CN108546293A - - 36 mutain of sperm antigen of people a kind of and its application - Google Patents
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Abstract
The present invention relates to 36 mutain of sperm antigen of people a kind of and its applications, by regarding a large amount of disease of immune system patients as research case, genetic test is carried out to case and is analyzed, determine the mutain of the sperm antigen 36 of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mutain of the sperm antigen 36 of the people, impulse is provided for the gene diagnosis of disease of immune system, and certain theoretical foundation is provided for the diagnosing and treating of disease.
Description
Technical field
The present invention relates to -36 mutain of sperm antigen of a kind of genetic engineering field more particularly to a kind of people and its answer
With.
Background technology
Sperm antigen ingredient is sufficiently complex, and sperm antigen -36 (sperm antigen-36) is exactly one of which.Sperm is anti-
Original further includes itself structural antigens, blood group antigens tissue compatible in addition to a variety of glycoprotein antigens for being present in sperm film surface
A variety of enzyme antigens etc. inside property antigen and sperm.
The sperm antigen gene mutation of people can cause to seriously affect to human health.To certain immune system diseases
Disease finds that the sperm antigen -36 of patient occurs during lupus erythematosus and immunity nephrotic progress gene sequencing
Mutation, therefore, the detection being mutated to the sperm antigen -36 of people is to judging whether human body has centainly with disease of immune system
Impulse.
Invention content
Present invention aims at -36 mutain of sperm antigen of people of offer a kind of and its applications.
Technical solution of the present invention includes：
In a first aspect, providing a kind of -36 mutain of sperm antigen of people, amino acid sequence such as SEQ ID NO:1 institute
Show.
Second aspect provides a kind of encoding gene of -36 mutain of sperm antigen 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 -36 mutain of sperm antigen of the people, just with the sperm antigen -36 of people
The comparison result of the nucleotide sequence of normal albumen determines.
Preferably, the nucleotide probe is SEQ ID NO:Base sequence shown in 3.
Fourth aspect provides a kind of monoclonal antibody of -36 mutain of sperm antigen of specific recognition people, coding
Amino acid sequence be SEQ ID NO:Shown in 1.
5th aspect provides a kind of ELISA kit for detecting the antibody of -36 mutain of sperm antigen of people,
The ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, the sperm for detecting object of said monoclonal antibody
- 36 albumen of antigen, 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 mutation egg of sperm antigen -36 detecting people based on any of the above-described ELISA kit
The method of white antibody, 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, incubation at room temperature；
B, -36 albumen of sperm antigen for detecting object is diluted to various concentration gradient using the sample diluting liquid, and
- 36 albumen of sperm antigen of various concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, 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, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
Preferably, further comprise：Blank control is tested.
The present invention provides -36 mutain of sperm antigen of people a kind of and its applications, and a large amount of disease of immune system are suffered from
Person carries out genetic test to case and analyzes, determine the mutain of the sperm antigen -36 of people as research case, according to
- 36 mutain of sperm antigen of the people prepares genetic chip, monoclonal antibody and ELISA kit, is disease of immune system
Gene diagnosis provide impulse, provide certain theoretical foundation for the diagnosing and treating of 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 another comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 3 is another comparison result schematic diagram that the embodiment of the present invention one provides；
Fig. 4 is a kind of genetic chip layout that the embodiment of the present invention one provides；
Fig. 5 is a kind of colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 6 is another colour developing result schematic diagram provided by Embodiment 2 of the present invention；
Fig. 7 is the standard curve that the offer of the embodiment of the present invention five is protein content；
Fig. 8 is the Westernblot testing result schematic diagrames that the embodiment of the present invention five provides；
Fig. 9 is the standard curve that the offer of the embodiment of the present invention six is protein content；
Figure 10 is the Westernblot 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 coding base of -36 mutain of sperm antigen of people
Cause, nucleotide sequence such as SEQ ID NO:Shown in 2；Correspondingly, determine that the sperm antigen -36 of people is prominent according to the encoding gene
Become albumen, amino acid sequence such as SEQ ID NO:Shown in 1.
Secondly, according to -36 mutain of sperm antigen and its encoding gene of people, genetic chip is realized as follows
Preparation.
