CN109734790A - People Agrin antigen, people's Agrin antibody assay kit and the preparation method and application thereof - Google Patents

Abstract

The present invention provides people Agrin antigens, ELISA detection kit and the preparation method and application thereof.The people Agrin antigen includes SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, amino acid sequence shown in SEQ ID NO:6 and SEQ ID NO:7, additionally provide seven kinds respectively containing above-mentioned seven kinds of people Agrin antigen detection kit and a kind simultaneously contain above-mentioned seven kinds of people Agrin antigen detection kit.The people's Agrin detection kit detects the Agrin antibody in human serum, and high specificity, reaction sensitivity is high, high-throughput, at low cost, can diagnose to myasthenia gravis illness, be suitable for large-scale promotion application.

Description

People Agrin antigen, people's Agrin antibody assay kit and the preparation method and application thereof

Technical field

The present invention relates to biopharmaceutical technology more particularly to people Agrin antigens, people's Agrin antibody assay kit And the preparation method and application thereof.

Background technique

Myasthenia gravis (Myasthenia gravis, MG), is common Neuromuscular junction (neuromuscular Junction, NMJ) disease.In Most patients, MG may be from one and itself exempt to acetylcholinergic receptor (AChRs) Epidemic disease reaction, can detect the autoantibody of AChRs in the MG patient of 80%-85%.However, AChR antibody is about 20% MG patient in be not detected.Evidence suggests, the patient of these " seronegativities " can produce NMJ is formed or Keep very crucial protein antibodies.The collectin Agrin discharged from motor neuron, is integrated to LDL receptor Relevant albumen 4 (LRP4), activated receptor tyrosine kinase MuSK instruct the formation of NMJ.The serum reverse of about 40%-70% Negative patient is answered to have MuSK autoantibody.The MG patient of remaining 6%-12% has double seronegativities anti-AChR and MuSK antibody It answers.Collectin Agrin is the heparan sulfate proteoglycan released from kinesitherapy nerve terminal.It adjusts neuromuscular and connects Formation, maintenance and the regeneration of point, and collectin function is interfered, it can lead to Phrenic nerve diaphragm deficiency in a small number of patients MG Collectin antibody can be detected with and without for AChR MuSK or LRP4 antibody.Aggregation Protein antibodies can be detected only in patient MG, and prompting these antibody is specific for MG.At present also The relevant report of nobody's Agrin antibody assay kit.

Summary of the invention

It is an object of the invention to overcome the defect of the prior art, provide a kind of people Agrin antigen and preparation method thereof, People's Agrin antibody assay kit and the preparation method and application thereof, in the detection kit energy specific detection human serum LRP4 antibody has diagnostic significance to myasthenia gravis, is quick on the draw, it is at low cost, be suitable for large-scale promotion application.

The present invention is implemented as follows:

It is an object of the present invention to a kind of people Agrin antigens, include any of the following segment or seven kinds of segments:

Segment 1:Agrin-40, amino acid sequence is as shown in SEQ ID NO.1；

Segment 2:Agrin-411, amino acid sequence is as shown in SEQ ID NO.2；

Segment 3:Agrin-701, amino acid sequence is as shown in SEQ ID NO.3；

Segment 4:Agrin-1103, amino acid sequence is as shown in SEQ ID NO.4；

Segment 5:Agrin-1281, amino acid sequence is as shown in SEQ ID NO.5；

Segment 6:Agrin-1635, amino acid sequence is as shown in SEQ ID NO.6；

Segment 7:Agrin-1864, amino acid sequence is as shown in SEQ ID NO.7.

The second object of the present invention is to provide people's preparation method of Agrin antigen, include the following steps:

Step 1, by Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 Gene chemical synthesis is carried out respectively with the DNA sequence dna of 7 segments of Agrin-1864, after design primer PCR amplification, is separately connected into table Up to carrier, recombinant expression plasmid is constructed；

The recombinant plasmid transformed built is entered expression bacterium, building recombinant expression engineering bacteria by step 2；

The inducing expression and purifying of step 3, people's Agrin antigen.

Specifically, the primer pair of 7 segments described in the step 1 is respectively as follows:

Agrin-40-P1: nucleotide sequence is as shown in SEQ ID NO.8；

Agrin-40-P2: nucleotide sequence is as shown in SEQ ID NO.9；

Agrin-411-P1: nucleotide sequence is as shown in SEQ ID NO.10；

Agrin-411-P2: nucleotide sequence is as shown in SEQ ID NO.11；

Agrin-701-P1: nucleotide sequence is as shown in SEQ ID NO.12；

Agrin-701-P2: nucleotide sequence is as shown in SEQ ID NO.13；

Agrin-1103-P1: nucleotide sequence is as shown in SEQ ID NO.14；

Agrin-1103-P2: nucleotide sequence is as shown in SEQ ID NO.15；

Agrin-1281-P1: nucleotide sequence is as shown in SEQ ID NO.16；

Agrin-1281-P2: nucleotide sequence is as shown in SEQ ID NO.17；

Agrin-1635-P1: nucleotide sequence is as shown in SEQ ID NO.18；

Agrin-1635-P2: nucleotide sequence is as shown in SEQ ID NO.19；

Agrin-1864-P1: nucleotide sequence is as shown in SEQ ID NO.20；

Agrin-1864-P2: nucleotide sequence is as shown in SEQ ID NO.21.

Specifically, in the step 1 PCR amplification reaction system are as follows: H₂O 38.7ul；Buffer 5ul；dNTP 3ul； Upper primer 1ul；Lower primer 1ul；DNA 1ul；Taq E 0.3ul；Amplification program are as follows: 94 degree of denaturation 5min；94 degree of denaturation 45sec, 57 degree of 150sec, 72 degree of 90sec, 32 circulations；72 degree of extension 10min.

