CN107937375B - The arginase mutain of people a kind of and its application - Google Patents
The arginase mutain of people a kind of and its application Download PDFInfo
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Abstract
The present invention relates to the arginase mutain of people a kind of and its applications, by regarding a large amount of cancer patients as research case, genetic test is carried out to case and is analyzed, determine the mutain of the arginase of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mutain of the arginase of the people, for the impulse that the gene diagnosis of cancer provides, the early diagnosis, early discovery and associated treatment of cancer are realized.
Description
Technical field
The present invention relates to the arginase mutains and its application of a kind of genetic engineering field more particularly to a kind of people.
Background technology
Arginase is the reaction enzymes that catalyzing hydrolysis L-arginine generates ornithine and urea, and general be contained in generates urea
In the liver of animal, kidney, spermary.Research finds being related closely for arginase and cancer.The arginase of people occurs
Mutation can impact human health, the study found that certain cancers, (cancer includes lung cancer, liver cancer, cancer of pancreas and white
Blood disease) patient peripheral blood carry out gene sequencing during, find patient arginase be mutated, it is therefore, right
The detection of the arginase mutation of people has certain impulse to judging whether human body suffers from cancer.
Invention content
Present invention aims at the arginase 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 arginase mutain of people is provided, amino acid sequence such as SEQ ID NO:Shown in 1.
Second aspect provides a kind of encoding gene of the arginase mutain 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 arginase mutain of the people, the normal albumen of arginase with people
Nucleotide sequence comparison result determine.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of monoclonal antibody of the arginase mutain of specific recognition people, coding
Amino acid sequence is SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA kit for detecting the antibody of the arginase mutain of people, described
ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, the arginase for detecting object of said monoclonal antibody
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.
6th aspect provides a kind of arginase mutain detecting people based on any of the above-described ELISA kit
Antibody method, 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 arginase albumen for detecting object is diluted to various concentration gradient using the sample diluting liquid, and will not
Arginase albumen with 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, washs and dries；
D, the ELIAS secondary antibody is added, and is incubated at room temperature；
E, the liquid in each hole is got rid of, the ELISA ELISA Plates cleaning solution is added, washs and dries；
F, the developing solution is added, room temperature is protected from light incubation, and the terminate liquid is added and terminates reaction；
G, 450nm measures OD values in microplate reader.
Preferably, further comprise：Blank control is tested.
The present invention provides the arginase mutain of people a kind of and its applications, using a large amount of cancer patients as research disease
Example carries out genetic test to case and analyzes, the mutain of the arginase of people determined, according to the arginine of the people
Enzyme mutant albumen prepares genetic chip, monoclonal antibody and ELISA kit, is the guide work that the gene diagnosis of cancer provides
With realizing the early diagnosis of cancer, early find and associated treatment.
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 Westernblot 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 Westernblot testing result schematic diagrames that the embodiment of the present invention six provides；
Fig. 9 is the standard curve that the offer of the embodiment of the present invention seven is protein content；
Figure 10 is the Westernblot testing result schematic diagrames that the embodiment of the present invention seven 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 encoding gene of the arginase mutain of people,
Its nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, determine that the arginase of people is mutated egg according to the encoding gene
In vain, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the arginase mutain and its encoding gene of people, genetic chip is realized as follows
It prepares.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe according to the nucleotide sequence of the arginase mutain of people, with
The comparison result of the nucleotide sequence of the normal albumen of arginase of people determines, and according to the design principle of following probe, if
Count out the nucleotide probe of the specificity of the arginase mutain 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 4；
3. G+C contents are 40%-60%, the specificity that non-specific hybridization ensures hybridization is reduced；
4. nucleotide probe intramolecule, which stablizes secondary structure pairing bases longs, is less than 4bp, can ensure in this way will not
Hybridization efficiency is influenced because of the secondary structure of nucleotide probe internal stability；
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 arginase mutain of people is corresponding with the normal albumen of the arginase of people
The comparison result of amino acid sequence please refers to Fig.1, wherein the Query sequences in Fig. 1 are that the arginase mutain of people corresponds to
Amino acid sequence, Sbjct sequences are the corresponding amino acid sequence of the normal albumen of arginase of people, Query sequences and Sbjct
Sequence between sequence is comparison result, as can be seen from FIG. 1, the arginase mutain of people relative to people arginase just
Normal albumen, several place's amino acid sequences are mutated, and several places are lacked, and are inserted at one, according to SEQ ID
NO:The comparison result of nucleotide sequence and Fig. 1 shown in 2, in order to the arginase of specific recognition object to be detected
Whether it is mutated, then when choosing nucleotide probe, can be designed according to any one of following several modes mode
Nucleotide probe：
(1) will include a nucleotide sequence of mutated site nucleotide as nucleotide probe.
