifuge tube
In.
(4) isometric isopropanol is added in the centrifuge tube of supernatant in storing step (3), it is abundant gently overturns centrifuge tube
Mixing liquid is placed at room temperature for 10min, 12000r/min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(5) it is cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min centrifuges 15min, carefully sucks all supernatants, super
Dry 15min in net platform is added 10 μ L DEPC and handles water dissolution.
(6) products therefrom is RNA can influence the labeling effciency and chip hybridization knot of probe if the purity of total serum IgE is not high
Fruit.So using QIAGENKit purifies total serum IgE.
2, 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 the centrifuge tube of 1.5mL：
| Total serum IgE | The most 6.5 μ L of 2 μ g |
| T7 Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
Wherein, the addition X μ L of RNase-free Water are to subtract T7 Promotor according to 11.5 μ L of total volume
The 5 μ L of addition of primer, then subtract the addition of total serum IgE and calculate and get.
(2) 10min, and ice bath 5min are kept the temperature at 65 DEG C, and 5 × First Strand B μ ffer are preheated at 65 DEG C
5min。
(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 are added after mixing after being denaturalized in the RNA of ice bath.
(5) pipette tips mixing is used to centrifuge later.
(6) 40 DEG C of reaction 2h.
3, 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 (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) configuration Transcription mix
(2) 60 μ L Transcription mix and mixing is added.
(3) 60 DEG C of hot lid in PCR instrument, 40 DEG C of reaction 2h.
4, 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, 350 μ L Buffer RLT are added and mix well.
(2) 250 μ L absolute ethyl alcohols are added, Tip mix well.
(3) total 700 solution of the μ L containing total serum IgE are transferred in the RNeasy pillars being sleeved in 2mL centrifuge tubes >=8000g from
Heart 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washings 15-30s discards filter
It crosses liquid and discards the casing of filtered solution and 2mL by RNeasy in >=8000g centrifuge washings 2min with 500 μ L Buffer RPE again
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) it is primary to repeat step (5).
5, cRNA concentration mensurations
With spectrophotometric analysis cRNA concentration.It needs to measure the light absorption value at 260nm and 280nm to determine the dense of sample
Degree and purity, A260/A280 should be purer cRNA (ratio 1.9-2.1 also can) close to 2.0.
6, cRNA fluorescents mark；
(1) 4 μ g of above-mentioned cRNA are taken and are concentrated into 6.6 μ L.
(2) add 10 μ L DMSO mixings.
(3) add the sodium bicarbonate (NaHCO that the 0.3M pH of 3.4 μ L are 9.03) and mixing.
(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.
7, 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.
8, cRNA sample fragments and 4 × 44K of chip hybridization microarrays
(1) according to the form below prepares fragmentation mixed liquor and then carries out fragmentation in 60 DEG C of warm bath 30min.
| Cy3 cRNA green fluorescences | 875ng |
| 10×Blocking Agent | 11μL |
| 25×Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ L 2 × GEx Hybridization Buffer are added.
(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.
9, chip washs
Washing lotion 1 (1L) configures：
| DEPC-H2O | 700mL |
| 20×SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 2 (1L) configures：
| DEPC-H2O | 997mL |
| 20×SSPE | 3.0mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 3：Stabilization and Drying Solution
(1) it takes out chip and washs 1min in washing lotion 1；
(2) chip is put into washing lotion 2 again and washs 1min (37 DEG C)；
(3) chip is finally washed into 30s in washing lotion 3.
10, chip scanning
Chip is scanned in scanner, the uroporphyrinogen decarboxylase albumen of detection object is determined according to scanning result
Whether it is mutated.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control is green
Color fluorescence shows that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, then showing to detect
The uroporphyrinogen decarboxylase albumen of object is mutated.In result shown in Fig. 4, negative control is colorless fluorescent, positive right
According to for green fluorescence, showing that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then table
The uroporphyrinogen decarboxylase albumen of bright detection object does not mutate.
Embodiment three, the preparation 1 of monoclonal antibody of uroporphyrinogen decarboxylase mutain of specific recognition people, basis
Base sequence (such as SEQ ID NO of the uroporphyrinogen decarboxylase mutain of people:Shown in 2) design sense primer such as SEQ ID
NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (F)：ggttttccgg agctga
Downstream primer (R)：atgtcatcag ggtccat
2, the DNA of detection object is that template carries out PCR amplification
| 10×Buffer | 5uL |
| dNTP | 2uL |
| Ex Taq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (F) | 3uL |
| Primer (R) | 3uL |
| Total system | 20uL |
DNA to detect object carries out PCR amplification as template, and the uroporphyrinogen decarboxylase mutating protein gene for obtaining people is complete
Full wafer section, and pMD19-T Vector (Takara companies) are connected, it is sequenced.Then it is prepared by special biotech firm anti-
Body is a kind of humanization or Chimeric antibodies.Wherein, the monoclonal antibody prepared can be with SEQ ID NO:Amino shown in 1
Acid sequence is specifically bound.The antibody of preparation is measured into content using ELISA method.