Specifically, in the step 3 inducing expression specific steps are as follows: 7 recombinant bacteriums are inoculated in LB culture solution respectively In when shaking bacterium to OD600 to 0.6-0.8, induced 4-6 hours by the IPTG that 24mg/ml concentration is added in 1:1000.

Specifically, the purification condition in the step 3 after inducing expression is: sample-loading buffer: 0.5M NaCl, 20mM Na₂HPO₃, 10mM imidazoles；Combination buffer: 0.5M NaCl, 20mM Na₂HPO₃, 20mM imidazoles；Elution buffer: 0.5M Nacl、20mM Na₂HPO₃, 500mM imidazoles.

The third object of the present invention is to provide a kind of expression vector of people Agrin antigen, and the expression vector is 7, The nucleotide sequence in the expression area of 7 expression vectors is respectively as follows: such as SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, shown in SEQ ID NO.28.

The fourth object of the present invention is to provide a kind of expression engineering bacteria of people Agrin antigen, which is 7, institute State the expression vector that 7 engineering bacterias separately include 7 people Agrin antigen.

The fifth object of the present invention is to provide people's Agrin antibody assay kit, the ELISA detection kit packet It includes:

(A) it is coated with the ELISA ELISA Plate of people's Agrin antigen；The people Agrin antigen include Agrin-40, Any one in Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864 Or seven kinds；

(B) standard female serum: the serum of Agrin negative antibody；

(C) standard positive serum: the serum of Agrin antibody positive；

(D) ELIAS secondary antibody of horseradish peroxidase-labeled: anti-human IGG, IGM and IGA；

(E) sample diluting liquid, coating buffer, confining liquid, ELISA ELISA Plate cleaning solution, antibody diluent, developing solution and Terminate liquid.

Wherein, described people Agrin antigen A grin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin- 1281, the peridium concentration of Agrin-1635 and Agrin-1864 is respectively 200ng/ml, 200ng/ml, 250ng/ml, 250ng/ Ml, 300ng/ml, 150ng/ml and 150ng/ml.

The sixth object of the present invention is to provide people's detection method of Agrin antibody, and the detection method includes following Step:

S1, preparation detection ELISA ELISA Plate: coating buffer will add after people Agrin antigen diluent described in claim 1 It is adsorbed into ELISA Plate, the dry coating washing lotion of sky adds coating to be closed with confining liquid；The people Agrin antigen include Agrin-40, Any one in Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864 Or seven kinds；

Respectively as primary antibody, sample-adding is incubated into ELISA ELISA Plate hole for S2, serum to be checked, negative serum, positive serum It educates；

The incubation of S3, ELIAS secondary antibody: ELISA ELISA Plate, cleaning solution is added in the ELIAS secondary antibody of horseradish peroxidase-labeled Washing, drying；

S4, developing solution is added, room temperature is protected from light incubation, and terminate liquid is added and terminates reaction；It is surveyed under 450nm wavelength in microplate reader Determine OD value.

The seventh object of the present invention is to provide the detection kit in diagnosing myasthenia myasthenia (MG) disease Using.

The invention has the advantages that:

The present invention provides people Agrin antigen, people Agrin antibody assay kit and detection methods, are prepared first People Agrin antigen (Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864), indirect ELISA detection then is carried out to sample serum using people Agrin antigen as envelope antigen, which can With for checkout and diagnosis, to myasthenia gravis (MG) disease, high specificity, high sensitivity, stability is good.

Detailed description of the invention

Fig. 1 is the cleavage map of the recombinant plasmid for the building that the embodiment of the present invention 1 provides；Wherein, (A) is to include Agrin-40 Recombinant plasmid cleavage map；It (B) is the cleavage map of the recombinant plasmid comprising Agrin-411；It (C) is to include Agrin-701's The cleavage map of recombinant plasmid；It (D) is the cleavage map of the recombinant plasmid comprising Agrin-1103；It (E) is to include Agrin-1281's The cleavage map of recombinant plasmid；It (F) is the cleavage map of the recombinant plasmid comprising Agrin-1635；It (G) is to include Agrin-1864's The cleavage map of recombinant plasmid；Wherein 1 swimming lane is the plasmid of non-digestion, and 2 swimming lanes are the band through corresponding endonuclease digestion；(F) For Marker histogram, which is 1Kb ladder；

Fig. 2 be the embodiment of the present invention 2 provide Agrin-40, Agrin-411, Agrin-701, Agrin-1103, The purified product electrophoretogram of Agrin-1281, Agrin-1635 and Agrin-1864 segment.

Specific embodiment

Embodiment 1 constructs recombinant expression plasmid and engineering bacteria

1, collectin Agrin is a kind of heparan sulfate proteoglycan, is made of multiple structural domains, these structural domains Including 9 kinases inhibitor structural domains, 4 epidermal growth factor-like structural domains and 1 adhesion molecule G homeodomain. Mature Agrin albumen is made of 2038 amino acid.It includes hydrophilic that the amino acid sequence of Agrin albumen is passed through in this research Property, the analysis such as surface accessibility, and combine the modification feature of its space conformation and each structural domain, select mature Agrin egg The segment in 7 white regions to obtain respectively.The present inventor passes through a series of research, obtains seven kinds of people's Agrin antigens, Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864.

2, PCR amplification people Agrin antigen gene

1, the DNA sequence dna of 7 segments is subjected to gene chemical synthesis respectively, design primer (taking restriction enzyme site) PCR amplification, if Meter PCR primer is as shown in table 1, and the part of underscore is restriction endonuclease site.

