CN109824773A - 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people is mutated and application - Google Patents

Abstract

The present invention relates to the mutation of 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people and applications, by using a large amount of liver cancer patients as research case, genetic test is carried out to case and is analyzed, determine the mutain of 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people, genetic chip, monoclonal antibody and ELISA kit are prepared according to the mutain of 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of the people, impulse is provided for the gene diagnosis of liver cancer, and certain theoretical basis is provided for the diagnosing and treating of kinds cancer.

Description

8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people is mutated and application

Technical field

The present invention relates to a kind of genetic engineering field more particularly to the 8 class eggs of epidermal growth factor receptor kinase substrate of people White 3 mutation and application.

Background technique

The formation and development of many growth factors and tumour has close relationship.EGF-R ELISA (epidermal Growth factor receptor, EGFR) itself there is tyrosine kinase activity, once it can with epidermal growth factor sub-portfolio There is correlation gene in active cell core, so that cell division be promoted to be proliferated.Epidermal growth factor recipient tyrosine kinase in recent years As the novel targets of oncotherapy, and inhibiting epidermal growth factor recipient tyrosine kinase then becomes a research for the treatment of tumour Hot spot.The epidermal growth factor receptor kinase mutation of people can cause to seriously affect to human health.Suffer to certain liver cancer During the peripheral blood of person carries out gene sequencing, 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of discovery patient is sent out Mutation is given birth to, therefore, to the detection of 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people mutation to whether judging human body There is certain impulse with associated cancer.

Summary of the invention

It is an object of that present invention to provide the mutation of 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people and applications.

Technical solution of the present invention includes:

In a first aspect, providing 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 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 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain 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 nucleosides of 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of people described above Acid sequence, it is true with the comparison result of the nucleotide sequence of the normal albumen of 8 albuminoid of epidermal growth factor receptor kinase substrate 3 of people It is fixed.

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 a kind of reagent, contains the EGF-R ELISA for detecting above-mentioned people in the reagent The primer pair of 8 albuminoid of kinase substrate, 3 mutain.

Preferably, the primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream Primer is the base sequence as shown in any in SEQ ID NO:8 ~ NO:12；Downstream primer is such as SEQ ID NO:23 ~ NO:37 In it is any shown in base sequence；

Or,

Its described primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as SEQ In ID NO:8 ~ NO:17 it is any shown in base sequence；Downstream primer is as shown in any in SEQ ID NO:28 ~ NO:37 Base sequence；

Or,

Its described primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as SEQ In ID NO:8 ~ NO:22 it is any shown in base sequence；Downstream primer is as shown in any in SEQ ID NO:33 ~ NO:37 Base sequence.

5th aspect provides 8 albuminoid 3 of the epidermal growth factor receptor kinase substrate mutation egg of specific recognition people a kind of White monoclonal antibody, is prepared by mentioned reagent, can be with amino acid sequencespecific knot shown in SEQ ID NO:1 It closes.

6th aspect, provides a kind of for detecting 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of people ELISA kit, the ELISA kit include: the ELISA ELISA Plate for being coated with said monoclonal antibody, ELIAS secondary antibody, Sample diluting liquid, coating buffer, ELISA ELISA Plate cleaning solution, developing solution and terminate liquid.

Preferably, the ELIAS secondary antibody is the Goat anti-Human IgG of diluted HRP horseradish peroxidase-labeled.

The present invention provides the mutation of 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people and applications, by a large amount of liver cancer Patient carries out genetic test to case and analyzes, determine the epidermal growth factor receptor kinase substrate 8 of people as research case The mutain of albuminoid 3 prepares gene according to 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of the people Chip, monoclonal antibody and ELISA kit provide impulse for the gene diagnosis of liver cancer, be kinds cancer diagnosis and It treats and certain theoretical basis is provided.

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.

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 according to be application No. is " 201710429915.8 ", patent name " detection method of mutain a kind of and Detection method described in the Chinese invention patent of device " determines 8 albuminoid of epidermal growth factor receptor kinase substrate of people The encoding gene of 3 mutains, nucleotide sequence is as shown in SEQ ID NO:2；Correspondingly, it is determined according to the encoding gene 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of people, amino acid sequence is as shown in SEQ ID NO:1.