1, the design of nucleotide probe
(1) design of nucleotide probe：Nucleotide probe is according to the nucleotides sequence of -36 mutain of sperm antigen of people
Row determine with the comparison result of the nucleotide sequence of the normal albumen of sperm antigen -36 of people, and according to the design of following probe
Principle designs the nucleotide probe of the specificity of -36 mutain of sperm antigen 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 -36 mutain of sperm antigen of people and the normal albumen of sperm antigen -36 of people
The comparison result of corresponding amino acid sequence please refers to Fig.1, wherein the Query sequences in Fig. 1 are that the sperm antigen -36 of people is prominent
Becoming the corresponding amino acid sequence of albumen, Sbjct sequences are the corresponding amino acid sequences of the normal albumen of sperm antigen -36 of people,
Sequence between Query sequences and Sbjct sequences is comparison result, as can be seen from FIG. 1, -36 mutain of sperm antigen of people
The normal albumen of sperm antigen -36 relative to people, has partial amino-acid series to be mutated.According to SEQ ID NO:Shown in 2
Nucleotide sequence and Fig. 1 comparison result, in order to specific recognition object to be detected sperm antigen -36 whether
Be mutated, then when choosing nucleotide probe, can by include mutated site nucleotide a nucleotide sequence
As 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, designs one kind preferably nucleotide probe such as SEQ ID NO in the manner described above:Shown in 3, which is：
aagggtacac aggcaaga
(2) synthesis of nucleotide probe：Above-mentioned designed nucleotide probe is synthesized by nucleotide sequence.
When -36 mutain of sperm antigen of people is compared in NCBI, the G-6-P of macaque in comparison
The normal albumen of isomerase, comparison result is referring to FIG. 2, Query sequences are -36 mutains pair of sperm antigen of people in Fig. 2
The amino acid sequence answered, Sbjct sequences are the corresponding amino acid sequences of the normal albumen of glucose-6-phosphate isomerase of macaque,
Sequence between Query sequences and Sbjct sequences is comparison result.
Meanwhile it during comparing -36 mutain of sperm antigen of people in NCBI, obtaining and East Africa baboon
The normal albumen of glucose-6-phosphate isomerase comparison result, referring to FIG. 3, Query sequences are that the sperm of people is anti-in Fig. 3
The corresponding amino acid sequence of -36 mutains of original, Sbjct sequences are the normal eggs of glucose-6-phosphate isomerase of East Africa baboon
White corresponding amino acid sequence, the sequence between Query sequences and Sbjct sequences are comparison result.
2, the preparation for the genetic chip whether sperm antigen -36 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 4.In Fig. 4, 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 number that negative internal reference Quality Control probe includes can be with nucleotide probes
Base number is identical, can also be different.In the embodiment of the present invention, the negative internal reference probe sequence needed for genetic chip can be：
tgatgctgat aattgcat。
Positive internal control Quality Control probe：It is one section of other genetic fragment for having homology with detection gene, it is anti-in the sperm of people
In former -36 mutains, select sequence corresponding from nucleotide probe different, and can be with the number phase of nucleotide probe base
Together, one section of nucleotide sequence that can also be different from the number of nucleotide probe base is as positive internal control Quality Control probe.It is preferred that
Nucleotide number positive internal control Quality Control probe identical with the number of nucleotide probe base is chosen on ground.In the embodiment of the present invention,
Positive internal control probe sequence needed for genetic chip can be：gatgccaaca aggaccgc.
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 at room temperature for 3-5min, be placed in a centrifuge,
12000r/min, 4 DEG C of centrifugation 15min, carefully draws supernatant in centrifuge tube, the supernatant of absorption is transferred to clean centrifuge tube
In.
(4) isometric isopropanol is added in the centrifuge tube of supernatant in storing step (3), it is abundant gently overturns centrifuge tube
Mixing liquid is placed at room temperature for 10min, 12000r/min, 4 DEG C of centrifugation 15min, carefully sucks all supernatants.
(5) it 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 |
| T7 Promotor primer | 5μL |
| RNase-free Water | XμL |
| Total volume | 11.5μL |
Wherein, the addition X μ L of RNase-free Water are to subtract 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, and 5 × First Strand B μ ffer are preheated at 65 DEG C
5min。
(3) following cDNA synthetic systems are configured：
| 5×First Strand Buffer | 4μL |
| 0.1M DTT | 2μL |
| 10mM dNTP mix | 1μL |
| MMLV RT | 1μL |
| RNase OUT | 0.5μL |
| Total volume | 8.5μL |
(4) above-mentioned 8.5 μ L are added after mixing after being denaturalized in the RNA of ice bath.
(5) pipette tips mixing is used to centrifuge later.
(6) 40 DEG C of reaction 2h.