Table 1

2, as shown in table 2 with PCR amplification system, thermal cycler parameters: 94 DEG C, 5min → (94 DEG C, 45S, → 57 DEG C, 150S,→72℃,90S)×32→72℃,10min.Amplified production connects for subsequent digestion enzyme.

Table 2

4, after PCR amplification, agarose gel electrophoresis recycle amplified band, digestion, enzyme connect, respectively by 7 DNA fragmentations connect into PET-28a expression vector establishment recombinant expression plasmid.Recombinant plasmid runs glue cleavage map and sees Figure of description 1.

5, recombinant plasmid transformed BL21 (DE3) bacterium that will be built constructs recombinant bacterium, result verification after the sequencing of recombinant bacterium The construction of recombinant plasmid success of the present embodiment.

The expression and purifying of 2 antigen of embodiment

1, inducing expression experiment is carried out with the recombinant protein expression engineering bacteria being built into.7 recombinant bacteriums are inoculated in respectively 600ml LB culture solution (ingredient: 10g sodium chloride/liter, 10g peptone/liter and 5g yeast extract/liter), 37 degree of 200RPM shake bacterium When to OD600 to 0.6-0.8, induced 4 hours by the IPTG that 24mg/ml concentration is added in 1:1000.Bacterium is received in centrifugation, prepares purifying.

2, the filler for purifying selection is the Ni Sepharose of GE (article No. is 17-0729-10), is distinguished according to its specification It is formulated as follows solution:

Sample-loading buffer A:0.5M NaCl+20mM Na₂HPO₃+ 10mM imidazoles.

Combination buffer B:0.5M NaCl+20mM Na₂HPO₃+ 20mM imidazoles

Elution buffer C:0.5M NaCl, 20mM Na₂HPO₃, 500mM imidazoles；

And the solution for purifying inclusion body:

Purify sample-loading buffer a:0.5M NaCl, the 20mM Na of inclusion body antigen₂HPO₃, 20mM imidazoles, 8M urea；

Combination buffer b:0.5MNaCl, 20mM Na₂HPO₃, 20mM imidazoles, 8M urea；

Elution buffer c:0.5M NaCl, 20mM Na₂HPO₃, 300mM imidazoles, 8M urea.

3, the bacterium that 7 kinds are collected by centrifugation is uniformly dispersed with sample-loading buffer A, (250W, super 3s are spaced 3s to ultrasound, whole 20min), centrifugation (12000RPM, 15min, 4 DEG C) for the first time, obtains the supernatant solution of the purpose antigen containing higher concentration.Forgive Body antigen need to be uniform with sample-loading buffer a redisperse by the precipitating of first time centrifugation, then (250W, super 3s are spaced 3s, entirely to ultrasound Journey 20min), be centrifuged (12000RPM, 15min, 4 DEG C) again, discard precipitating, obtain the solution of the purpose antigen containing higher concentration. The solution of 7 kinds of destination proteins is subjected to loading, washing, elution to filled column respectively, respectively obtains 7 destination proteins.Purifying Good inclusion body antigen needs renaturation, using to renaturation inclusion body antigen same volume containing different urea concentrations (4.5M, 3.5M, 2.5M, 1.5M, 0.5M, 0M) renaturation buffer continue dialysis renaturation, every kind of renaturation buffer is dialysed 4 hours.

Soluble antigen (Agrin-609) and complete inclusion body antigen (Agrin-40, Agrin-411, the Agrin- of renaturation 701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864), carry out the SDS-PAGE of 12% concentration gel Electrophoresis detects seven destination protein purity and is respectively as follows: 93.5%, 94.9%, 93.4%, 94.5%, 94.1%, 94.8%, and 95.1%.See Figure of description 2.It is stored for future use after every kind of antigen measuring concentration.

3 people Agrin antibody assay kit of embodiment and application method

1, coated elisa plate

(1) coating buffer: NaCl 8.5g, Na₂HPO₄·12H₂O 30.8g, KH₂PO₄2.2g adds ddH₂O to 1000ml is adjusted PH to 7.4.

(2) coating washing lotion: NaCl 8.0g, KH₂PO₄0.24g、Na₂HPO₄·12H₂O 2.9g、KCl 0.2g、 TWEEN200.5ml adds to ddH₂O to 1000ml, is adjusted to PH7.4.

(3) method for coating: 7 Agrin antigen fragments are coated with 0.1M PBS (PH7.4) coating buffer respectively In ELISA Plate hole, 4 spend night, wherein Agrin-25, Agrin-344, Agrin-530, Agrin-824 and Agrin-1048's Peridium concentration is respectively 200ng/ml, 200ng/ml, 250ng/ml, 150ng/ml and 250ng/ml.Every hole in 96 hole elisa Plates Add 100 μ L, sets 2-8 DEG C and adsorb 24 hours.Sky goes coating buffer, and coating is used washing lotion board-washing 3 times.

2, it closes

(1) coating confining liquid: Na₂HPO₄·12H₂O 3.582g, NaH₂PO₄·2H₂O 1.561g, NaCl 9.0g, BSA20g, xylose 10g adjust pH to 7.2, are settled to 1000ml.

(2) " locked in " operation: the dry coating washing lotion of sky is closed 2 hours, or set 2-8 DEG C with 37 degree of 1.5%BSA confining liquid Overnight.Natural drying sealing is spare after removing confining liquid.

3, the incubation (primary antibody incubation) of yin and yang attribute serum

Serum to be detected is diluted by 10-100 times, is separately added into the lath of 7 kinds of antigen that (every kind of antigen has by excellent The specific serum diluting multiple changed), and positive control and negative control is added, 37 degree are incubated for 1 hour, are washed with cleaning solution The formula of plate, the cleaning solution is as follows:

Cleaning solution (0.15M): NaCl 8.0g, KH₂PO₄0.24g、Na₂HPO₄·12H₂O 2.9g、KCl 0.2g、 TWEEN20 0.5ml, adds to ddH₂O to 1000ml, is adjusted to PH7.4.

