CN109022384A - The mutation of tRNA (guanine-N (7))-transmethylase and application - Google Patents
The mutation of tRNA (guanine-N (7))-transmethylase and application Download PDFInfo
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Abstract
The present invention relates to the mutation of tRNA (guanine-N (7))-transmethylase and applications, by using a large amount of lung cancer, leukaemic as research case, genetic test is carried out to case and is analyzed, determine the mutain of tRNA (guanine-N (7))-transmethylase of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to tRNA (guanine-N (7))-transmethylase mutain of the people, gene diagnosis for lung cancer, leukaemia provides impulse, realizes the diagnosing and treating of related disease.
Description
Technical field
The present invention relates to a kind of genetic engineering field more particularly to tRNA (guanine-N (7))-transmethylase mutation and
Using.
Background technique
TRNA (guanine-N (7))-transmethylase (tRNA (guanine-N (7) -)-methyltransferase) is urged
Change the formation of N (7) methyl guanine on 46 sites of tRNA.TRNA (m~7G46) transmethylase (TrmB) is exactly that one kind is urged
Change enzyme of the methylation reaction to be modified tRNAs transcript precursor.
TRNA (guanine-N (7))-transmethylase mutation of people can cause adverse effect to human health.Right
Certain diseases during the peripheral blood including lung cancer, leukaemic carries out gene sequencing, find the tRNA (bird of patient
Purine-N (7))-transmethylase is mutated, therefore, to tRNA (guanine-N (7))-transmethylase mutation of people
Detection to judge human body whether suffer from related disease have certain impulse.
Summary of the invention
It is an object of that present invention to provide the mutation of tRNA (guanine-N (7))-transmethylase and applications.
Technical solution of the present invention includes:
In a first aspect, providing tRNA (guanine-N (7))-transmethylase mutain of people a kind of, amino acid sequence
Column are as shown in SEQ ID NO:1.
Second aspect provides a kind of coding base of the mutain of tRNA (guanine-N (7))-transmethylase of people
Cause, nucleotide sequence is as shown in SEQ ID NO:2.
The third aspect provides a kind of genetic chip, comprising: solid phase carrier and fixed nucleotide probe on this carrier；
The nucleotide probe is according to the nucleotides sequence of tRNA (guanine-N (7))-transmethylase mutain of people described above
Column are determined with the comparison result of the nucleotide sequence of tRNA (guanine-N (7)) normal albumen of-transmethylase of people.
Preferably, the nucleotide probe is base sequence shown in SEQ ID NO:3；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:4；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:5.
Fourth aspect provides tRNA (guanine-N (7))-transmethylase mutain of specific recognition people a kind of
Monoclonal antibody, can be in conjunction with amino acid sequencespecific shown in SEQ ID NO:1.
5th aspect provides a kind of resisting for tRNA (guanine-N (7))-transmethylase mutain for detecting people
The ELISA kit of body, the ELISA kit include: the ELISA ELISA Plate for being coated with said monoclonal antibody, enzyme mark two
Anti-, tRNA (guanine-N (7))-transmethylase of test object, sample diluting liquid, coating buffer, ELISA ELISA Plate are washed
Wash liquid, developing solution and terminate liquid.
Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeled.
The present invention provides the mutation of tRNA (guanine-N (7))-transmethylase and applications, by a large amount of lung cancer, leukaemia
Patient carries out genetic test to case and analyzes as research case, determines that tRNA (guanine-N (7))-methyl of people turns
The mutain for moving enzyme, prepares genetic chip, list according to tRNA (guanine-N (7))-transmethylase mutain of the people
Clonal antibody and ELISA kit, the gene diagnosis for lung cancer, leukaemia provide impulse, realize the diagnosis of related disease
And treatment.
The above description is only an overview of the technical scheme 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, the following is a detailed description of the preferred embodiments of the present invention and the accompanying drawings.
Detailed description of the invention
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 diagram 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 diagram that the embodiment of the present invention six provides.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment one, genetic chip
It is possible, firstly, to 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 tRNA (guanine-N (7))-methyl transfer of people
The encoding gene of enzyme mutant albumen, nucleotide sequence is as shown in SEQ ID NO:2；Correspondingly, it is determined according to the encoding gene
TRNA (guanine-N (7))-transmethylase mutain of people out, amino acid sequence is as shown in SEQ ID NO:1.
