CN108424886A - A kind of auxiliary protein synthase mutain of the deoxidation of people and its application - Google Patents
A kind of auxiliary protein synthase mutain of the deoxidation of people and its application Download PDFInfo
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- CN108424886A CN108424886A CN201810456133.8A CN201810456133A CN108424886A CN 108424886 A CN108424886 A CN 108424886A CN 201810456133 A CN201810456133 A CN 201810456133A CN 108424886 A CN108424886 A CN 108424886A
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- C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
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Abstract
The present invention relates to a kind of auxiliary protein synthase mutain of deoxidation of people and its applications, by regarding a large amount of liver cancer and Patients with Fatty Liver as research case, genetic test is carried out to case and is analyzed, determine the mutain of the auxiliary protein synthase of the deoxidation of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the auxiliary protein synthase mutain of the deoxidation of the people, impulse is provided for the gene diagnosis of liver cancer and fatty liver, and is provided fundamental basis for the diagnosing and treating of relevant disease.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to a kind of auxiliary protein synthase mutain of deoxidation of people and
It is applied.
Background technology
The oxidation that the auxiliary protein synthase of deoxidation (deoxyhypusine synthase) can be catalyzed spermidine NAD dependences is split
Change, and be catalyzed on the ε amino groups for the special lysine residue that spermidine butylamine is transferred to eIF-5A precursor proteins, in formation
Between the auxiliary protein residues of product deoxidation.The auxiliary protein synthase mutation of deoxidation of people can impact human health.To liver
During the peripheral blood of cancer and Patients with Fatty Liver carries out gene sequencing, find that the auxiliary protein synthase of deoxidation of patient has occurred
Therefore mutation has centainly the detection of the deoxidation of people auxiliary protein synthase mutation to judging whether human body suffers from relevant disease
Impulse.
Invention content
Present invention aims at a kind of auxiliary protein synthase mutain of deoxidation of people of offer and its applications.
Technical solution of the present invention includes：
In a first aspect, providing a kind of auxiliary protein synthase mutain of the deoxidation of people, amino acid sequence such as SEQ ID
NO:Shown in 1.
Second aspect provides a kind of encoding gene of the mutain of the auxiliary protein synthase of the deoxidation of people, nucleotides sequence
Row 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 auxiliary protein synthase mutain of deoxidation of people described above, the deoxidation with people
The comparison result of the nucleotide sequence of the auxiliary normal albumen of protein synthase determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of monoclonal antibody of the auxiliary protein synthase mutain of the deoxidation 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 examinations for detecting the antibody of the auxiliary protein synthase mutain of deoxidation of people
Agent box, the ELISA kit include：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection object of said monoclonal antibody
The auxiliary protein synthase albumen of deoxidation, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and termination
Liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
The present invention provides a kind of auxiliary protein synthase mutain of deoxidation of people and its applications, by a large amount of liver cancer and fat
Hepatopath carries out genetic test to case and analyzes, determine the mutation of the auxiliary protein synthase of the deoxidation of people as research case
Albumen prepares genetic chip, monoclonal antibody and ELISA kit according to the auxiliary protein synthase mutain of the deoxidation of the people,
Impulse is provided for the gene diagnosis of liver cancer and fatty liver, and basis is provided for the diagnosing and treating of relevant disease.
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；
Fig. 7 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Fig. 8 is the Western blot testing result schematic diagrames that the embodiment of the present invention six provides.