Secondly, being pressed according to 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain and its encoding gene of people The preparation of genetic chip is realized according to such as under type.

1, the design of nucleotide probe

(1) design of nucleotide probe: nucleotide probe is prominent according to 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people The white nucleotide sequence of a kink of preserved egg, the nucleotide sequence with the normal albumen of 8 albuminoid of epidermal growth factor receptor kinase substrate 3 of people Comparison result determine, and according to the design principle of following probe, design the epidermal growth factor receptor kinase for people The nucleotide probe of the specificity of 8 albuminoid of substrate, 3 mutain.

Wherein, the principle of nucleotide probe design is as follows:

Nucleotide probe Tm value should be close to the average Tm value of whole gene group, 5 DEG C of fluctuation up and down；

The duplicate single base of nucleotide probe intramolecular is continuously no more than 4；

G+C content is 40%-60%, reduces the specificity that non-specific hybridization guarantees hybridization；

4 bp should be less than with the bases longs of probe molecule internal stability secondary structure pairing in nucleotide probe sequences, in this way Can guarantee that hybridization efficiency will not be influenced because of the secondary structure of nucleotide probe internal stability；

Similitude through Homology search and other sequences is less than 40%；

Continuously it is no more than 20 bases with the homologous fragment of non-probe alternative sequence by contrast.

Wherein, the corresponding amino acid sequence of 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of people and people The comparison result of the corresponding amino acid sequence of the normal albumen of 8 albuminoid 3 of epidermal growth factor receptor kinase substrate please refer to figure 1, wherein the Query sequence in Fig. 1 is the corresponding ammonia of 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of people Base acid sequence, Sbjct sequence are the corresponding amino acid of the normal albumen of 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people Sequence, the sequence between Query sequence and Sbjct sequence are comparison result, as can be seen from FIG. 1, the epidermal growth factor receptor of people Epidermal growth factor receptor kinase substrate 8 albuminoid 3 normal albumen of 8 albuminoid of body kinase substrate, 3 mutain relative to people, Some amino acid sequence is lacked, and has several places to be mutated.According to nucleotide shown in SEQ ID NO:2 The comparison result of sequence and Fig. 1, in order to the epidermal growth factor receptor kinase substrate of specific recognition object to be detected Whether 8 albuminoids 3 are mutated, then when choosing nucleotide probe, it can be according to any one of following several modes Mode designs nucleotide probe:

It will include a nucleotide sequence of mutated site nucleotide as nucleotide probe.

Several nucleotide sequence (sequences of base sequence are respectively selected before deletion sites and after deletion sites It is constant), generate 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: ttcagccaag ccagga；

The nucleotide probe as shown in SEQ ID NO:4 are as follows: aagctcacct gaga；

The nucleotide probe as shown in SEQ ID NO:5 are as follows: agaagctgga ggttccaa.

(2) above-mentioned designed nucleotide probe the synthesis of nucleotide probe: is synthesized by nucleotide sequence.

2, the system for the genetic chip whether 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of detection people mutates It is standby

In order to guarantee the quality of test object sample, it is right also blank control, positive control and feminine gender need to be designed on genetic chip According to.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 control, zero For 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 of any genetic fragment is free from as the contamination monitoring index in chip fabrication process.

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, raw in the epidermis of people In 8 albuminoid of growth factor receptor body kinase substrate, 3 mutain, select corresponding from nucleotide probe sequence different, and can be with The number of nucleotide probe base is identical, one section of nucleotide sequence conduct that can also be different from the number of nucleotide probe base Positive internal control Quality Control probe.Preferably, nucleotide number positive internal control matter identical with the number of nucleotide probe base is chosen Control probe.In the embodiment of the present invention, positive internal control probe sequence needed for genetic chip can be with are as follows: agccatttac ttgcac.

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 1-3 mL of acquisition testing object.

(2) the 1.5 mL EP pipe for taking DEPC to handle, as detected sample processing tube, in detected sample treatment The 300 μ L of blood of test object is added in pipe, adds 700 μ L of Trizol, mixes well, be placed at room temperature for 10 min.