3, aaUTP marks cRNA synthesis
The configuration of NTP：
| 100mM ATP | 250μL |
| 100mM GTP | 250μL |
| 100mM CTP | 250μL |
| 100mM UTP | 187.5μL |
| RNase free H2O | 62.5μL |
| Total volume | 1000μL |
It is spare to be distributed into 10 pipes, notes：40 DEG C of heat preservation 1min before 50%PEG (polyethylene glycol) use.Simultaneously by following behaviour
Make configuration Transcriptionmix；
(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 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
Cross liquid discard the casing of filtered solution and 2mL in >=8000g centrifuge washings 2min with 500 μ L Buffer RPE again will
RNeasymini 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) above-mentioned cRNA4 μ g 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 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 |
| 10×Blocking Agent | 11μL |
| 25×Fragmentation Buffer | 2.2μL |
| Nuclease-free water | XμL |
| Total volume | 55μL |
(2) 55 μ 2 × GEx of L HybridizationBuffer 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 4 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, determines that -36 albumen of sperm antigen of detection object is according to scanning result
It is no to be mutated.Please refer to Fig. 5 and Fig. 6, in result shown in fig. 5, 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 detection pair
- 36 albumen of sperm antigen of elephant is mutated.In result shown in fig. 6, negative control is colorless fluorescent, and positive control is green
Color fluorescence shows that the sample quality of acquisition testing object is that there is no problem, and experimental group redgreen fluorescence, then showing to detect
- 36 albumen of sperm antigen of object does not mutate.
Embodiment three, specific recognition people -36 mutain of sperm antigen monoclonal antibody preparation
1, according to base sequence (such as SEQ ID NO of -36 mutain of the sperm antigen of people:Shown in 2) design upstream draw
Object such as SEQ ID NO:Shown in 4, and, downstream primer such as SEQ ID NO:Shown in 5：
Sense primer (F)：atggccgctc tcacccgg
Downstream primer (R)：tcttgcctgt gtaccctt
2, the DNA of detection object is that template carries out PCR amplification
| 10×Buffer | 5uL |
| dNTP | 2uL |
| Ex Taq | 1uL |
| ddH2O | 5uL |
| Template DNA | 1uL |
| Primer (F) | 3uL |
| Primer (R) | 3uL |
| Total system | 20uL |
DNA to detect object carries out PCR amplification as template, and -36 mutating protein gene of sperm antigen for obtaining people is complete
Segment, and pMD19-TVector (Takara companies) is connected, it is sequenced.Then antibody is prepared by special biotech firm, is
A kind of humanization or Chimeric antibodies.The antibody of preparation is measured into content using ELISA method.
Example IV, -36 mutain of sperm antigen 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, -36 albumen of sperm antigen for detecting object, sample diluting liquid, coating buffer solution, the washing of ELISA ELISA Plates
Liquid, 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, using patients with SLE as detection object, and it is red using ELISA kit to detect this
Whether -36 albumen of sperm antigen of spot lupus 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, -36 albumen of sperm antigen of patients with SLE is diluted to various concentration gradient using sample diluting liquid, and
- 36 albumen of sperm antigen 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=0.99907, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire patients with SLE
The concentration of -36 albumen of sperm antigen.Utilize sperm antigen-in the ELISA method detection patients with SLE blood serum sample of foundation
The content of 36 albumen.It is used in combination Western blot to be identified.Referring to FIG. 7, for the standard curve of light absorption value.Wherein, X-axis is
Light absorption value, Y-axis are 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)
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；
(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 -36 mutain monoclonal antibody of sperm antigen of the standby people of corporation, and secondary antibody is horseradish peroxide
The Goat anti-Human IgG of compound enzyme label.
J, Western blot Analysis of test results：It is shown according to Western blot experimental procedures as a result, such as Fig. 8 institutes
Show, wherein the Marker in Fig. 8 is used to characterize the size of labelled protein.Compared with blank control, only near experimental group 36KD
There is apparent band, wherein -36 mutant protein molecules amount of sperm antigen is about 36KD, shows the essence of the patients with SLE
- 36 albumen of sub- antigen is implicitly present in mutation.
Embodiment six
In embodiments of the present invention, using immunity nephrotic as detection object, and should using ELISA kit detection
Whether -36 albumen of sperm antigen of immunity nephrotic 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, -36 albumen of sperm antigen for detecting object is diluted to various concentration gradient using sample diluting liquid, and will not
- 36 albumen of sperm antigen with 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=0.99854, therefore, this
It measures effective.The wavelength that detection obtains is substituted into standard curve for the light absorption value at 450nm can acquire immunity kidney in sample
The concentration of -36 albumen of sperm antigen of patient.Using in ELISA method detection immunity nephrotic's blood serum sample of foundation
The content of -36 albumen of sperm antigen.It is used in combination Westernblot to be identified.Referring to FIG. 9, for the standard curve of light absorption value.Its
In, X-axis is light absorption value, and Y-axis is corresponding concentration.