(2) before deletion sites with respectively selected after deletion sites several nucleotide sequences (base sequence it is suitable
Sequence is constant), generate nucleotide probe；
(3) whole nucleotides sequence column-generation nucleotide probe of insertion position is chosen；
(4) before insertion position, and/or, after insertion position, respectively select several nucleotide sequences, the selection
Several sequences (sequence of base sequence position is constant) be inserted into whole nucleotide sequence generate nucleotide probe jointly；
(5) several nucleotide sequences are selected before insertion position, if being selected at the starting position of insertion position
Dry nucleotide sequence, generates nucleotide probe；
(6) several nucleotide sequences are selected after insertion position, if being selected at the end position of insertion position
Dry 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, in the manner described above (1) design one kind preferably nucleotide probe such as SEQ ID NO:Shown in 3, the nucleotide probe
For：gtgatgtgaa ggattatggg g
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether arginase 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, ☆ is 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 digit that negative internal reference Quality Control probe includes can be with nucleotide probes
Digit is identical.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 arginine of people
In enzyme mutant albumen, select sequence corresponding from nucleotide probe different, and digit can be identical as the digit of nucleotide probe,
One section of nucleotide sequence that can also be different from nucleotide probe digit is as positive internal control Quality Control probe.Preferably, core is chosen
Thuja acid digit positive internal control Quality Control probe identical with the digit of nucleotide probe.In the embodiment of the present invention, needed for genetic chip
Positive internal control probe sequence can be：attgactacc ttaacccacc t.
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) the 1.5mLEP pipes that DEPC is handled is taken to be added in being detected sample processing tube to be detected sample processing tube
The 300 μ L of blood for detecting object, add 700 μ L of Trizol, mix well, be placed at room temperature for 10Min.
(3) chloroform of 200 μ L is added, covers tightly centrifuge tube lid, 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, gently overturns centrifuge tube and mixes well liquid, be 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 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 1.5mL centrifuge tubes：
| 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 |
10 minutes ice baths of (2) 65 DEG C of heat preservations 5 minutes, preheat 5 × First Strand B μ ffer at 65 DEG C 5 points in advance
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 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：
It is spare to be distributed into 10 pipes, notes：40 DEG C of heat preservation 1min before 50%PEG uses.Following operative configuration is pressed simultaneously
Transcription mix；
(1) Transcriptionmix is configured
| RNase-free Water | 5.7μL |
| 4×Transcription Buffer | 20μL |
| NTP | 16μL |
| 0.1M DTT | 6μL |
| 50%PEG | 6.4μL |
| aa-UTP(25mM) | 4μL |
| Inorganic Pyrophosphatase | 0.6μL |
| T7 RNA Polymerase | 0.8μL |
| Total volume | 60μL |
(2) 60 μ l Transcriptionmix 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 BufferRLT 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 15-30sec, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=15-30sec of 8000g centrifuge washings abandons
Going filtered solution 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) it is primary to repeat step (5).
5, cRNA concentration mensurations
With spectrophotometric analysis RNA concentration.Need 260 and 280nm measure absorbance come determine sample concentration and
Purity A260/A280 should be purer RNA (ratio 1.9-2.1 also can) close to 2.0.
6, cRNA fluorescents mark；
(1) above-mentioned cRNA4 μ g are taken and are concentrated into 6.6 μ L.
(2) add 10 μ L DMSO mixings.
(3) add 0.3M sodium bicarbonates (NaHCO3) pH9.0 and mixing of 3.4 μ L.
(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 chip hybridization 4x44Kmicroarrays
(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 |
| 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.