Operation: serum to be checked makees serum dilution with PBS liquid, by 1:400 times of dilution proportion, is added in coating plate hole, 100 holes μ L/.It directly draws standard positive serum or standard female serum is added in coating plate hole, 100 holes μ L/.Set ELISA Plate in 37 DEG C, 30min.

4, the incubation of ELIAS secondary antibody

The secondary antibody (anti-human IGG, IGM and IGA) of certain density horseradish peroxidase-labeled is added, into ELISA Plate hole 100 holes μ L/ are added, set 37 DEG C, 15min, board-washing.

5, it develops the color

Substrate solution A50 μ L, 50 μ L of substrate solution B, jog mixing, 37 DEG C of reaction 15min are added.

(1) substrate solution A: sodium acetate 13.6g, citric acid 1.6g, 30% hydrogen peroxide 0.3ml, distilled water add to 500ml

(2) substrate solution B: 0.2g TMB is taken to be dissolved in 20mlDMSO, disodium ethylene diamine tetraacetate 0.2g, citric acid 0.95g, glycerol 50ml, distilled water add to 500ml.

6, it terminates:

Terminate liquid: 2mol/L H₂SO₄。

Every hole is added 50ul terminate liquid and is terminated after colour developing.

7, read plate:

OD450 value is measured with microplate reader.

It should be noted that serum is detected through the lath of 7 kinds of antigen, the OD450 value of any of which lath reaches sun Property value when, then determine the serum be to Agrin it is positive, to the antigen fragment at the positive.Serum individual exists to Agrin in vivo And the antibody of the Agrin segment.7 kinds of detection kits that this 7 kinds of antigens are prepared into, which can be used separately or combine, to be made With.

People Agrin antibody assay kit provided by the invention can also be used for preparing immune colloidal gold detection test paper strip, exempt from The various forms of products such as epidemic disease fluorescence, immunoturbidimetry, chemiluminescence.

The application of 4 people's Agrin antibody assay kit of embodiment

One, it applies

1, this research detects 613 parts of neurological autoimmune diseases patients serums according to embodiment 3, this 613 parts of serum take off marrow polyneuritis neuropathy CIDP, Guillain Barre GBS and myasthenia gravis MG from chronic inflammatory Equal patients, wherein chronic inflammatory takes off marrow polyneuritis neuropathy CIDP patients serum and verifies in hospital through clinical detection It is the serum of CIDP antibody positive, this research also has detected 300 parts of normal human serum samples with batch.

2, experimental result such as following table (only listing 60 parts of sample datas), this testing result is shown, Agrin-1635 antibody Detection kit.MG patients serum positive rate up to 25%, CIDP positive patient sera positive rate up to 10%, normal The positive rate of human serum is up to 5%, critical value 0.49.

Table 3- sample data testing result

4, in addition, Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 Also indicate that in the positive ratio of MG patients serum be 3 times or more of normal person with Agrin-1864 detection.As a result it is counted with SPSS Software 17.0 is analyzed, carries out one-way analysis of variance, and carry out multiple comparative test with Student-Newman-Keuls.As a result It is as follows:

Table 4- testing result statistical analysis table

Note: the Student-Newman- of the One-WayANOVA program of detection data SPSS17.0 statistical analysis software The Keuls method of inspection carries out Multiple range test analysis, with significant difference (P between the group with different alphabetical shoulders number (a, b, c) in column <0.05)。

Two, the technical indicator of 7 kinds of ELISA detection kits

1, critical value: detection patients serum and normal human serum are carried out according to embodiment 3, it is determined that test validity is sentenced Determine method, the calculation method of critical value (CUT OFF), sample yin and yang attribute determination method.

(1) Agrin-40:

Test validity determines: Positive control wells average value >=0.53；Negative control average value≤0.28；

Critical value=negative control hole average value+0.13=0.41；

Feminine gender determines: sample OD value < critical value person (0.41) is Agrin-40 negative antibody；

The positive determines: sample OD value >=critical value person (0.41) is Agrin-40 antibody positive.

(2) Agrin-411:

Test validity determines: Positive control wells average value >=0.53；Negative control average value≤0.29；

Critical value=negative control hole average value+0.14=0.43；

Feminine gender determines: sample OD value < critical value person (0.43) is Agrin-411 negative antibody；

The positive determines: sample OD value >=critical value person (0.43) is Agrin-411 antibody positive.

(3) Agrin-701:

Test validity determines: Positive control wells average value >=0.53；Negative control average value≤0.29；

Critical value=negative control hole average value+0.14=0.43；

Feminine gender determines: sample OD value < critical value person (0.43) is Agrin-701 negative antibody；

The positive determines: sample OD value >=critical value person (0.43) is Agrin-701 antibody positive.

(4) Agrin-1103:

Test validity determines: Positive control wells average value >=0.53；Negative control average value≤0.25；

Critical value=negative control hole average value+0.14；

Feminine gender determines: sample OD value < critical value person (0.39) is Agrin-1103 negative antibody；

The positive determines: sample OD value >=critical value person (0.39) is Agrin-1103 antibody positive.

(5) Agrin-1281:

Test validity determines: Positive control wells average value >=0.53；Negative control average value≤0.26；

Critical value=negative control hole average value+0.14=0.40；

Feminine gender determines: sample OD value < critical value person (0.40) is Agrin-1281 negative antibody；

The positive determines: sample OD value >=critical value person is (0.40) Agrin-1281 antibody positive.