(3) chloroform of 140 μ L is added, covers tightly pipe lid, firmly shakes, be placed at room temperature for 3-5 min, be placed in a centrifuge, 12000 r/ min, 4 DEG C of 15 min of centrifugation, it is careful to draw supernatant in centrifuge tube, the supernatant of absorption is transferred to clean centrifugation Guan Zhong.

(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, is placed at room temperature for 10 min, 12000 r/ min, 4 DEG C of 15 min of centrifugation carefully suck all supernatants.

(5) it is cleaned once with the ethyl alcohol of 1 mL 75%, 7500 r/ min, 4 DEG C of 15 min of centrifugation are carefully sucked on all Clearly, 10 μ L DEPC processing water dissolution is added in dry 15 min in super-clean bench.

(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 purifying total serum IgE using QIAGEN RNeasy Kit.

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 1.5 mL:

Total serum IgE	The most 6.5 μ L of 2 μ g
		T7 Promotor primer	5 µL
RNase-free Water	X µL
		Total volume	11.5 µL

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) 10 min, and 5 min of ice bath are kept the temperature at 65 DEG C, by 5 × First Strand B μ ffer at 65 DEG C Preheat 5 min.

(3) following cDNA synthetic system is configured:

5×First Strand Buffer	4 µL
		0.1 M DTT	2 µL
10 mM dNTP mix	1 µL
		MMLV RT	1 µL
RNase OUT	0.5 µL
		Total volume	8.5 µL

(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 2 h of reaction.

3, aaUTP marks cRNA synthesis

The configuration of NTP:

100 mM ATP	250 µL
		100 mM GTP	250 µL
100 mM CTP	250 µL
		100 mM UTP	187.5 µL
RNase free H2O	62.5 µL
		Total volume	1000 µL

It is spare to be distributed into 10 pipes, note: 50% PEG(polyethylene glycol) 40 DEG C of 1 min of heat preservation before use.Simultaneously by following operation Configure Transcription mix；

(1) Transcription mix is configured

RNase-free Water	5.7 µL
		4×Transcription Buffer	20 µL
NTP	16 µL
		0.1 M DTT	6 µL
50% PEG	6.4 µL
		Aa-UTP(25 mM)	4 µL
Inorganic Pyrophosphatase	0.6 µL
		T7 RNA Polymerase	0.8 µL
Total volume	60 µL

(2) 60 μ L Transcription mix are added and mix.

(3) 60 DEG C of hot lid in PCR instrument, 40 DEG C of 2 h of reaction.

4, cRNA is purified

QIAGEN RNeasy Mini kit purifies cRNA, and specific method can be found in the operation that QIAGEN company provides with kit Handbook.

(1) 20 μ L RNase 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 and cover in the RNeasy pillar in 2 mL centrifuge tubes >=8000 G is centrifuged 15-30 s, discards filtered solution.

(4) >=8000 g centrifuge washing 15-30 ss abandoning is drawn in 500 μ L Buffer RPE to RNeasy mini pillars Filtered solution is gone to be incited somebody to action again with 500 μ L Buffer RPE in the casing that 2 min of >=8000 g centrifuge washing discards filtered solution and 2 mL RNeasy mini pillar is transferred in a 1.5 new mL Eppendorf pipes.

(5) water for drawing 30 μ L RNase free stands 1 min, 1 min of >=8000 g centrifugation elution.

(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 260 nm and 280 nm to determine the concentration of sample And purity, A260/A280 should close to 2.0 for purer cRNA(ratio 1.9-2.1 can also).

6, cRNA fluorescent marks；

(1) it takes above-mentioned 4 μ g of cRNA and is concentrated into 6.6 μ L.

(2) plus 10 μ L DMSO are mixed.

(3) add the sodium bicarbonate (NaHCO that the 0.3 M 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 1 h of heat preservation.

(5) 25 DEG C of 15 min of heat preservation after adding the 4 M 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 30 min of warm bath.

Cy3 cRNA green fluorescence	875 ng
		10×Blocking Agent	11 µL
25×Fragmentation Buffer	2.2 µL
		Nuclease-free water	X µL
Total volume	55 µL

(2) 55 μ L 2 × GEx Hybridization Buffer are added.

(3) it takes 100 μ L hybridization solutions to be added drop-wise on chip ware, while is added dropwise respectively in sky as shown in Figure 2 by chip layout On white, negative, positive, experimental group position.It covers chip and is sealed in hybridizing box, 60 DEG C of rollings hybridize 16 h.