Secondly, according to tRNA (guanine-N (7))-transmethylase mutain and its encoding gene of people, according to such as
The preparation of under type realization genetic chip.
1, the design of nucleotide probe
(1) design of nucleotide probe: nucleotide probe is prominent according to tRNA (guanine-N (7))-transmethylase of people
The white nucleotide sequence of a kink of preserved egg, the ratio with the nucleotide sequence of tRNA (guanine-N (7)) normal albumen of-transmethylase of people
Result is determined, and according to the design principle of following probe, tRNA (guanine-N (7))-methyl designed for people turns
Move the nucleotide probe of the specificity of enzyme mutant albumen.
Wherein, the principle of nucleotide probe design is as follows:
1. nucleotide probe Tm value should be close to the average Tm value of whole gene group, 5 DEG C of fluctuation up and down；
2. the duplicate single base of nucleotide probe intramolecular is continuously no more than 4；
3. G+C content is 40%-60%, the specificity that non-specific hybridization guarantees 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 guarantee 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, tRNA (guanine-N (7))-corresponding amino acid sequence of transmethylase mutain of people and people
The comparison result of tRNA (guanine-N (7)) corresponding amino acid sequence of the normal albumen of-transmethylase referring to FIG. 1, wherein,
Query sequence in Fig. 1 is tRNA (guanine-N (7))-corresponding amino acid sequence of transmethylase mutain of people,
Sbjct sequence is tRNA (guanine-N (7)) corresponding amino acid sequence of the normal albumen of-transmethylase of people, can according to Fig. 1
Know, tRNA (guanine-N (7))-transmethylase mutain of people turns relative to tRNA (guanine-N (7))-methyl of people
The normal albumen of enzyme is moved, has amino acid sequence at one to be inserted into.According to nucleotide sequence shown in SEQ ID NO:2, and
The comparison result of Fig. 1, in order to specific recognition object to be detected tRNA (guanine-N (7))-transmethylase whether
It is mutated, then when choosing nucleotide probe nucleosides can be designed according to any mode in following several modes
Acid 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, it the nucleotide sequence according to shown in SEQ ID NO:2 and is set according to above-mentioned nucleotide probe
Principle is counted, at least can be designed that following several preferably nucleotide probes in the manner described above:
The nucleotide probe as shown in SEQ ID NO:3 are as follows: ttggggtgag ttgggat；
The nucleotide probe as shown in SEQ ID NO:4 are as follows: tttgcctatg caggg；
The nucleotide probe as shown in SEQ ID NO:5 are as follows: gtgcatttag ggact.
(2) above-mentioned designed nucleotide probe the synthesis of nucleotide probe: is synthesized by nucleotide sequence.
2, the preparation for the genetic chip whether tRNA (guanine-N (7))-transmethylase of detection people mutates
In order to guarantee the quality of test object sample, blank control, positive control and yin need to be also designed on genetic chip
Property control.Wherein, the layout of the genetic chip can be at least 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: being 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, negative internal reference probe sequence needed for genetic chip can be with are as follows:
tgatgctgat aattgcat。
Positive internal control Quality Control probe: being one section of other genetic fragment for having homology with detection gene, in the tRNA of people
In (guanine-N (7))-transmethylase mutain, select sequence corresponding from nucleotide probe different, and can be with core
The number of thuja 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 sun
Property 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
Probe.In the embodiment of the present invention, positive internal control probe sequence needed for genetic chip can be with are as follows: gtctggagat ccgggtg.
It should be noted that clicking and entering negative internal reference in the deposition process of genetic chip according to the layout of genetic chip and visiting
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 pipe for taking DEPC to handle, as detected sample processing tube, is added test object in pipe
300 μ L of blood, add 700 μ L of Trizol, mix well, be placed at room temperature for 10min.
(3) chloroform of 140 μ L is added, covers tightly pipe lid, firmly shakes, be placed at room temperature for 3-5min, be placed in a centrifuge,
12000r/min, 4 DEG C of centrifugation 15min, it is careful to draw supernatant in centrifuge tube, the supernatant of absorption is transferred to clean centrifuge tube
In.