(6) Agrin-1635:

Test validity determines: Positive control wells average value >=0.53；Negative control average value≤0.36；

Critical value=negative control hole average value+0.13=0.49；

Feminine gender determines: sample OD value < critical value person (0.49) is Agrin-1635 negative antibody；

The positive determines: sample OD value >=critical value person (0.49) is Agrin-1635 antibody positive.

(7) Agrin-1864:

Test validity determines: Positive control wells average value >=0.53；Negative control average value≤0.21；

Critical value=negative control hole average value+0.13=0.34；

Feminine gender determines: sample OD value < critical value person (0.34) is Agrin-1864 negative antibody；

The positive determines: sample OD value >=critical value person (0.34) is Agrin-1864 antibody positive.

2, specific: in addition to positive serum, other test samples are feminine gender.These statistics indicate that, it is provided by the invention Cross reaction is not present between kit and other serum antibodies.

3, sensitivity: 12800 times of positive serum dilutions can detect (i.e. 1 μ L serum is added in 12800 μ L sample dilutions, Take wherein 100 μ L addition sample detection hole) it can detect.

4, stability: being placed in 37 DEG C of no less than 2 days kit 20 parts of samples of synchronous detection stored with 4 DEG C for kit, Its coincidence rate is 100%.

5, precision: taking 7 kinds of serum specimens of concentration in gradient, dilute respectively, respectively with batch measurement 10 times, variation within batch Coefficient is to be below 4%；Same 7 parts of serum, measures 10 times again every other day, and variation is below 5%；Meet kit precision to want It asks.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Sequence table

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Gly Pro Cys Val Cys Asp Phe Ser Cys Gln Ser Val Pro Gly Ser Pro

1 5 10 15

Val Cys Gly Ser Asp Gly Val Thr Tyr Ser Thr Glu Cys Glu Leu Lys

20 25 30

Lys Ala Arg Cys Glu Ser Gln Arg Gly Leu Tyr Val Ala Ala Gln Gly

35 40 45

Ala Cys Arg Gly Pro Thr Phe Ala Pro Leu Pro Pro Val Ala Pro Leu

50 55 60

His Cys Ala Gln Thr Pro Tyr Gly Cys Cys Gln Asp Asn Ile Thr Ala

65 70 75 80

Ala Arg Gly Val Gly Leu Ala Gly Cys Pro Ser Ala Cys Gln Cys Asn

85 90 95

Pro His Gly Ser Tyr Gly Gly Thr Cys Asp Pro Ala Thr Gly Gln Cys

100 105 110

Ser Cys Arg Pro Gly Val Gly Gly Leu Arg Cys Asp Arg Cys Glu Pro

115 120 125

Gly Phe Trp Asn Phe Arg Gly Ile Val Thr Asp Gly Arg Ser Gly Cys

130 135 140

Thr Pro Cys Ser Cys Asp Pro Gln Gly Ala Val Arg Asp Asp Cys Glu

145 150 155 160

Gln Met Thr Gly Leu Cys Ser Cys Lys Pro Gly Val Ala Gly Pro Lys

165 170 175

Cys Gly Gln Cys Pro Asp Gly Arg Ala Leu Gly Pro Ala Gly Cys Glu

180 185 190

Ala Asp Ala Ser Ala Pro Ala Thr Cys Ala Glu Met Arg Cys Glu Phe

195 200 205

Gly Ala Arg Cys Val Glu Glu Ser Gly Ser Ala His Cys Val Cys Pro

210 215 220

Met Leu Thr Cys Pro Glu Ala Asn Ala Thr Lys Val Cys Gly Ser Asp

225 230 235 240

Gly Val Thr Tyr Gly Asn Glu Cys Gln Leu Lys Thr Ile Ala Cys Arg

245 250 255

Gln Gly Leu Gln Ile Ser Ile Gln Ser Leu Gly Pro Cys Gln Glu Ala

260 265 270

Val Ala Pro Ser Thr His Pro Thr Ser Ala Ser Val Thr Val Thr Thr

275 280 285

Pro Gly Leu Leu Leu Ser Gln Ala Leu Pro Ala Pro Pro Gly Ala Leu

290 295 300

Pro Leu Ala Pro Ser Ser Thr Ala His Ser Gln Thr Thr Pro Pro Pro

305 310 315 320

Ser Ser Arg Pro Arg Thr Thr Ala Ser Val Pro Arg Thr Thr Val Trp

325 330 335

Pro Val Leu Thr Val Pro Pro Thr Ala Pro Ser Pro Ala Pro Ser Leu

340 345 350

Val Ala Ser Ala Phe Gly Glu Ser Gly Ser Thr Asp Gly Ser Ser Asp

355 360 365

Glu Glu Leu Ser Gly Asp Gln Glu Ala Ser Gly Gly Gly Ser Gly Gly

370 375 380

Leu Glu Pro Leu Glu Gly Ser Ser Val Ala Thr Pro Gly Pro Pro Val

385 390 395 400

<210> 4

<211> 180

<212> PRT

<213>people Agrin (Agrin-1103)