9, chip washs

Washing lotion 1(1 L) configuration:

DEPC-H2O	700 mL
		20×SSPE	300 mL
20%N-Lauroylsarcosine	0.25 mL

Washing lotion 2(1 L) configuration:

DEPC-H2O	997 mL
		20×SSPE	3.0 mL
20%N-Lauroylsarcosine	0.25 mL

Washing lotion 3:Stabilization and Drying Solution

(1) it takes out chip and washs 1 min in washing lotion 1；

(2) chip is put into washing lotion 2 again and washs 1 min(37 DEG C)；

(3) chip is finally washed into 30 s in washing lotion 3.

10, chip scanning

Chip is scanned in scanner, the epidermal growth factor receptor kinase bottom of test object is determined according to scanning result Whether 8 albuminoid of object, 3 albumen 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 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 albumen of test object is mutated.It is shown in Fig. 4 As a result in, negative control is colorless fluorescent, and positive control is green fluorescence, shows that the sample quality of acquisition testing object is that do not have Problem, and experimental group redgreen fluorescence, then showing 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of test object Albumen does not mutate.

Embodiment three, specific recognition people 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain Dan Ke The preparation of grand antibody

1, the determination of reagent

Drawing containing 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain for detecting above-mentioned people in the reagent Object pair.

Wherein, according to the ORF(such as SEQ ID of the normal albumen of 8 albuminoid of the epidermal growth factor receptor kinase substrate of people 3 Shown in NO:6), it can determine the corresponding alkali of ORF of the normal albumen of 8 albuminoid of epidermal growth factor receptor kinase substrate 3 of people Basic sequence (as shown in SEQ ID NO:7).

It is possible to further according to the base sequence of 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of people It arranges (as shown in SEQ ID NO:2), and the normal egg of 8 albuminoid of epidermal growth factor receptor kinase substrate 3 of the people according to people The corresponding base sequence of white ORF (as shown in SEQ ID NO:7), the primer pair designed include following any upstream primers with The combination of following any downstream primers: upstream primer is the base sequence as shown in any in SEQ ID NO:8 ~ NO:12；Downstream Primer is the base sequence as shown in any in SEQ ID NO:23 ~ NO:37；Or, the primer pair designed includes following any The combination of upstream primer and following any downstream primers: upstream primer is the alkali as shown in any in SEQ ID NO:8 ~ NO:17 Basic sequence；Downstream primer is the base sequence as shown in any in SEQ ID NO:28 ~ NO:37；Or, the primer pair packet designed Include the combination of following any upstream primers Yu following any downstream primers: upstream primer is as appointed in SEQ ID NO:8 ~ NO:22 Base sequence shown in one；Downstream primer is the base sequence as shown in any in SEQ ID NO:33 ~ NO:37:

As shown in SEQ ID NO:8: tccatcaccg tgcaggagcc gggcctgcca

As shown in SEQ ID NO:9: ggcactagca ctctgctctt ccagtgccag

As shown in SEQ ID NO:10: gaagtggggg cagagcgact gaagaccagc

As shown in SEQ ID NO:11: ctgcagaagg ctctggagga agagctggag

As shown in SEQ ID NO:12: caaagacctc gacttggagg ccttcagcca

As shown in SEQ ID NO:13: aaggagacaa gtgcccctga gctcgtacac

As shown in SEQ ID NO:14: atcctcttca agtccctgaa cttcatcctg

As shown in SEQ ID NO:15: gccaggtgcc ctgaggctgg cctagcagcc

As shown in SEQ ID NO:16: caagtgatct cacccctcct cacccctaaa

As shown in SEQ ID NO:17: gctatcaacc tgctacagtc ctgtctaagc

As shown in SEQ ID NO:18: acacacaacc atgaccctca gcctggggac

As shown in SEQ ID NO:19: cccaactcca ggccctccag ccccaaacct

As shown in SEQ ID NO:20: gcccagccag ccctgaaaat gcaagtcttg

As shown in SEQ ID NO:21: tacgagtttg aagctaggaa cccacgggaa

As shown in SEQ ID NO:22: ctgactgtgg tccagggaga gaagctggag

As shown in SEQ ID NO:23: ggatggtggg aggcctgcag tgtaagggga

As shown in SEQ ID NO:24: ccatggactg tgctctaggg tcctctggtg

As shown in SEQ ID NO:25: gggctgttct ggagggatcc ccggctccag

As shown in SEQ ID NO:26: atagcgtgcc tgctccatag ggagcggcct

As shown in SEQ ID NO:27: ttccatagca ggccccctcc atctgtcctg

As shown in SEQ ID NO:28: ctggtatcct aagggtgctt ggctggaggg

As shown in SEQ ID NO:29: ctctggaagt tgccagtcat ctgagaatgt

As shown in SEQ ID NO:30: gggttggtag ggcaggggct catcgcctgt

As shown in SEQ ID NO:31: ccagtcggcc cggctagtgg tccaggctgg

As shown in SEQ ID NO:32: gcccaacccc atccaaaggt tactctcagg

As shown in SEQ ID NO:33: acatagcatc tgtagctccc caggtcttat

As shown in SEQ ID NO:34: gcgaagtagc tggctccccg tcagggaccc

As shown in SEQ ID NO:35: aagtgtcctc accgtggcag tggagaagtt

As shown in SEQ ID NO:36: ctctgcctgc agccagtctg tgacctcttc

As shown in SEQ ID NO:37: aggcctcgag ctaagtcgaa gcattggaac

In the present embodiment, it is used to detect the 8 class egg of epidermal growth factor receptor kinase substrate of above-mentioned people in reagent containing one The primer pair of white 3 mutain.

For the primer pair (primer (F) and primer (R)) of each combination producing, following PCR amplification steps are executed.

2, the DNA of test object is that template carries out PCR amplification

10×Buffer	5 uL
		dNTP	2 uL
Ex Taq	1 uL
		ddH2O	5 uL
Template DNA	1 uL
		Primer (F)	3 uL
Primer (R)	3 uL
		Total system	20 uL

PCR amplification is carried out by template of the DNA of test object, obtains 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people The complete segment of mutating protein gene, and connect pMD19-T Vector(Takara company), 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, 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain for detecting people ELISA kit

In the present embodiment, the composition of ELISA kit are as follows: be coated with monoclonal antibody described in embodiment three ELISA ELISA Plate, ELIAS secondary antibody, 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 albumen of test object, sample diluting liquid, coating buffering Liquid, 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 diluted HRP(horseradish peroxidase) label Goat anti-Human IgG；Wherein, extension rate can To 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 2 mol/L.

Embodiment five

In embodiments of the present invention, using liver cancer patient as test object, and the liver cancer patient is detected using ELISA kit Whether 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 albumen 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 2.0 ug/ ML, and the monoclonal antibody after dilution is loaded into the hole of ELISA ELISA Plate, it is incubated at room temperature 1-2 h.

B, 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 albumen of liver cancer patient is diluted using sample diluting liquid It is loaded respectively at various concentration gradient, and by 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 albumen of various concentration gradient Into the hole of ELISA ELISA Plate, and it is incubated at room temperature 1-2 h；

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 1-2 h；

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 10-20 min, and the terminate liquid is added and terminates reaction；

G, it is the light absorption value at 450 nm 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=0.99933, therefore, this survey It is fixed effective.The wavelength that will test, which substitutes into standard curve for the light absorption value at 450 nm, can acquire the epidermis life of liver cancer patient The concentration of 8 albuminoid of growth factor receptor body kinase substrate, 3 albumen.Liver cancer patient blood serum sample is detected using the ELISA method of foundation The content of 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 albumen.And it is identified with Western blot.It please refers to Fig. 5 is 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

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:15 mL)

ddH2O	5.9 mL
		30% acrylamide mixed liquor	5.9 mL
1.5 mol/L Tris(PH 8.8)	3.8 mL
		10% SDS	0.15 mL
10% ammonium persulfate	0.15 mL
		TEMED	0.006 mL

B. upper layer spacer gel concentration preparation system (Total Volum:8 mL)

ddH2O	5.5 mL
		30% acrylamide mixed liquor	1.3 mL
1.0 mol/L Tris(PH 6.8)	1.0 mL
		10% SDS	0.08 mL
10% ammonium persulfate	0.08 mL
		TEMED	0.008 mL

(3) lower layer's separation gel of respective volume is added in each offset plate, adds the sterile ddH of 1 mL₂O flattens separation gel, After gelling to be separated is solid, remaining moisture in plastic plate is blotted with filter paper, upper layer is then added, glue is concentrated, waited after being inserted into comb Upper layer concentration gelling is solid.