Specific 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 auxiliary protein synthase mutain of the deoxidation of people
Encoding gene, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, determine that the deoxidation of people is auxiliary according to the encoding gene
Protein synthase mutain, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to the auxiliary protein synthase mutain of the deoxidation of people and its encoding gene, base is realized as follows
Because of the preparation of chip.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the nucleosides of the auxiliary protein synthase mutain of deoxidation of people
Acid sequence determines with the comparison result of the nucleotide sequence of the normal albumen of the auxiliary protein synthase of deoxidation of people, and according to as follows
The design principle of probe designs the nucleotide probe of the specificity of the auxiliary protein synthase mutain of deoxidation 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. 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 the auxiliary protein synthase mutain of the deoxidation of people is synthesized with the auxiliary albumen of the deoxidation of people
The comparison result of the corresponding amino acid sequence of the normal albumen of enzyme please refers to Fig.1, wherein the Query sequences in Fig. 1 are the deoxidations of people
The corresponding amino acid sequence of auxiliary protein synthase mutain, Sbjct sequences are the normal albumen of the auxiliary protein synthase of deoxidation of people
Corresponding amino acid sequence, the sequence between Query sequences and Sbjct sequences are comparison result, and as can be seen from FIG. 1, people's is de-
Deoxidation auxiliary protein synthase normal albumen of the auxiliary protein synthase mutain of oxygen relative to people, inserts a part of amino acid sequence
Row, according to SEQ ID NO:The comparison result of nucleotide sequence and Fig. 1, to be detected in order to specific recognition shown in 2
Whether the auxiliary protein synthase of deoxidation of object is mutated, then when choosing nucleotide probe, it can be according to following several
Any one of mode 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：aaccctgagt ggtgtt
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether auxiliary protein synthase of deoxidation 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 nucleotide number that negative internal reference Quality Control probe includes can be with nucleotide
The base number of probe is identical, also can be different.In the embodiment of the present invention, negative internal reference probe sequence needed for genetic chip can be with
For：tgatgctgat aattgcat.
Positive internal control Quality Control probe：It is one section of other genetic fragment for having homology with detection gene, it is auxiliary in the deoxidation of people
In protein synthase mutain, select sequence corresponding from nucleotide probe different, and can be with 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 visited as positive internal control Quality Control
Needle.Preferably, nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen.The present invention is real
It applies in example, the positive internal control probe sequence needed for genetic chip can be：ggaggcgcca gcgggg.
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 1.5mL EP pipes that DEPC is handled are taken, as detected sample processing tube, in detected sample processing tube
The middle 300 μ L of blood that detection object is added, 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 in a centrifuge, 12000r/min, 4 DEG C of centrifugations
15min, it is careful to draw supernatant in centrifuge tube, the supernatant of absorption is transferred in clean centrifuge tube.
(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 being cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min, 4 DEG C of centrifugation 15min carefully suck all supernatants,
The dry 15min in super-clean bench 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 |
| T7Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
Wherein, the addition X μ L of RNase-free Water are to subtract T7Promotor 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, in advance by 5 × First Strand B μ ffer at 65 DEG C
Preheat 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 L RNase 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.
| Cy3cRNA 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 auxiliary protein synthase egg of deoxidation of detection object is determined according to scanning result
Whether it is mutated in vain.It please refers to Fig.3 and Fig. 4, in result shown in Fig. 3, negative control redgreen fluorescence, positive control is
Green fluorescence shows that the sample quality of acquisition testing object is that there is no problem, and experimental group is green fluorescence, then showing to examine
The auxiliary protein synthase albumen of deoxidation for surveying object is mutated.In result shown in Fig. 4, negative control is colorless fluorescent, sun
Property control be green fluorescence, show that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, that
Show that the auxiliary protein synthase albumen of deoxidation for detecting object does not mutate.