<400> 4

Ala Ser Cys Tyr Asn Ser Ala Leu Gly Cys Cys Ser Asp Gly Lys Thr

1 5 10 15

Pro Ser Leu Asp Ala Glu Gly Ser Asn Cys Pro Ala Thr Lys Val Phe

20 25 30

Gln Gly Val Leu Glu Leu Glu Gly Val Glu Gly Gln Glu Leu Phe Tyr

35 40 45

Thr Pro Glu Met Ala Asp Pro Lys Ser Glu Leu Phe Gly Glu Thr Ala

50 55 60

Arg Ser Ile Glu Ser Thr Leu Asp Asp Leu Phe Arg Asn Ser Asp Val

65 70 75 80

Lys Lys Asp Phe Arg Ser Val Arg Leu Arg Asp Leu Gly Pro Gly Lys

85 90 95

Ser Val Arg Ala Ile Val Asp Val His Phe Asp Pro Thr Thr Ala Phe

100 105 110

Arg Ala Pro Asp Val Ala Arg Ala Leu Leu Arg Gln Ile Gln Val Ser

115 120 125

Arg Arg Arg Ser Leu Gly Val Arg Arg Pro Leu Gln Glu His Val Arg

130 135 140

Phe Met Asp Phe Asp Trp Phe Pro Ala Phe Ile Thr Gly Ala Thr Ser

145 150 155 160

Gly Ala Ile Ala Ala Gly Ala Thr Ala Arg Ala Thr Thr Ala Ser Arg

165 170 175

Leu Pro Ser Ser

180

<210> 5

<211> 229

<212> PRT

<213>people Agrin (Agrin-1281)

<400> 5

Ser Ser Ala Val Thr Pro Arg Ala Pro His Pro Ser His Thr Ser Gln

1 5 10 15

Pro Val Ala Lys Thr Thr Ala Ala Pro Thr Thr Arg Arg Pro Pro Thr

20 25 30

Thr Ala Pro Ser Arg Val Pro Gly Arg Arg Pro Pro Ala Pro Gln Gln

35 40 45

Pro Pro Lys Pro Cys Asp Ser Gln Pro Cys Phe His Gly Gly Thr Cys

50 55 60

Gln Asp Trp Ala Leu Gly Gly Gly Phe Thr Cys Ser Cys Pro Ala Gly

65 70 75 80

Arg Gly Gly Ala Val Cys Glu Lys Val Leu Gly Gly Asp His Pro Cys

85 90 95

Leu Pro Asn Pro Cys His Gly Gly Ala Pro Cys Gln Asn Leu Glu Ala

100 105 110

Gly Arg Phe His Cys Gln Cys Pro Pro Gly Arg Val Gly Pro Thr Cys

115 120 125

Ala Asp Glu Lys Ser Pro Cys Gln Pro Asn Pro Cys His Gly Ala Ala

130 135 140

Pro Cys Arg Val Leu Pro Glu Gly Gly Ala Gln Cys Glu Cys Pro Leu

145 150 155 160

Gly Arg Glu Gly Thr Phe Cys Gln Thr Ala Ser Gly Gln Asp Gly Ser

165 170 175

Gly Ala Gly His Pro Cys Thr Arg Ala Ser Gly His Pro Cys Leu Asn

180 185 190

Gly Ala Ser Cys Val Pro Arg Glu Ala Ala Tyr Val Cys Leu Cys Pro

195 200 205

Gly Gly Phe Ser Gly Pro His Cys Glu Lys Gly Leu Val Glu Lys Ser

210 215 220

Ala Gly Asp Val Asp

225

<210> 6

<211> 188

<212> PRT

<213>people Agrin (Agrin-1635)

<400> 6

Pro Phe Leu Ala Asp Phe Asn Gly Phe Ser His Leu Glu Leu Arg Gly

1 5 10 15

Leu His Thr Phe Ala Arg Asp Leu Gly Glu Lys Met Ala Leu Glu Val

20 25 30

Val Phe Leu Ala Arg Gly Pro Ser Gly Leu Leu Leu Tyr Asn Gly Gln

35 40 45

Lys Thr Asp Gly Lys Gly Asp Phe Val Ser Leu Ala Leu Arg Asp Arg

50 55 60

Arg Leu Glu Phe Arg Tyr Asp Leu Gly Lys Gly Ala Ala Val Ile Arg

65 70 75 80

Ser Arg Glu Pro Val Thr Leu Gly Ala Trp Thr Arg Val Ser Leu Glu

85 90 95

Arg Asn Gly Arg Lys Gly Ala Leu Arg Val Gly Asp Gly Pro Arg Val

100 105 110

Leu Gly Glu Ser Pro Lys Ser Arg Lys Val Pro His Thr Val Leu Asn

115 120 125

Leu Lys Glu Pro Leu Tyr Val Gly Gly Ala Pro Asp Phe Ser Lys Leu

130 135 140

Ala Arg Ala Ala Ala Val Ser Ser Gly Phe Asp Gly Ala Ile Gln Leu

145 150 155 160

Val Ser Leu Gly Gly Arg Gln Leu Leu Thr Pro Glu His Val Leu Arg

165 170 175

Gln Val Asp Val Thr Ser Phe Ala Gly His Pro Cys

180 185

<210> 7

<211> 204

<212> PRT

<213>people Agrin (Agrin-1864)