2, albumen loading electrophoresis:

(1) comb is pulled up, duct label is carried out, 5 × SDS electrophoretic buffer is added, then to SDS- is added in protein sample PAGE albumen sample-loading buffer (5 ×), boiling water bath heat 3-5 min, loading；

(2) electrophoresis first is carried out with 80 V voltages, after running concentration glue to band, uses 100 V voltages instead and run about 100 min.

3, transferring film and incubation:

(1) first pvdf membrane is activated in methyl alcohol about 30 s.Gel, foam-rubber cushion and pvdf membrane are delayed in electrotransfer in advance respectively 30 min are balanced in fliud flushing.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 120 min is run with the electric current of 150 mA in ice；

(2) it takes the film out, is put into 5% skim milk at once after the completion of, gently room temperature closes 1 h on shaking table；

(3) PBST cleaning closed film, cleans 3 times, every time 10 min；

(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, 10 min every time；

(6) secondary antibody (being diluted with 3% milk with 1:5000-1:10000) is incubated at room temperature 2 h of film；

(7) film 3 times are cleaned with PBST, every time 10 min；

(8) it after Pierce ECL Western Blotting Substrate kit A, B liquid mixes in equal volume, drips dropwise It is added on pvdf membrane；

(9) exposure image in FluorChem E FE0511, experiment are analyzed with Image J.

Wherein, primary antibody is 8 albuminoid of epidermal growth factor receptor kinase substrate, the 3 mutain Dan Ke of the standby people of corporation Grand antibody, 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.It is only attached in 137 KD of experimental group compared with blank control Closely there is obvious band, wherein 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutant protein molecules amount is about 137 KD shows that 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 albumen of the liver cancer 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.

Claims (9)

1. a kind of mutain of 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people, which is characterized in that its amino acid Sequence is as shown in SEQ ID NO:1.

2. a kind of encoding gene of 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of people, which is characterized in that Its 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 nucleosides of nucleotide probe 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of people according to claim 2 Acid sequence, it is true with the comparison result of the nucleotide sequence of the normal albumen of 8 albuminoid of epidermal growth factor receptor kinase substrate 3 of people It is fixed.

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 reagent, which is characterized in that contain the epidermal growth factor for detecting people described in claim 1 in the reagent The primer pair of 8 albuminoid of receptor kinase substrate, 3 mutain.

6. reagent according to claim 5, which is characterized in that

The primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as SEQ ID In NO:8 ~ NO:12 it is any shown in base sequence；Downstream primer is the base as shown in any in SEQ ID NO:23 ~ NO:37 Sequence；

Or, the primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as SEQ In ID NO:8 ~ NO:17 it is any shown in base sequence；Downstream primer is as shown in any in SEQ ID NO:28 ~ NO:37 Base sequence；

Or, the primer pair includes the combination of following any upstream primers Yu following any downstream primers: upstream primer is such as SEQ In ID NO:8 ~ NO:22 it is any shown in base sequence；Downstream primer is as shown in any in SEQ ID NO:33 ~ NO:37 Base sequence.

7. a kind of monoclonal antibody of 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of specific recognition people, It is characterized in that, be prepared by reagent such as described in claim 5 or 6, it can be with amino acid sequence shown in SEQ ID NO:1 Column specific binding.

8. it is a kind of for detecting the ELISA kit of 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of people, It is characterized in that, the ELISA kit includes: ELISA ELISA Plate, the enzyme for being coated with monoclonal antibody described in claim 7 Mark secondary antibody, sample diluting liquid, coating buffer, ELISA ELISA Plate cleaning solution, developing solution and terminate liquid.

9. being used to detect 8 albuminoid of epidermal growth factor receptor kinase substrate, 3 mutain of people according to claim 8 The ELISA kit of antibody, which is characterized in that the ELIAS secondary antibody is the goat of diluted HRP horseradish peroxidase-labeled Anti-human igg.