Embodiment three, specific recognition people the auxiliary protein synthase mutain of deoxidation monoclonal antibody preparation
1, according to base sequence (such as SEQ ID NO of the auxiliary protein synthase mutain of the deoxidation of people:Shown in 2) in design
Swim primer such as SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (F)：atggaaggtt ccctggaa
Downstream primer (R)：ctctgtgttc tgctcca
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, obtains the auxiliary protein synthase mutating protein gene of deoxidation of people
Complete segment, 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, the auxiliary protein synthase mutain of deoxidation 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 auxiliary protein synthase albumen of deoxidation for detecting object, sample diluting liquid, coating buffer solution, ELISA enzyme marks
Plate cleaning solution, developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition at least can be a kind of following parameter：
ELISA ELISA Plates can be the ELISA enzyme marks in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) 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 liver cancer patient as detection object, and detects the liver cancer using ELISA kit and suffers from
Whether the auxiliary protein synthase albumen of deoxidation 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 auxiliary protein synthase albumen of the deoxidation of liver cancer patient is diluted to various concentration gradient using sample diluting liquid, and
The auxiliary protein synthase albumen of the deoxidation of various 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=0.99786, 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 deoxidation of liver cancer patient
The concentration of auxiliary protein synthase albumen.It is closed using the auxiliary albumen of deoxidation in the ELISA method detection liver cancer patient blood serum sample of foundation
At the content of zymoprotein.It is used in combination Westernblot to be identified.Referring to FIG. 5, for the standard curve of light absorption value.Wherein, X-axis
For light absorption value, 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)
| ddH2O | 5.5mL |
| 30% acrylamide mixed liquor | 1.3mL |
| 1.0mol/L Tris(PH6.8) | 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；
After the isometric mixing of Pierce ECL Western Blotting Substrate kit A, B liquid, it is added dropwise dropwise
On pvdf membrane；
(9) exposure image in FluorChem E FE0511, experiment Image J analyses.
Wherein, primary antibody is the auxiliary protein synthase mutain monoclonal antibody of deoxidation of the standby people of corporation, and secondary antibody is peppery
The Goat anti-Human IgG of root peroxidase labelling.
J, Western blot Analysis of test results：It is shown according to Western blot experimental procedures as a result, such as Fig. 6 institutes
Show, wherein the Marker in Fig. 6 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 54KD
There is apparent band, wherein the molecular weight of the auxiliary protein synthase mutain of deoxidation is about 54KD, shows the liver cancer patient
The auxiliary protein synthase albumen of deoxidation is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, using Patients with Fatty Liver as detection object, and the fat is detected using ELISA kit
Whether the auxiliary protein synthase albumen of deoxidation of hepatopath 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 auxiliary protein synthase albumen of the deoxidation for detecting object is diluted to various concentration gradient using sample diluting liquid, and
The auxiliary protein synthase albumen of the deoxidation of various 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=0.99779, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire fatty liver trouble in sample
The concentration of the auxiliary protein synthase albumen of deoxidation of person.It is detected in Patients with Fatty Liver blood serum sample and is taken off using the ELISA method of foundation
The content of the auxiliary protein synthase albumen of oxygen.It is used in combination Western blot to be identified.Referring to FIG. 7, the standard for light absorption value is bent
Line.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot are identified
J, Western blot 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 54KD, wherein the molecular weight of the auxiliary protein synthase mutain of deoxidation is about 54KD, shows the fat
