CN108251407A - The 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people - Google Patents
The 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people Download PDFInfo
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- C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
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Abstract
The present invention relates to the 5 phosphoric acid hydroxyl L lysine phosphoroclastic cleavage enzyme mutant albumen of people a kind of, using several phosphoric acid oxylysine patient with urine disease as research case, genetic test is carried out to case and is analyzed, determine the mutain of 5 phosphoric acid hydroxyl L lysine phosphoroclastic cleavage enzymes, genetic chip, monoclonal antibody and ELISA kit are prepared according to the 5 phosphoric acid hydroxyl L lysine phosphoroclastic cleavage enzyme mutant albumen, the gene diagnosis that disease is urinated for phosphoric acid oxylysine provides impulse, realizes the Clinics and Practices of phosphoric acid oxylysine urine disease.
Description
Technical field
A kind of a kind of 5- phosphoric acid hydroxy-L-lysine phosphoric acid the present invention relates to genetic engineering field more particularly to people is split
Solve enzyme mutant albumen.
Background technology
It is urinated with PHYKPL (5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavages enzyme) relevant disease including phosphoric acid oxylysine
Disease, relevant approach are lysine degradations.It is examined to whether the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzymes of people are mutated
It surveys, has certain impulse to judging whether human body urinates disease with phosphoric acid oxylysine.
Invention content
Present invention aims at provide a kind of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people.
Technical solution of the present invention includes：
In a first aspect, provide the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people a kind of, amino acid sequence
Row such as SEQ ID NO:Shown in 1.
Second aspect provides a kind of encoding gene of the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people,
Its 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 according to the nucleotide sequence of the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of the people,
It is determined with the comparison result of the nucleotide sequence of the normal albumen of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzymes of people.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of specific recognition people a kind of
Monoclonal antibody, coding amino acid sequence be SEQ ID NO:Shown in 1.
5th aspect provides a kind of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen for being used to detect people
The ELISA kit of antibody, the ELISA kit include：It is coated with ELISA ELISA Plates, the enzyme mark of said monoclonal antibody
Secondary antibody, the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavages zymoprotein for detecting object, sample diluting liquid, coating buffer solution, ELISA
ELISA Plate cleaning solution, developing solution and terminate 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- phosphoric acid hydroxyls-L- based on any of the above-described ELISA kit detection people and relies
The method of the antibody of propylhomoserin phosphoroclastic cleavage enzyme mutant albumen, including：
A, said monoclonal antibody is diluted, and the monoclonal after dilution is resisted using the coating buffer solution
Body is loaded into the hole of ELISA ELISA Plates, and is incubated at room temperature；
B, the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavages zymoprotein for detecting object is diluted using the sample diluting liquid
Into various concentration gradient, and by the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage zymoproteins of various concentration gradient be loaded respectively to
In the hole of ELISA ELISA Plates, and it 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, wavelength is measured in microplate reader as the light absorption value at 450nm.
Preferably, further comprise：Blank control is tested.
The present invention provides the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people a kind of, by several phosphorus
Sour oxylysine patient with urine disease carries out case genetic test and analyzes, determine 5- phosphoric acid hydroxyls-L- as research case
The mutain of lysine phosphoroclastic cleavage enzyme prepares base according to the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen
Because of chip, monoclonal antibody and ELISA kit, the gene diagnosis that disease is urinated for phosphoric acid oxylysine provides impulse, real
The Clinics and Practices of existing phosphoric acid oxylysine urine disease.
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 implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after attached drawing is coordinated 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 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. is " 201710429915.8 ", patent name
Detection method described in the Chinese invention patent of method and device " determines that the 5- phosphoric acid hydroxy-L-lysine phosphoric acid of people is split
Solve the encoding gene of enzyme mutant albumen, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, it is true according to the encoding gene
Make the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen and its encoding gene of people, according to such as
Under type realizes the preparation of genetic chip.
1st, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is dashed forward according to the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzymes of people
Become the nucleotide sequence of albumen, with the nucleotide sequence of the normal albumen of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzymes of people
Comparison result determines, and according to the design principle of following probe, design the 5- phosphoric acid hydroxy-L-lysine phosphoric acid for people
The nucleotide probe of the specificity of lyases mutain.