<400> 7

Ser Ala Gly Asp Val Asp Thr Leu Ala Phe Asp Gly Arg Thr Phe Val

1 5 10 15

Glu Tyr Leu Asn Ala Val Thr Glu Ser Glu Leu Ala Asn Glu Ile Pro

20 25 30

Val Pro Glu Thr Leu Asp Ser Gly Ala Leu His Glu Lys Ala Leu Gln

35 40 45

Ser Asn His Phe Glu Leu Ser Leu Arg Thr Glu Ala Thr Gln Gly Leu

50 55 60

Val Leu Trp Ser Gly Lys Ala Thr Glu Arg Ala Asp Tyr Val Ala Leu

65 70 75 80

Ala Ile Val Asp Gly His Leu Gln Leu Ser Tyr Asn Leu Gly Ser Gln

85 90 95

Pro Val Val Leu Arg Ser Thr Val Pro Val Asn Thr Asn Arg Trp Leu

100 105 110

Arg Val Val Ala His Arg Glu Gln Arg Glu Gly Ser Leu Gln Val Gly

115 120 125

Asn Glu Ala Pro Val Thr Gly Ser Ser Pro Leu Gly Ala Thr Gln Leu

130 135 140

Asp Thr Asp Gly Ala Leu Trp Leu Gly Gly Leu Pro Glu Leu Pro Val

145 150 155 160

Gly Pro Ala Leu Pro Lys Ala Tyr Gly Thr Gly Phe Val Gly Cys Leu

165 170 175

Arg Asp Val Val Val Gly Arg His Pro Leu His Leu Leu Glu Asp Ala

180 185 190

Val Thr Lys Pro Glu Leu Arg Pro Cys Pro Thr Pro

195 200

<210> 8

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

gacggatcca catgcccgga gcgcgcgc 28

<210> 9

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

agctcgagtc agccacggcg ggacagg 27

<210> 10

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

gacggatccc gtccccgctg ctcctgc 27

<210> 11

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

agctcgagtc agtcctccga gtcctc 26

<210> 12

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

gacggatccg ggccgtgtgt ctgtgac 27

<210> 13

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

agctcgagtc agacaggtgg cccag 25

<210> 14

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

gacggatccg ccagctgcta caatagc 27

<210> 15

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

agctcgagtt agctactcgg cagacg 26

<210> 16

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

gacggatcca gcagcgcagt gacacctc 28

<210> 17

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

agctcgagtt agtccacatc accggc 26

<210> 18

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

gacggatccc ccttcctggc tgacttc 27

<210> 19

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

agctcgagtc agcaggggtg acctgc 26

<210> 20

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

gacggatcct cagcggggga cgtggat 27

<210> 21

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

agctcgagtc atggggtggg gcaggg 26

<210> 22

<211> 1146

<212> DNA

<213>people Agrin (Agrin-30)

<400> 22

acatgcccgg agcgcgcgct ggagcggcgc gaggaggagg cgaacgtggt gctcaccggg 60

acggtggagg agatcctcaa cgtggacccg gtgcagcaca cgtactcctg caaggttcgg 120

gtctggcggt acttgaaggg caaagacctg gtggcccggg agagcctgct ggacggcggc 180

aacaaggtgg tgatcagcgg ctttggagac cccctcatct gtgacaacca ggtgtccact 240

ggggacacca ggatcttctt tgtgaaccct gcacccccat acctgtggcc agcccacaag 300

aacgagctga tgctcaactc cagcctcatg cggatcaccc tgcggaacct ggaggaggtg 360

gagttctgtg tggaagataa acccgggacc cacttcactc cagtgcctcc gacgcctcct 420

gatgcgtgcc ggggaatgct gtgcggcttc ggcgccgtgt gcgagcccaa cgcggagggg 480

ccgggccggg cgtcctgcgt ctgcaagaag agcccgtgcc ccagcgtggt ggcgcctgtg 540

tgtgggtcgg acgcctccac ctacagcaac gaatgcgagc tgcagcgggc gcagtgcagc 600

cagcagcgcc gcatccgcct gctcagccgc gggccgtgcg gctcgcggga cccctgctcc 660

aacgtgacct gcagcttcgg cagcacctgt gcgcgctcgg ccgacgggct gacggcctcg 720

tgcctgtgcc ccgcgacctg ccgtggcgcc cccgagggga ccgtctgcgg cagcgacggc 780

gccgactacc ccggcgagtg ccagctcctg cgccgcgcct gcgcccgcca ggagaatgtc 840

ttcaagaagt tcgacggccc ttgtgacccc tgtcagggcg ccctccctga cccgagccgc 900

agctgccgtg tgaacccgcg cacgcggcgc cctgagatgc tcctacggcc cgagagctgc 960

cctgcccggc aggcgccagt gtgtggggac gacggagtca cctacgaaaa cgactgtgtc 1020

atgggccgat cgggggccgc ccggggtctc ctcctgcaga aagtgcgctc cggccagtgc 1080

cagggtcgag accagtgccc ggagccctgc cggttcaatg ccgtgtgcct gtcccgccgt 1140

ggctga 1146

<210> 23

<211> 873

<212> DNA

<213>people Agrin (Agrin-411)

<400> 23

cgtccccgct gctcctgcga ccgcgtcacc tgtgacgggg cctacaggcc cgtgtgtgcc 60

caggacgggc gcacgtatga cagtgattgc tggcggcagc aggctgagtg ccggcagcag 120

cgtgccatcc ccagcaagca ccagggcccg tgtgaccagg ccccgtcccc atgcctcggg 180

gtgcagtgtg catttggggc gacgtgtgct gtgaagaacg ggcaggcagc gtgtgaatgc 240

ctgcaggcgt gctcgagcct ctacgatcct gtgtgcggca gcgacggcgt cacatacggc 300

agcgcgtgcg agctggaggc cacggcctgt accctcgggc gggagatcca ggtggcgcgc 360

aaaggaccct gtgaccgctg cgggcagtgc cgctttggag ccctgtgcga ggccgagacc 420

gggcgctgcg tgtgcccctc tgaatgcgtg gctttggccc agcccgtgtg tggctccgac 480

gggcacacgt accccagcga gtgcatgctg cacgtgcacg cctgcacaca ccagatcagc 540

ctgcacgtgg cctcagctgg accctgtgag acctgtggag atgccgtgtg tgcttttggg 600

gctgtgtgct ccgcagggca gtgtgtgtgt ccccggtgtg agcacccccc gcccggcccc 660

gtgtgtggca gcgacggtgt cacctacggc agtgcctgcg agctacggga agccgcctgc 720

ctccagcaga cacagatcga ggaggcccgg gcagggccgt gcgagcaggc cgagtgcggt 780

tccggaggct ctggctctgg ggaggacggt gactgtgagc aggagctgtg ccggcagcgc 840

ggtggcatct gggacgagga ctcggaggac tga 873

<210> 24

<211> 1203

<212> DNA

<213>people Agrin (Agrin-701)