The auxiliary protein synthase albumen of deoxidation of fat hepatopath 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>A kind of auxiliary protein synthase mutain of the deoxidation of people and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 222
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Glu Gly Ser Leu Glu Arg Glu Ala Pro Ala Gly Ala Leu Ala Ala
1 5 10 15
Val Leu Lys His Ser Ser Thr Leu Pro Pro Glu Ser Thr Gln Val Arg
20 25 30
Gly Tyr Asp Phe Asn Arg Gly Val Asn Tyr Arg Ala Leu Leu Glu Ala
35 40 45
Phe Gly Thr Thr Gly Phe Gln Ala Thr Asn Phe Gly Arg Ala Val Gln
50 55 60
Gln Val Asn Ala Met Ile Glu Lys Lys Leu Glu Pro Leu Ser Gln Asp
65 70 75 80
Glu Asp Gln His Ala Asp Leu Thr Gln Ser Arg Arg Pro Leu Thr Ser
85 90 95
Cys Thr Ile Phe Leu Gly Tyr Thr Ser Asn Leu Ile Ser Ser Gly Ile
100 105 110
Arg Glu Thr Ile Arg Tyr Leu Val Gln His Asn Met Val Asp Val Leu
115 120 125
Val Thr Thr Ala Gly Gly Val Glu Glu Asp Leu Ile Lys Cys Leu Ala
130 135 140
Pro Thr Tyr Leu Gly Glu Phe Ser Leu Arg Gly Lys Glu Leu Arg Glu
145 150 155 160
Asn Gly Ile Asn Arg Glu Pro Val Val Leu Gly Arg Gly Ala Gly Pro
165 170 175
Arg Val Leu Gly Pro Asp Ala Tyr Met Pro Pro Val Leu Arg Ile Gly
180 185 190
Asn Leu Leu Val Pro Asn Glu Asn Tyr Cys Lys Phe Glu Asp Trp Leu
195 200 205
Met Pro Ile Leu Asp Gln Met Val Met Glu Gln Asn Thr Glu
210 215 220
<210> 2
<211> 672
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggaaggtt ccctggaacg ggaggcgcca gcgggggcgc tggccgccgt gctaaagcac 60
agctcgacgt tgccgcccga aagcacccag gtccggggct acgacttcaa ccgcggtgtg 120
aattaccgcg cactgctgga ggccttcggc accaccggct tccaagcaac caacttcggg 180
cgcgctgtac agcaagtcaa tgccatgatc gagaagaagc tggaaccact gtcacaggat 240
gaagaccagc acgcggacct gacccagagc cgccgcccac ttaccagctg caccattttc 300
ctgggatata catccaacct catcagttca ggcatccgtg agaccattcg ctaccttgtg 360
cagcacaaca tggtggacgt attggtgacc acagctggcg gcgtggagga agacctcatc 420
aagtgcctgg cgcccacata cttgggcgag tttagcctca gggggaagga gctccgggag 480
aacgggatca ataggtgaga accctgagtg gtgttgggca ggggagctgg accaagggtc 540
ctggggcctg atgcctacat gcctcctgtt ctcaggatcg gaaacctgct ggtgcccaat 600
gagaattact gcaagtttga ggactggctg atgcccattc tggaccagat ggtgatggag 660
cagaacacag ag 672
<210> 3
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
aaccctgagt ggtgtt 16
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggaaggtt ccctggaa 18
<210> 5
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ctctgtgttc tgctcca 17
Claims (7)
1. a kind of auxiliary protein synthase mutain of the deoxidation of people, 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 auxiliary protein synthase mutain of the deoxidation 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 auxiliary protein synthase mutain of deoxidation of people according to claim 2, it is de- with people
The comparison result of the nucleotide sequence of the normal albumen of the auxiliary protein synthase of oxygen 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 auxiliary protein synthase mutain of the deoxidation of specific recognition people, which is characterized in that it can
With SEQ ID NO:Amino acid sequencespecific shown in 1 combines.
6. a kind of ELISA kit for detecting the antibody of the auxiliary protein synthase mutain of deoxidation of people, which is characterized in that
The ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection of monoclonal antibody described in claim 5
The auxiliary protein synthase albumen of deoxidation, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and the end of object
Only liquid.
7. being used to detect the ELISA reagents of the antibody of the auxiliary protein synthase mutain of deoxidation of people according to claim 6
Box, which is characterized in that the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
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Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810456133.8A CN108424886A (en) | 2018-05-14 | 2018-05-14 | A kind of auxiliary protein synthase mutain of the deoxidation of people and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108424886A true CN108424886A (en) | 2018-08-21 |
Family
ID=63162946
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810456133.8A Withdrawn CN108424886A (en) | 2018-05-14 | 2018-05-14 | A kind of auxiliary protein synthase mutain of the deoxidation of people and its application |
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| Country | Link |
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| CN (1) | CN108424886A (en) |
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2018
- 2018-05-14 CN CN201810456133.8A patent/CN108424886A/en not_active Withdrawn
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Application publication date: 20180821 |