(4) isometric isopropanol is added in the centrifuge tube of supernatant in storing step (3), it is abundant gently overturns centrifuge tube
Liquid is mixed, 10min, 12000r/min, 4 DEG C of centrifugation 15min is placed at room temperature for, carefully sucks all supernatants.
(5) it is cleaned once with the ethyl alcohol of 1mL 75%, 7500r/min is centrifuged 15min, all supernatants is carefully sucked, super
10 μ L DEPC processing water dissolution is added in dry 15min in net platform.
(6) products therefrom will affect the labeling effciency and chip hybridization knot of probe if the purity of total serum IgE is not high for RNA
Fruit.So using QIAGENKit purifies total serum IgE.
2, the first chain 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 |
| T7Promotorprimer | 5μL |
| RNase-freeWater | XμL |
| Total volume | 11.5μL |
Wherein, the additional amount X μ L of RNase-free Water is to subtract T7 Promotor according to 11.5 μ L of total volume
The 5 μ L of additional amount of primer, then subtract the additional amount 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 system is 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 is added after mixing after being denaturalized in the RNA of ice bath.
(5) it is centrifuged after being mixed with pipette tips.
(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, note: 40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcription mix；
(1) Transcriptionmix is configured
(2) 55 μ L 2 × GEx Hybridization Buffer are 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 purifies cRNA, and specific method can be found in what QIAGEN company provided with kit
Operation manual.
(1) 20 μ LRNase free water are added, 350 μ L Buffer RLT are added and mix well.
(2) 250 μ L dehydrated alcohols are added, Tip mix well.
(3) total 700 solution of the μ L containing total serum IgE are transferred to cover in the RNeasy pillar in 2mL centrifuge tube >=8000g from
Heart 15-30s, discards filtered solution.
(4) draw 500 μ L Buffer RPE to RNeasy mini pillars it is interior >=8000g centrifuge washing 15-30s discards
Filtered solution again with 500 μ L Buffer RPE the casing that >=8000g centrifuge washing 2min discards filtered solution and 2mL will
RNeasymini pillar is transferred in a new 1.5mL Eppendorf pipe.
(5) water for drawing 30 μ L RNase free stands 1min, >=8000g centrifugation elution 1min.
(6) it is primary that step (5) are repeated.
5, cRNA concentration mensuration
With spectrophotometric analysis cRNA concentration.Need 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 can also) close to 2.0.
6, cRNA fluorescent marks；
(1) it takes above-mentioned cRNA4 μ g and is concentrated into 6.6 μ L.
(2) plus 10 μ L DMSO are mixed.
(3) add the sodium bicarbonate (NaHCO that the 0.3M pH of 3.4 μ L is 9.03) and mix.
(4) above-mentioned 20 μ L cRNA mixture is added in fluorescent dye Cy3 and is mixed.25 DEG C of heat preservation 1h.
(5) 25 DEG C of heat preservation 15min after adding the 4M Hydroxylamine of 9 μ L to mix.
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 fragment 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 fluorescence | 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 of rollings hybridize 16h.
9, chip washs
Washing lotion 1 (1L) configuration:
| DEPC-H2O | 700mL |
| 20×SSPE | 300mL |
| 20%N-Lauroylsarcosine | 0.25mL |
Washing lotion 2 (1L) configuration:
| 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 1min (37 DEG C) in washing lotion 2 again；
(3) chip is finally washed into 30s in washing lotion 3.
10, chip scanning
Chip is scanned in scanner, the tRNA (guanine-N of test object is determined according to scanning result
(7)) whether-transmethylase is mutated.Please refer to Fig. 3 and Fig. 4, in result shown in Fig. 3, negative control redgreen is glimmering
Light, positive control are green fluorescence, show that the sample quality of acquisition testing object is that there is no problem, and experimental group is that green is glimmering
Light, then showing that tRNA (guanine-N (7))-transmethylase of test object is mutated.In result shown in Fig. 4,
Negative control is colorless fluorescent, and positive control is green fluorescence, shows that the sample quality of acquisition testing object is that there is no problem,
And experimental group redgreen fluorescence, then showing that tRNA (guanine-N (7))-transmethylase of test object does not mutate.