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%-70%, the specificity that non-specific hybridization ensures hybridization is reduced；
4. nucleotide probe intramolecule stablizes secondary structure pairing bases longs less than 4bp, can ensure in this way 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- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people and people
The comparison result of the corresponding amino acid sequence of the normal albumen of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzymes please refers to Fig.1,
In, the Query sequences in Fig. 1 are the corresponding amino acid sequences of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people
Row, Sbjct sequences are the corresponding amino acid sequences of the normal albumen of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzymes of people, Query
Sequence between sequence and Sbjct sequences is comparison result, and as can be seen from FIG. 1, the 5- phosphoric acid hydroxy-L-lysine phosphoric acid of people is split
5- phosphoric acid hydroxyl-L- lysine phosphoroclastic cleavage enzyme normal albumen of the enzyme mutant albumen relative to people is solved, is occurred at a position
Missing.According to SEQ ID NO:The comparison result of nucleotide sequence and Fig. 1 shown in 2, is treated in order to specific recognition
Whether the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzymes of detection object are mutated, then when choosing nucleotide probe,
Nucleotide probe can be designed according to any one of following several ways mode：Several nucleosides are selected before deletion sites
Acid sequence with selecting several nucleotide sequences after deletion sites, 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, it is as follows to design a kind of preferably nucleotide probe in the manner described above：
Nucleotide probe：attctggtgc gtat
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2nd, the preparation of genetic chip whether the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzymes of detection people mutate
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, ☆ is 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 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 aattgcatag。
Positive internal control Quality Control probe：It is one section of other genetic fragment for having homology with detection gene, in the 5- phosphoric acid of people
In hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen, select sequence corresponding from nucleotide probe different, and can be with nucleosides
The number of acid probe base is identical, and one section of nucleotide sequence that can also be different from the number of nucleotide probe base is as positive
Internal reference Quality Control probe.Preferably, the positive internal control Quality Control identical with the number of nucleotide probe base of nucleotide number is chosen to visit
Needle.In the embodiment of the present invention, the positive internal control probe sequence needed for genetic chip can be：aacggctcat cagc.
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
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
1st, sample treatment
(1) the blood 1-3mL of acquisition testing object.
(2) the 1.5mL EP pipes that DEPC is handled are taken, as detected sample processing tube, detection object is added in pipe
300 μ L of blood, add 700 μ L of Trizol, abundant mixing is placed at room temperature for 10min.
(3) chloroform of 140 μ L is added in, pipe lid is covered tightly, 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 step (3) is stored, 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 centrifugation 15min carefully suck all supernatants, super
Dry 15min in net 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 QIAGENKit purifies total serum IgE.
2nd, the first chains of cDNA and the second chain one-step synthesis method
(1) take 2 μ g RNA that following reaction solution is configured in the centrifuge tube of 1.5mL：
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 calculating of total serum IgE and get.
(2) 10min, and ice bath 5min are kept the temperature at 65 DEG C, 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 in after mixing after being denaturalized in the RNA of ice bath.
(5) it is 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 (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) configuration Transcription mix
(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 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washings 15-30s discards
Filtered solution discards the casing of filtered solution and 2mL by RNeasy with 500 μ L Buffer RPE in >=8000g centrifuge washings 2min 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).
5th, 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.
6th, cRNA fluorescents mark；
(1) above-mentioned 4 μ g of cRNA are taken and are concentrated into 6.6 μ L.
(2) add 10 μ L DMSO mixings.
(3) sodium bicarbonate (NaHCO that the 0.3M pH for adding 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 1 h of heat preservation.
(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.
| Cy3 cRNA green fluorescences | 875ng |
| 10×Blocking Agent | 11μL |
| 25×Fragmentation Buffer | 2.2μL |
| Nuclease-free water | X μL |
| Total volume | 55μL |
(2) 55 μ L 2 × 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
Washing lotion 1 (1L) is configured：
| DEPC-H2O | 700mL |
| 20*SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 2 (1L) is configured：
| 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.
10th, chip scanning
Chip is scanned in scanner, the 5- phosphoric acid hydroxyls-L- for determining detection object according to scanning result relies ammonia
Whether acid phosphoric acid cracking zymoprotein is mutated.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen
Fluorescence, positive control are green fluorescence, and the sample quality for showing acquisition testing object is that there is no problem, and experimental group is green
Fluorescence, then the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage zymoproteins for showing to detect object are mutated.It is shown in Fig. 4
As a result in, negative control is colorless fluorescent, and positive control is green fluorescence, and the sample quality for showing acquisition testing object is that do not have
Problem, and experimental group redgreen fluorescence, then show to detect the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage zymoproteins of object
It does not mutate.