Example IV, uroporphyrinogen decarboxylase mutain for detecting people antibody ELISA kit
In the present embodiment, the group of ELISA kit becomes：It is coated with the ELISA enzymes of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, the uroporphyrinogen decarboxylase albumen for detecting object, sample diluting liquid, coating buffer solution, ELISA ELISA Plates
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) labels；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 benzidines；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
Research method：Men and women leukaemic totally 22 is chosen, as detection object, and utilizes ELISA kit
Whether the uroporphyrinogen decarboxylase albumen for detecting 22 patients is mutated.
By taking wherein a certain position patient as an example, the detection method to the patient includes：
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 uroporphyrinogen decarboxylase albumen of patient is diluted to various concentration gradient using sample diluting liquid, and will be different
The uroporphyrinogen decarboxylase albumen of concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, and is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature 1-2h；
E, the liquid in each hole is got rid of, ELISA ELISA Plate cleaning solutions are added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated 10-20min, and the terminate liquid is added and terminates reaction；
G, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
H, standard curve is made：Using standard concentration as abscissa, the light absorption value that standard items measure is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measures effective when ＞ 0.99；Wherein it is possible to use ELISA Calc
The Software on Drawing standard curve can know regression coefficient R according to the ELISA Calc softwares2=0.99970, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire the uroporphyrinogen of patient
The concentration of decarboxylase protein.Contained using uroporphyrinogen decarboxylase albumen in the ELISA method detection patient serum sample of foundation
Amount.It is used in combination Western blot to be identified.Referring to FIG. 5, corresponding to the standard curve of light absorption value for the patient.Wherein, X-axis is
Light absorption value, Y-axis are corresponding concentration.
I, Western blot are identified
1, glue
(1) it cleans glass plate and dries；
(2) reference molecule cloning process prepares SDS-PAGE glue：
A. lower layer'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 layer 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 layer's separation gel of respective volume is added in each offset plate, adds the sterile ddH of 1mL2O flattens separation
Glue blots remaining moisture in plastic plate after gelling to be separated is solid with filter paper, and upper layer is then added and concentrates glue, after being inserted into comb
Wait for upper layer concentration gelling solid.
2, albumen loading electrophoresis：
(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffers is added, then to being added in protein sample
SDS-PAGE albumen sample-loading buffer (5 ×), boiling water bath heat 3-5min, loading；
(2) electrophoresis first is carried out with 80V voltages, after waiting for that band ran concentration glue, uses 100V voltages instead and run about 100min.
3, 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 sequence 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, about 120min is run with the electric current of 150mA in ice；
(2) it takes the film out after the completion of, is put into 5% skim milk at once, gently room temperature closes 1h on shaking table；
(3) PBST cleans closed film, cleans 3 times, each 10min；
(4) primary antibody for being diluted to corresponding multiple is added, 4 DEG C of incubator overnights are incubated；
(5) primary antibody is recycled, PBST cleans film 3 times, each 10min；
(6) secondary antibody is (with 3% milk with 1:5000-1:10000 dilutions) incubation at room temperature film 2h；
(7) film is cleaned 3 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 Image J analyses.
Wherein, primary antibody is the uroporphyrinogen decarboxylase mutain monoclonal antibody of the standby people of corporation, and secondary antibody is horseradish
The Goat anti-Human IgG of 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 45KD
There is apparent band, wherein the molecular weight of uroporphyrinogen decarboxylase mutain is about 45KD, shows the uroporphyrin of the patient
Former decarboxylase protein is implicitly present in mutation.
According to the studies above method, the probability that uroporphyrinogen decarboxylase albumen mutates in 22 patients is 58%, by
This can determine that the abrupt climatic change of uroporphyrinogen decarboxylase albumen there is certain guide to make the gene diagnosis of leukaemia
With.