Embodiment three, specific recognition people tRNA (guanine-N (7))-transmethylase mutain monoclonal it is anti-
The preparation of body
1, according to the base sequence of the tRNA of people (guanine-N (7))-transmethylase mutain (such as SEQ ID NO:
Shown in 2) upstream primer is designed as shown in SEQ ID NO:6, and, downstream primer is as shown in SEQ ID NO:7:
Upstream primer (F): gtggaactgt caccgctg
Downstream primer (R): gctggtttgg gaggtcac
2, the DNA of test 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 |
PCR amplification is carried out by template of the DNA of test object, obtains tRNA (guanine-N (7))-transmethylase of people
The complete segment of mutating protein gene, and pMD19-T Vector (Takara company) is connected, it is sequenced.Then by special life
Object corporation is a kind of humanization or Chimeric antibodies for antibody.Wherein, the monoclonal antibody prepared can be with SEQ ID NO:
Amino acid sequencespecific shown in 1 combines.The antibody of preparation is measured into content using ELISA method.
The antibody of example IV, tRNA (guanine-N (7))-transmethylase mutain for detecting people
ELISA kit
In the present embodiment, the composition of ELISA kit are as follows: be coated with the ELISA enzyme of monoclonal antibody described in embodiment three
Target, ELIAS secondary antibody, tRNA (guanine-N (7))-transmethylase of test object, sample diluting liquid, coating buffer,
ELISA ELISA Plate cleaning solution, developing solution and terminate liquid.
Wherein, the parameter of above-mentioned each composition can be at least a kind of following parameter:
ELISA ELISA Plate can be the ELISA enzyme mark in 96 holes；
ELIAS secondary antibody can be the Goat anti-Human IgG of diluted HRP (horseradish peroxidase) label；Wherein, dilution times
Number can be 8000 times.
Coating buffer can be 1 × PBS, pH:7.4；
ELISA ELISA Plate 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, using patients with lung cancer as test object, and the lung cancer is detected using ELISA kit and is suffered from
Whether tRNA (guanine-N (7))-transmethylase 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, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plate, it is incubated at room temperature 1-2h.
B, tRNA (guanine-N (7))-transmethylase of patients with lung cancer is diluted to using sample diluting liquid different dense
Gradient is spent, and the tRNA of various concentration gradient (guanine-N (7))-transmethylase is loaded respectively to ELISA ELISA Plate
Kong Zhong, and it is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plate 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 solution is added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated for 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, make standard curve: using standard concentration as abscissa, the light absorption value of standard items measurement is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measurement is 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 software2=0.99863, therefore, this
Measurement is effective.The wavelength that will test, which substitutes into standard curve for the light absorption value at 450nm, can acquire the tRNA of patients with lung cancer
The concentration of (guanine-N (7))-transmethylase.Utilize tRNA in the ELISA method detection Serum of Patients with Lung Cancer sample of foundation
The content of (guanine-N (7))-transmethylase.And it is identified with Westernblot.Referring to FIG. 5, being the mark of light absorption value
Directrix curve.Wherein, X axis is light absorption value, 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)
| 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 with filter paper after gelling to be separated is solid, upper layer is then added, glue is concentrated, after being inserted into comb
Wait upper layer concentration gelling solid.
2, albumen loading electrophoresis:
(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffer 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 voltage, after running concentration glue to band, uses 100V voltage 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.It is clipped by filter paper-glue-film-filter paper sequence, is put into transferring film device according to principle of the black flour to black flour
In, it is added refrigerator (ice bag), about 120min is run with the electric current of 150mA in ice；
(2) it takes the film out, is put into 5% skim milk at once after the completion of, 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 shaking tables are incubated overnight；
(5) primary antibody is recycled, PBST is cleaned film 3 times, each 10min；
(6) secondary antibody (being diluted with 3% milk with 1:5000-1:10000) is incubated at room temperature film 2h；
(7) film 3 times are cleaned with PBST, each 10min；
(8) after Pierce ECL Western Blotting Substrate kit A, B liquid mixes in equal volume, 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 that tRNA (guanine-N (7))-transmethylase mutain monoclonal of the standby people of corporation is anti-
Body, secondary antibody are the Goat anti-Human IgGs of horseradish peroxidase-labeled.