Embodiment three, specific recognition people 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen monoclonal
The preparation of antibody
1st, according to base sequence (such as SEQ ID of the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people
NO: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 (P)：atggccgcag accagc
Downstream primer (F)：ctcccggtag gggcccc
2nd, the DNA of detection object carries 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- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavages of people
The complete segment of enzyme mutant protein gene, and pMD19-T Vector (Takara companies) are connected, it is sequenced.Then by special
Biotech firm prepares antibody, is a kind of humanization or Chimeric antibodies.The antibody of preparation is measured into content using ELISA method.
Example IV, for detecting the antibody of the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people
ELISA kit
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- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavages zymoprotein for detecting object, sample diluting liquid, coating buffering
Liquid, 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 benzidine；
Terminate liquid can be the sulfuric acid solution of 2mol/L.
Embodiment five
In embodiments of the present invention, using phosphoric acid oxylysine patient with urine disease as detection object, and ELISA reagents are utilized
Box detect the phosphoric acid oxylysine patient with urine disease 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage zymoproteins whether have occurred it is prominent
Become, this method can at least include following a kind of：
A, monoclonal antibody prepared by embodiment three is diluted, such as is diluted to using the coating buffer solution
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, using sample diluting liquid by the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavages of phosphoric acid oxylysine patient with urine disease
Zymoprotein is diluted to various concentration gradient, and by the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage zymoproteins of various concentration gradient
It 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 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, wavelength is measured in microplate reader as the light absorption value at 450nm.
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 during ＞ 0.99；According to ELISA Calc Software on Drawing
Go out standard curve, which can be obtained according to the ELISA Calc softwares2=0.99916, therefore, this measures effective.It will inspection
The wavelength measured substitutes into standard curve for the light absorption value at 450nm can acquire the 5- phosphorus of phosphoric acid oxylysine patient with urine disease
The concentration of sour hydroxy-L-lysine phosphoroclastic cleavage zymoprotein.Utilize the ELISA method detection phosphoric acid oxylysine urine disease of foundation
The content of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage zymoprotein in patient serum sample.And it is reflected with Western blot
It is fixed.Fig. 5 is please referred to, is the standard curve of light absorption value.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot are identified
1st, glue
(1) it cleans glass plate and dries；
(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(PH8.8) | 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)
(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 for upper strata concentration gelling solid.
2nd, albumen loading electrophoresis：
(1) comb is pulled up, duct label 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 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 in, 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) 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, PBST cleanings film 3 times, each 10min are recycled；
(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 are analyzed with Image J.
Wherein, primary antibody is the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant protein monoclonals of the standby people of corporation
Antibody, secondary antibody are the Goat anti-Human IgGs of horseradish peroxidase-labeled.
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 38KD
There is apparent band, wherein, the molecular weight of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen is about 38KD, table
The 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage zymoproteins of the bright phosphoric acid oxylysine patient with urine disease are implicitly present in mutation.
The above is only the preferred embodiment of the present invention, 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 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 155
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ala Ala Asp Gln Arg Pro Lys Ala Asp Thr Leu Ala Leu Arg Gln
1 5 10 15
Arg Leu Ile Ser Ser Ser Cys Arg Leu Phe Phe Pro Glu Asp Pro Val
20 25 30
Lys Ile Val Arg Ala Gln Gly Gln Tyr Met Tyr Asp Glu Gln Gly Ala
35 40 45
Glu Tyr Ile Asp Cys Ile Ser Asn Val Ala His Val Gly His Cys His
50 55 60
Pro Leu Val Val Gln Ala Ala His Glu Gln Asn Gln Val Leu Asn Thr
65 70 75 80
Asn Ser Arg Tyr Leu His Asp Asn Ile Val Asp Tyr Ala Gln Arg Leu
85 90 95
Ser Glu Thr Leu Pro Glu Gln Leu Cys Val Phe Tyr Phe Leu Asn Ser
100 105 110
Gly Ala Tyr His Gly His Leu Ser Ser Leu Ile Asp Ile Ser Pro Tyr
115 120 125
Lys Phe Arg Asn Leu Asp Gly Gln Lys Glu Trp Val His Val Ala Pro
130 135 140
Leu Pro Asp Thr Tyr Arg Gly Pro Tyr Arg Glu
145 150 155
<210> 2
<211> 465
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggccgcag accagcgccc gaaggccgac accctggccc tgaggcaacg gctcatcagc 60
tcttcctgca gactcttttt tcccgaggat cctgttaaga ttgtccgggc ccaagggcag 120
tacatgtacg atgaacaggg ggcagaatac atcgattgca tcagcaatgt ggcgcacgtt 180
gggcactgcc accctctcgt ggtccaagca gcacatgagc agaaccaggt gctcaacacc 240
aacagccggt acctgcatga caacatcgtg gactatgcgc agaggctgtc agagaccctg 300
ccggagcagc tctgtgtgtt ctatttcctg aattctggtg cgtatcacgg ccacctgagc 360
tccctgattg acatcagtcc ctacaagttc cgcaacctgg atggccagaa ggagtgggtc 420
cacgtggcac ctctcccaga cacctaccgg ggcccctacc gggag 465
<210> 3
<211> 14
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
attctggtgc gtat 14
<210> 4
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggccgcag accagc 16
<210> 5
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ctcccggtag gggcccc 17
Claims (9)
1. the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of a kind of people, which is characterized in that its amino acid sequence is such as
SEQ ID NO:Shown in 1.