(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
(1 liter) configuration of washing lotion 1：
| DEPC-H2O | 700mL |
| 20*SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
(1 liter) configuration of washing lotion 2：
| DEPC-H2O | 997mL |
| 20*SSPE | 3.0mL |
| 20%N-Lauroylsarcosine | 0.25mL |
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.
10, chip scanning
Chip is scanned in scanner, determines whether the arginase albumen of detection object is sent out according to scanning result
Mutation is given birth to.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control is that green is glimmering
Light 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 object
Arginase albumen be mutated.In result shown in Fig. 4, negative control is colorless fluorescent, and positive control is that green is glimmering
Light shows that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then showing to detect object
Arginase albumen do not mutate.
Embodiment three, specific recognition people arginase mutain monoclonal antibody preparation
1, according to base sequence (such as SEQ ID NO of the arginase mutain of people:Shown in 2) design sense primer is such as
SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (P)：atgagcgcca agtccagaac
Downstream primer (F)：cttaggtggg ttaaggtagt
2, the DNA of detection object is that template carries out PCR amplification
DNA to detect object carries out PCR amplification as template, obtains the arginase mutating protein gene complete slice of people
Section, and pMD19-TVector (Takara companies) is connected, it is sequenced.Then antibody is prepared by special biotech firm, is one
Kind humanization or Chimeric antibodies.The antibody of preparation is measured into content using ELISA method.
Example IV, arginase 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, detect the arginase albumen of 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
In embodiments of the present invention, it using patients with lung cancer as detection object, and detects the lung cancer using ELISA kit and suffers from
Whether the arginase albumen of person is mutated, and this method at least may include following one kind：
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 arginase albumen of patients with lung cancer is diluted to various concentration gradient using sample diluting liquid, and will be different dense
The arginase albumen of degree 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, 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 when ＞ 0.99；Wherein it is possible to soft using ELISA Calc
Part draws the standard curve, can know regression coefficient R according to the ELISA Calc softwares2=0.9995, therefore, this measurement
Effectively.Bring the OD450 values that detection obtains into concentration that standard curve can acquire the arginase albumen of patients with lung cancer.It utilizes
The content of arginase albumen in the ELISA method detection Serum of Patients with Lung Cancer sample of foundation.It is used in combination Westernblot to reflect
It is fixed.Referring to FIG. 5, for the standard curve of OD values.Wherein, X-axis is OD values, and Y-axis is corresponding concentration.
I, Westernblot is 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(PH8.8) | 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)
(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 close membrane 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 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) it after the isometric mixing of Pierce ECLWestern Blotting Substrate kit A, B liquid, drips dropwise
It is added on pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment Image J analyses.
Wherein, primary antibody is the arginase mutain monoclonal antibody of the standby people of corporation, and secondary antibody is horseradish peroxidating
The Goat anti-Human IgG of object enzyme label.
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 73KD
There is apparent band, wherein arginase mutant protein molecules amount is about 73KD, shows the arginase egg of the patients with lung cancer
It is implicitly present in mutation in vain.
Embodiment six
In embodiments of the present invention, it using liver cancer patient as detection object, and detects the liver cancer using ELISA kit and suffers from
Whether the arginase albumen of person is mutated, and this method at least may include following one kind：
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 arginase albumen for detecting object is diluted to various concentration gradient using sample diluting liquid, and will be different dense
The arginase albumen of degree 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, 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 when ＞ 0.99；Wherein it is possible to soft using ELISA Calc
Part draws the standard curve, can know regression coefficient R according to the ELISA Calc softwares2=0.9959, therefore, this measurement
Effectively.By the obtained OD450 values of detection bring into standard curve can acquire liver cancer patient in sample arginase albumen it is dense
Degree.Utilize the content of arginase albumen in the ELISA method detection liver cancer patient blood serum sample of foundation.It is used in combination
Westernblot is identified.Referring to FIG. 7, for the standard curve of OD values.Wherein, X-axis is OD values, and Y-axis is corresponding dense
Degree.
I, Westernblot is identified
J, Westernblot Analysis of test results
Wherein, step i and step j are identical as in embodiment five, and details are not described herein, referring 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 73KD, wherein arginase mutant protein molecules amount is about 73KD, shows the essence of the liver cancer patient
Propylhomoserin zymoprotein is implicitly present in mutation.