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 36KD, wherein -36 mutant protein molecules amount of sperm antigen is about 36KD, shows the immunity kidney
- 36 albumen of sperm antigen of patient 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>- 36 mutain of sperm antigen of people a kind of and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 147
<212> PRT
<213>Species home sapiens (Homo sapiens)
<400> 1
Met Ala Ala Leu Thr Arg Asp Pro Gln Phe Gln Lys Leu Gln Gln Trp
1 5 10 15
Tyr Arg Glu His Arg Ser Glu Leu Asn Leu Arg Arg Leu Phe Asp Ala
20 25 30
Asn Lys Asp Arg Phe Asn His Phe Ser Leu Thr Leu Asn Thr Asn His
35 40 45
Gly His Ile Leu Val Asp Tyr Ser Lys Asn Leu Val Thr Glu Asp Val
50 55 60
Met Arg Met Leu Val Asp Leu Ala Lys Ser Arg Gly Val Glu Ala Ala
65 70 75 80
Arg Glu Arg Met Phe Asn Gly Glu Lys Ile Asn Tyr Thr Glu Gly Arg
85 90 95
Ala Val Leu His Val Ala Leu Arg Asn Arg Ser Asn Thr Pro Ile Leu
100 105 110
Val Asp Gly Lys Asp Val Met Pro Glu Val Asn Lys Val Leu Asp Lys
115 120 125
Met Lys Ser Phe Cys Gln Arg Val Arg Ser Gly Asp Trp Lys Gly Thr
130 135 140
Gln Ala Arg
145
<210> 2
<211> 441
<212> DNA
<213>Species home sapiens (Homo sapiens)
<400> 2
atggccgctc tcacccggga cccccagttc cagaagctgc agcaatggta ccgcgagcac 60
cgctccgagc tgaacctgcg ccgcctcttc gatgccaaca aggaccgctt caaccacttc 120
agcttgaccc tcaacaccaa ccatgggcat atcctggtgg attactccaa gaacctggtg 180
acggaggacg tgatgcggat gctggtggac ttggccaagt ccaggggcgt ggaggccgcc 240
cgggagcgga tgttcaatgg tgagaagatc aactacaccg agggtcgagc cgtgctgcac 300
gtggctctgc ggaaccggtc aaacacaccc atcctggtag acggcaagga tgtgatgcca 360
gaggtcaaca aggttctgga caagatgaag tctttctgcc agcgtgtccg gagcggtgac 420
tggaagggta cacaggcaag a 441
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
aagggtacac aggcaaga 18
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggccgctc tcacccgg 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
tcttgcctgt gtaccctt 18
Claims (9)
1. a kind of mutain of the sperm antigen -36 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 -36 mutain of sperm antigen 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
Nucleotide probe is according to the nucleotide sequence of -36 mutain of sperm antigen of the people, the normal egg of sperm antigen -36 with people
The comparison result of white nucleotide sequence 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 -36 mutain of sperm antigen of specific recognition people, which is characterized in that its ammonia encoded
Base acid sequence is SEQ ID NO:Shown in 1.
6. a kind of ELISA kit for detecting the antibody of -36 mutain of sperm antigen of people, which is characterized in that described
ELISA kit includes：It is coated with the ELISA ELISA Plates, ELIAS secondary antibody, detection object of monoclonal antibody described in claim 5
- 36 albumen of sperm antigen, sample diluting liquid, coating buffer solution, ELISA ELISA Plates cleaning solution, developing solution and terminate liquid.
7. it is used to detect the ELISA kit of the antibody of -36 mutain of sperm antigen of people according to claim 6,
It is characterized in that, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeleds.
8. a kind of antibody for -36 mutain of sperm antigen detecting people based on the ELISA kit of claim 6 or 7
Method, which is characterized in that including：
A, monoclonal antibody described in claim 5 is diluted using the coating buffer solution, and by the list after dilution
Clonal antibody is loaded into the hole of ELISA ELISA Plates, incubation at room temperature；
B, -36 albumen of sperm antigen for detecting object is diluted to various concentration gradient using the sample diluting liquid, and will not
- 36 albumen of sperm antigen with concentration gradient is loaded respectively into the hole of ELISA ELISA Plates, incubation 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, it is the light absorption value at 450nm that wavelength is measured in microplate reader.
9. the method for the antibody of -36 mutain of sperm antigen of ELISA kit detection people according to claim 8,
It is characterized in that, further comprises：Blank control is tested.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810261499.XA CN108546293A (en) | 2018-03-28 | 2018-03-28 | - 36 mutain of sperm antigen of people a kind of and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108546293A true CN108546293A (en) | 2018-09-18 |
Family
ID=63517343
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|---|---|---|---|
| CN201810261499.XA Pending CN108546293A (en) | 2018-03-28 | 2018-03-28 | - 36 mutain of sperm antigen of people a kind of and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108546293A (en) |
-
2018
- 2018-03-28 CN CN201810261499.XA patent/CN108546293A/en active Pending
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