A kind of 2. encoding gene of the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people, which is characterized in that its core
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- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of the people, with people
The comparison result of nucleotide sequence of the normal albumen of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzymes determine.
4. genetic chip according to claim 3, which is characterized in that the nucleotide probe is SEQID NO:Shown in 3
Base sequence.
5. a kind of monoclonal antibody of the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of specific recognition people,
It is characterized in that, the amino acid sequence of coding is SEQ ID NO:Shown in 1.
6. a kind of ELISA reagents of the antibody for the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen for being used to detect people
Box, which is characterized in that the ELISA kit includes：Be coated with monoclonal antibody described in claim 5 ELISA ELISA Plates,
ELIAS secondary antibody, detect the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavages zymoprotein of object, sample diluting liquid, coating buffer solution,
ELISA ELISA Plates cleaning solution, developing solution and terminate liquid.
7. it is used to detect the anti-of the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant albumen of people according to claim 6
The ELISA kit of body, which is characterized in that the ELIAS secondary antibody resists for the goat of diluted HRP horseradish peroxidase-labeleds
Human IgG.
8. a kind of 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavages based on the ELISA kit detection people of claim 6 or 7
The method of the antibody of enzyme mutant albumen, which is characterized in that including：
A, monoclonal antibody described in claim 5 is diluted, and by the list after dilution using the coating buffer solution
Clonal antibody is loaded into the hole of ELISA ELISA Plates, and is incubated at room temperature；
B, the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage zymoproteins for detecting object are diluted to not using the sample diluting liquid
Same concentration gradient, and the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage zymoproteins of various concentration gradient are loaded respectively to ELISA
In the hole of ELISA Plate, and it 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, wavelength is measured in microplate reader as the light absorption value at 450nm.
9. the 5- phosphoric acid hydroxy-L-lysine phosphoroclastic cleavage enzyme mutant eggs of ELISA kit detection people according to claim 8
The method of white antibody, which is characterized in that further comprise：Blank control is tested.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107403074A (en) * | 2017-06-09 | 2017-11-28 | 天津市湖滨盘古基因科学发展有限公司 | A kind of detection method and device of mutain |
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2018
- 2018-01-18 CN CN201810047357.3A patent/CN108251407A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107403074A (en) * | 2017-06-09 | 2017-11-28 | 天津市湖滨盘古基因科学发展有限公司 | A kind of detection method and device of mutain |
Non-Patent Citations (4)
| Title |
|---|
| MARIA VEIGA-DA-CUNHA ET AL.: "Molecular Identification of Hydroxylysine Kinase and of Ammoniophospholyases Acting on 5-Phosphohydroxy-L-lysine and Phosphoethanolamine", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| VEIGA-DA-CUNHA M ET AL.: "ACCESSION NO:NP_699204,5-phosphohydroxy-L-lysine phospho-lyase isoform 2 [Homo sapiens]", 《GENBANK》 * |
| 国家食品药品监督管理局人事司: "《临床检验仪器与体外诊断试剂》", 31 October 2010 * |
| 苏联麟 等: "基于UPLC-Q/TOF-MS 的醋制五味子对酒精性肝损伤大鼠胆汁代谢物的影响研究", 《中草药》 * |
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