Embodiment seven
In embodiments of the present invention, using Pancreas cancer patients as detection object, and the pancreas is detected using ELISA kit
Whether the arginase albumen of cancer patient is mutated, and this method at least may include following one kind：
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 arginase albumen of Pancreas cancer patients is diluted to various concentration gradient using sample diluting liquid, and will be different
The arginase 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, 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, 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 when ＞ 0.99；Wherein it is possible to soft using ELISA Calc
Part draws the standard curve, can know regression coefficient R according to the ELISA Calc softwares2=0.9962, therefore, this measurement
Effectively.The arginase albumen of Pancreas cancer patients in sample can be acquired by bringing detection obtained OD450 values into standard curve
Concentration.Utilize the content of arginase albumen in the ELISA method detection Pancreas cancer patients blood serum sample of foundation.It is used in combination
Westernblot is identified.Referring to FIG. 9, for the standard curve of OD values.Wherein, X-axis is OD values, and Y-axis is corresponding dense
Degree.
I, Westernblot is identified
J, Westernblot Analysis of test results
Wherein, step i and step j are identical as in embodiment five, and details are not described herein, referring to FIG. 10, in Figure 10
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 73KD, wherein arginase mutant protein molecules amount is about 73KD, shows the Pancreas cancer patients
Arginase albumen is implicitly present in mutation.
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 arginase mutain of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 293
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ser Ala Lys Ser Arg Thr Ile Gly Ile Ile Gly Ala Pro Phe Ser
1 5 10 15
Lys Gly Gln Ser Val Met Arg Ile Met Gly Thr Cys Pro Leu Leu Thr
20 25 30
Ser Leu Met Thr Val Pro Phe Lys Leu Arg Ile Gln Gly Leu Trp Glu
35 40 45
Lys Gln Ala Ser Ser Trp Leu Ala Arg Trp Gln Lys Ser Arg Arg Thr
50 55 60
Glu Glu Ser Ala Trp Cys Trp Ala Glu Thr Thr Val Trp Gln Leu Glu
65 70 75 80
Ala Ser Leu Ala Met Pro Gly Ser Thr Leu Ile Leu Glu Ser Ser Gly
85 90 95
Trp Met Leu Thr Leu Ile Ser Thr Leu His Gln Pro Gln Val Glu Thr
100 105 110
Cys Met Asp Asn Leu Tyr Leu Ser Ser Arg Asn Lys Glu Arg Phe Pro
115 120 125
Asp Val Pro Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp
130 135 140
Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile
145 150 155 160
Leu Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg
165 170 175
Leu Gly Ile Gly Lys Val Met Glu Glu Thr Leu Ser Tyr Leu Leu Gly
180 185 190
Arg Lys Lys Arg Pro Ile His Leu Ser Phe Asp Val Asp Gly Leu Asp
195 200 205
Pro Ser Phe Thr Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr
210 215 220
Tyr Arg Glu Gly Leu Tyr Ile Thr Glu Glu Ile Tyr Lys Thr Gly Leu
225 230 235 240
Leu Ser Gly Leu Asp Ile Met Glu Val Asn Pro Ser Leu Gly Lys Thr
245 250 255
Pro Glu Glu Val Thr Arg Thr Val Asn Thr Ala Val Ala Ile Thr Leu
260 265 270
Ala Cys Phe Gly Leu Ala Arg Glu Gly Asn His Lys Pro Ile Asp Tyr
275 280 285
Leu Asn Pro Pro Lys
290
<210> 2
<211> 894
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atgagcgcca agtccagaac catagggatt attggagctc ctttctcaaa gggacagagt 60
gtgatgtgaa ggattatggg gacctgccct ttgctgacat ccctaatgac agtccctttc 120
aaattgtgaa gaatccaagg tctgtgggaa aagcaagcga gcagctggct ggcaaggtgg 180
cagaagtcaa gaagaacgga agaatcagcc tggtgctggg cggagaccac agtttggcaa 240
ttggaagcat ctctggccat gccagggtcc accctgatct tggagtcatc tgggtggatg 300
ctcacactga tatcaacact ccactgacaa ccacaagtgg aaacttgcat ggacaacctg 360
tatctttcct cctgaaggaa ctaaaaggaa agattccccg atgtgccagg attctcctgg 420
gtgactccct gtatatctgc caaggatatt gtgtatattg gcttgagaga cgtggaccct 480
ggggaacact acattttgaa aactctaggc attaaatact tttcaatgac tgaagtggac 540
agactaggaa ttggcaaggt gatggaagaa acactcagct atctactagg aagaaagaaa 600
aggccaattc atctaagttt tgatgttgac ggactggacc catctttcac accagctact 660
ggcacaccag tcgtgggagg tctgacatac agagaaggtc tctacatcac agaagaaatc 720
tacaaaacag ggctactctc aggattagat ataatggaag tgaacccatc cctggggaag 780
acaccagaag aagtaactcg aacagtgaac acagcagttg caataacctt ggcttgtttc 840
ggacttgctc gggagggtaa tcacaagcct attgactacc ttaacccacc taag 894
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gtgatgtgaa ggattatggg g 21
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgagcgcca agtccagaac 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
cttaggtggg ttaaggtagt 20
Claims (3)
1. a kind of mutain of the arginase of people, which is characterized in that its amino acid sequence such as SEQ ID NO:Shown in 1.