J, Western blot Analysis of test results: showing according to Western blot experimental procedure as a result, such as Fig. 6 institute
Show, wherein the Marker in Fig. 6 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 58KD
There is obvious band, wherein the molecular weight of tRNA (guanine-N (7))-transmethylase mutain is about 58KD, is shown
TRNA (guanine-N (7))-transmethylase of the patients with lung cancer is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, using leukaemic as test object, and the white blood is detected using ELISA kit
Whether tRNA (guanine-N (7))-transmethylase of patient is mutated, and this method at least may include as next
Kind:
A, monoclonal antibody prepared by embodiment three is diluted using the coating buffer, for example, being diluted to
2.0ug/mL, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plate, it is incubated at room temperature 1-2h；
B, it is diluted to using tRNA (guanine-N (7))-transmethylase that sample diluting liquid will test object different dense
Gradient is spent, and the tRNA of various concentration gradient (guanine-N (7))-transmethylase is loaded respectively to ELISA ELISA Plate
Kong Zhong, and it is incubated at room temperature 1-2h；
C, the liquid in each hole is got rid of, the ELISA ELISA Plate 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 solution is added, washs and dries；
F, the developing solution is added, room temperature, which is protected from light, is incubated for 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, make standard curve: using standard concentration as abscissa, the light absorption value of standard items measurement is ordinate, makees bid
Directrix curve；Calculate standard curve regression coefficient R2, work as R2This measurement is 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 software2=0.99948, therefore, this
Measurement is effective.The wavelength that will test is that the light absorption value substitution standard curve at 450nm can acquire leukaemia trouble in sample
The concentration of tRNA (guanine-N (7))-transmethylase of person.Serum of leukaemia is detected using the ELISA method of foundation
The content of tRNA (guanine-N (7))-transmethylase in sample.And it is identified with Westernblot.Referring to FIG. 7, being
The standard curve of light absorption value.Wherein, X-axis is light absorption value, and Y-axis is corresponding concentration.
I, Western blot is identified
J, Western blot Analysis of test results
Wherein, step i and step j is 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 obvious band in 58KD, wherein the molecular weight of tRNA (guanine-N (7))-transmethylase mutain is about
58KD shows that tRNA (guanine-N (7))-transmethylase of the leukaemic 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>mutation of tRNA (guanine-N (7))-transmethylase and application
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 233
<212> PRT
<213>species home sapiens (Homo sapiens)
<400> 1
Val Glu Leu Ser Pro Leu Phe Pro Asp Thr Leu Ile Leu Gly Leu Glu
1 5 10 15
Ile Arg Val Lys Val Ser Asp Tyr Val Gln Asp Arg Ile Arg Ala Leu
20 25 30
Arg Ala Ala Pro Ala Gly Gly Phe Gln Asn Ile Ala Cys Leu Arg Ser
35 40 45
Asn Ala Met Lys His Leu Pro Asn Phe Phe Tyr Lys Gly Gln Leu Thr
50 55 60
Lys Met Phe Phe Leu Phe Pro Asp Pro His Phe Lys Arg Thr Lys His
65 70 75 80
Lys Trp Arg Ile Ile Ser Pro Thr Leu Leu Ala Glu Tyr Ala Tyr Val
85 90 95
Leu Arg Val Gly Val Ser Trp Asp Arg Arg Asp Asp Gly Val Ser Gly
100 105 110
Leu Leu Gly Ala Leu His Glu Phe Ser Gly Gly Ala Phe Arg Asp Ser
115 120 125
His His His Phe Leu His Leu Ala Ala Asp Val Ala Cys Ser Leu Leu
130 135 140
Ser Arg Val Ile Leu Pro Met Gln Gly Leu Val Tyr Thr Ile Thr Asp
145 150 155 160
Val Leu Glu Leu His Asp Trp Met Cys Thr His Phe Glu Glu His Pro
165 170 175
Leu Phe Glu Arg Val Pro Leu Glu Asp Leu Ser Glu Asp Pro Val Val
180 185 190
Gly His Leu Gly Thr Ser Thr Glu Glu Gly Lys Lys Val Leu Arg Asn
195 200 205
Gly Gly Lys Asn Phe Pro Ala Ile Phe Arg Arg Ile Gln Asp Pro Val
210 215 220
Leu Gln Ala Val Thr Ser Gln Thr Ser
225 230
<210> 2