The above is only a preferred embodiment of the present invention, it is not intended to restrict 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 uroporphyrinogen decarboxylase mutain of people and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 179
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Gly Phe Pro Glu Leu Lys Asn Asp Thr Phe Leu Arg Ala Ala Trp Gly
1 5 10 15
Glu Glu Thr Asp Tyr Thr Pro Val Trp Cys Met Arg Gln Ala Gly Arg
20 25 30
Tyr Leu Pro Glu Phe Arg Glu Thr Arg Ala Ala Gln Asp Phe Phe Ser
35 40 45
Thr Cys Arg Ser Pro Glu Ala Cys Cys Glu Leu Thr Leu Gln Val Arg
50 55 60
Gly Pro Gln Lys Arg Glu Arg Phe Met Leu Gln Ser Ala Thr Gln Pro
65 70 75 80
Val Ser Cys Phe Leu Gln Pro Leu Arg Arg Phe Pro Leu Asp Ala Ala
85 90 95
Ile Ile Phe Ser Asp Ile Leu Val Val Pro Gln Ala Leu Gly Met Glu
100 105 110
Val Thr Met Val Pro Gly Lys Gly Pro Ser Phe Pro Glu Pro Leu Arg
115 120 125
Glu Glu Gln Asp Leu Glu Arg Leu Arg Asp Pro Glu Val Val Ala Ser
130 135 140
Glu Leu Gly Tyr Val Phe Gln Ala Ile Thr Leu Thr Arg Gln Arg Leu
145 150 155 160
Ala Gly Arg Val Pro Leu Ile Gly Phe Ala Gly Ala Pro Trp Thr Leu
165 170 175
Met Thr Tyr
<210> 2
<211> 538
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
ggttttccgg agctgaagaa tgacacattc ctgcgagcag cctggggaga ggaaacagac 60
tacactcccg tttggtgcat gcgccaggca ggccgttact taccagagtt tagggaaacc 120
cgggctgccc aggacttttt cagcacgtgt cgctctcctg aggcctgctg tgaactgact 180
ctgcaggtga ggggtccaca aaagagggaa agatttatgc ttcagtctgc cacctagcaa 240
cctgtctcct gtttcctaca gccactgcgt cgcttccctc tggatgctgc catcattttc 300
tccgacatcc ttgttgtacc ccaggcactg ggcatggagg tgaccatggt acctggcaaa 360
ggacccagct tcccagagcc attaagagaa gagcaggacc tagaacgcct acgggatcca 420
gaagtggtag cctctgagct aggctatgtg ttccaagcca tcacccttac ccgacaacga 480
ctggctggac gtgtgccgct gattggcttt gctggtgccc catggaccct gatgacat 538
<210> 3
<211> 12
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tctgcaggtg ag 12
<210> 4
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ggttttccgg agctga 16
<210> 5
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
atgtcatcag ggtccat 17
Claims (7)
1. the uroporphyrinogen decarboxylase mutain of people a kind of, which is characterized in that its amino acid sequence such as SEQ ID NO:1 institute
Show.
2. a kind of encoding gene of the uroporphyrinogen decarboxylase mutain 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
The nucleotide sequence of the nucleotide probe uroporphyrinogen decarboxylase mutain of people according to claim 2, the urine porphin with people
The comparison result of the nucleotide sequence of the normal albumen of quinoline original decarboxylase 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. a kind of monoclonal antibody of the uroporphyrinogen decarboxylase mutain of specific recognition people, which is characterized in that it can be with
SEQ ID NO:Amino acid sequencespecific shown in 1 combines.
6. a kind of ELISA kit for detecting the antibody of the uroporphyrinogen decarboxylase mutain of people, which is characterized in that institute
Stating ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection pair of monoclonal antibody described in claim 5
Uroporphyrinogen decarboxylase albumen, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and the termination of elephant
Liquid.
7. it is used to detect the ELISA kit of the antibody of the uroporphyrinogen decarboxylase mutain of people according to claim 6,
It is characterized in that, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
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|---|---|---|---|
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| Publication Number | Publication Date |
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ID=63162905
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117568325A (en) * | 2024-01-15 | 2024-02-20 | 黑龙江新和成生物科技有限公司 | Recombinant bacterium for expressing mutant uroporphyrinogen decarboxylase |
-
2018
- 2018-05-14 CN CN201810455537.5A patent/CN108424902A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117568325A (en) * | 2024-01-15 | 2024-02-20 | 黑龙江新和成生物科技有限公司 | Recombinant bacterium for expressing mutant uroporphyrinogen decarboxylase |
| CN117568325B (en) * | 2024-01-15 | 2024-04-16 | 黑龙江新和成生物科技有限公司 | Recombinant bacterium for expressing mutant uroporphyrinogen decarboxylase |
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Application publication date: 20180821 |