2. a kind of encoding gene of the arginase mutain of people, which is characterized in that its nucleotide sequence such as SEQ ID NO:2
It is shown.
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 arginase mutain of people according to claim 2, just with the arginase of people
The comparison result of the nucleotide sequence of normal albumen determines；Wherein, the nucleotide probe is SEQ ID NO:Base sequence shown in 3
Row.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1345966A (en) * | 2000-09-26 | 2002-04-24 | 上海博德基因开发有限公司 | Novel poly peptide-human arginase 35.42 and polynucleotide for encoding said polypeptide |
| CN1364901A (en) * | 2001-01-10 | 2002-08-21 | 上海博德基因开发有限公司 | New polypeptide-arginase 8.91 and polynucleotide for encoding such polypeptide |
| CN1670197A (en) * | 2005-03-03 | 2005-09-21 | 中山大学 | Arginase of Japanese blood fluke, its coding nucleic acid and application |
| CN103184209A (en) * | 2011-12-27 | 2013-07-03 | 拜奥生物科技（上海）有限公司 | Homosapiensarginase and pegylation homosapiensarginase as well as applications thereof |
| CN105112391A (en) * | 2015-09-22 | 2015-12-02 | 浙江道尔生物科技有限公司 | Human-derived arginase mutant as well as preparation method and application thereof |
-
2017
- 2017-11-30 CN CN201711232021.6A patent/CN107937375B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1345966A (en) * | 2000-09-26 | 2002-04-24 | 上海博德基因开发有限公司 | Novel poly peptide-human arginase 35.42 and polynucleotide for encoding said polypeptide |
| CN1364901A (en) * | 2001-01-10 | 2002-08-21 | 上海博德基因开发有限公司 | New polypeptide-arginase 8.91 and polynucleotide for encoding such polypeptide |
| CN1670197A (en) * | 2005-03-03 | 2005-09-21 | 中山大学 | Arginase of Japanese blood fluke, its coding nucleic acid and application |
| CN103184209A (en) * | 2011-12-27 | 2013-07-03 | 拜奥生物科技（上海）有限公司 | Homosapiensarginase and pegylation homosapiensarginase as well as applications thereof |
| CN105112391A (en) * | 2015-09-22 | 2015-12-02 | 浙江道尔生物科技有限公司 | Human-derived arginase mutant as well as preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| arginase (EC 3.5.3.1)[Homo sapiens],Accession NO:AAA51776.1;Haraguchi,Y.et al.;《GenBank》;19941031;第1页 * |
| ID:EAW48053;Venter J.C.et al.;《EMBL》;20140516;第1-3页 * |
| 基因芯片技术及其在疾病诊断上的应用;戴克胜等;《江苏医药杂志》;20010430;第27卷(第4期);第292-294页 * |
| 精氨酸酶2在肝细胞癌组织中的表达及临床病理意义;肖锋等;《第二军医大学学报》;20130228;第34卷(第2期);第155-159页 * |
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