<211> 702
<212> DNA
<213>species home sapiens (Homo sapiens)
<400> 2
gtggaactgt caccgctgtt cccagacaca cttattctgg gtctggagat ccgggtgaag 60
gtctcagact atgtacaaga ccggattcgg gccctacgcg cagctcctgc aggtggcttc 120
cagaacatcg cctgtctccg tagcaatgcc atgaagcacc ttcctaactt cttctacaag 180
ggccagctga caaagatgtt cttcctcttc cccgacccac atttcaagcg gacaaagcac 240
aagtggcgaa tcatcagtcc caccctgcta gcagaatatg cctacgtgct aagagttggg 300
gtgagttggg atcggaggga tgatggggtg agtggcctac tcggggccct tcactaagaa 360
ttcagtgggg gtgcatttag ggactcacac caccacttcc ttcatctggc tgctgacgtt 420
gcatgcagcc tcctgtctag ggtgattttg cctatgcagg ggctggtgta taccataacc 480
gatgtgctgg agctacacga ctggatgtgc actcatttcg aagagcaccc actgtttgag 540
cgtgtgcctc tggaggacct gagtgaagac cccgttgtgg gacatctagg cacctcaact 600
gaggagggga agaaagttct acgtaatgga gggaagaatt tcccagccat cttccgaaga 660
atacaagatc ccgtcctcca ggcagtgacc tcccaaacca gc 702
<210> 3
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ttggggtgag ttgggat 17
<210> 4
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tttgcctatg caggg 15
<210> 5
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gtgcatttag ggact 15
<210> 6
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gtggaactgt caccgctg 18
<210> 7
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gctggtttgg gaggtcac 18
Claims (7)
1. a kind of tRNA of people (guanine-N (7))-transmethylase mutain, which is characterized in that its amino acid sequence is such as
Shown in SEQ ID NO:1.
2. a kind of encoding gene of the tRNA of people (guanine-N (7))-transmethylase mutain, which is characterized in that its core
Nucleotide sequence is as shown in SEQ ID NO:2.
3. a kind of genetic chip characterized by comprising solid phase carrier and fixed nucleotide probe on this carrier；It is described
The nucleotides sequence of nucleotide probe tRNA (guanine-N (7))-transmethylase mutain of people according to claim 2
Column are determined with the comparison result of the nucleotide sequence of tRNA (guanine-N (7)) normal albumen of-transmethylase of people.
4. genetic chip according to claim 3, which is characterized in that
The nucleotide probe is base sequence shown in SEQ ID NO:3；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:4；
Or, the nucleotide probe is base sequence shown in SEQ ID NO:5.
5. a kind of monoclonal antibody of the tRNA of specific recognition people (guanine-N (7))-transmethylase mutain, special
Sign is, can be in conjunction with amino acid sequencespecific shown in SEQ ID NO:1.
6. a kind of ELISA reagent of the antibody of tRNA (guanine-N (7))-transmethylase mutain for detecting people
Box, which is characterized in that the ELISA kit include: the ELISA ELISA Plate for being coated with monoclonal antibody described in claim 5,
ELIAS secondary antibody, tRNA (guanine-N (7))-transmethylase of test object, sample diluting liquid, coating buffer, ELISA enzyme
Target cleaning solution, developing solution and terminate liquid.
7. according to claim 6 for detecting the antibody of tRNA (guanine-N (7))-transmethylase mutain of people
ELISA kit, which is characterized in that the ELIAS secondary antibody be diluted HRP horseradish peroxidase-labeled Goat anti-Human
IgG。
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810747677.XA CN109022384A (en) | 2018-07-10 | 2018-07-10 | The mutation of tRNA (guanine-N (7))-transmethylase and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810747677.XA CN109022384A (en) | 2018-07-10 | 2018-07-10 | The mutation of tRNA (guanine-N (7))-transmethylase and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109022384A true CN109022384A (en) | 2018-12-18 |
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ID=64641541
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810747677.XA Pending CN109022384A (en) | 2018-07-10 | 2018-07-10 | The mutation of tRNA (guanine-N (7))-transmethylase and application |
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| Country | Link |
|---|---|
| CN (1) | CN109022384A (en) |
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2018
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