<400> 24

gggccgtgtg tctgtgactt cagctgccag agtgtcccag gcagcccggt gtgcggctca 60

gatggggtca cctacagcac cgagtgtgag ctgaagaagg ccaggtgtga gtcacagcga 120

gggctctacg tagcggccca gggagcctgc cgaggcccca ccttcgcccc gctgccgcct 180

gtggccccct tacactgtgc ccagacgccc tacggctgct gccaggacaa tatcaccgca 240

gcccggggcg tgggcctggc tggctgcccc agtgcctgcc agtgcaaccc ccatggctct 300

tacggcggca cctgtgaccc agccacaggc cagtgctcct gccgcccagg tgtggggggc 360

ctcaggtgtg accgctgtga gcctggcttc tggaactttc gaggcatcgt caccgatggc 420

cggagtggct gtacaccctg cagctgtgat ccccaaggcg ccgtgcggga tgactgtgag 480

cagatgacgg ggctgtgctc gtgtaagccc ggggtggctg gacccaagtg tgggcagtgt 540

ccagacggcc gtgccctggg ccccgcgggc tgtgaagctg acgcttctgc gcctgcgacc 600

tgtgcggaga tgcgctgtga gttcggtgcg cggtgcgtgg aggagtctgg ctcagcccac 660

tgtgtctgcc cgatgctcac ctgtccagag gccaacgcta ccaaggtctg tgggtcagat 720

ggagtcacat acggcaacga gtgtcagctg aagaccatcg cctgccgcca gggcctgcaa 780

atctctatcc agagcctggg cccgtgccag gaggctgttg ctcccagcac tcacccgaca 840

tctgcctccg tgactgtgac caccccaggg ctcctcctga gccaggcact gccggccccc 900

cccggcgccc tccccctggc tcccagcagt accgcacaca gccagaccac ccctccgccc 960

tcatcacgac ctcggaccac tgccagcgtc cccaggacca ccgtgtggcc cgtgctgacg 1020

gtgcccccca cggcaccctc ccctgcaccc agcctggtgg cgtccgcctt tggtgaatct 1080

ggcagcactg atggaagcag cgatgaggaa ctgagcgggg accaggaggc cagtgggggt 1140

ggctctgggg ggctcgagcc cttggagggc agcagcgtgg ccacccctgg gccacctgtc 1200

tga 1203

<210> 25

<211> 543

<212> DNA

<213>people Agrin (Agrin-1103)

<400> 25

gcttcctgct acaactccgc gttgggctgc tgctctgatg ggaagacgcc ctcgctggac 60

gcagagggct ccaactgccc cgccaccaag gtgttccagg gcgtcctgga gctggagggc 120

gtcgagggcc aggagctgtt ctacacgccc gagatggctg accccaagtc agaactgttc 180

ggggagacag ccaggagcat tgagagcacc ctggacgacc tcttccggaa ttcagacgtc 240

aagaaggatt ttcggagtgt ccgcttgcgg gacctggggc ccggcaaatc cgtccgcgcc 300

attgtggatg tgcactttga ccccaccaca gccttcaggg cacccgacgt ggcccgggcc 360

ctgctccggc agatccaggt gtccaggcgc cggtccttgg gggtgaggcg gccgctgcag 420

gagcacgtgc gatttatgga ctttgactgg tttcctgcgt ttatcacggg ggccacgtca 480

ggagccattg ctgcgggagc cacggccaga gccaccactg catcgcgcct gccgtcctct 540

taa 543

<210> 26

<211> 687

<212> DNA

<213>people Agrin (Agrin-1281)

<400> 26

tcctctgctg tgacccctcg ggccccgcac cccagtcaca caagccagcc cgttgccaag 60

accacggcag cccccaccac acgtcggccc cccaccactg cccccagccg tgtgcccgga 120

cgtcggcccc cggcccccca gcagcctcca aagccctgtg actcacagcc ctgcttccac 180

ggggggacct gccaggactg ggcattgggc gggggcttca cctgcagctg cccggcaggc 240

aggggaggcg ccgtctgtga gaaggtgctt ggcggggacc acccctgcct gcccaacccc 300

tgccatggcg gggccccatg ccagaacctg gaggctggaa ggttccattg ccagtgcccg 360

cccggccgcg tcggaccaac ctgtgccgat gagaagagcc cctgccagcc caacccctgc 420

catggggcgg cgccctgccg tgtgctgccc gagggtggtg ctcagtgcga gtgccccctg 480

gggcgtgagg gcaccttctg ccagacagcc tcggggcagg acggctctgg ggcaggtcac 540

ccctgcaccc gggcctcagg ccacccctgc ctcaatgggg cctcctgcgt cccgagggag 600

gctgcctatg tgtgcctgtg tcccggggga ttctcaggac cgcactgcga gaaggggctg 660

gtggagaagt cagcggggga cgtggat 687

<210> 27

<211> 567

<212> DNA

<213>people Agrin (Agrin-1635)

<400> 27

cccttcctgg ctgacttcaa cggcttctcc cacctggagc tgagaggcct gcacaccttt 60

gcacgggacc tgggggagaa gatggcgctg gaggtcgtgt tcctggcacg aggccccagc 120

ggcctcctgc tctacaacgg gcagaagacg gacggcaagg gggacttcgt gtcgctggca 180

ctgcgggacc gccgcctgga gttccgctac gacctgggca agggggcagc ggtcatcagg 240

agcagggagc cagtcaccct gggagcctgg accagggtct cactggagcg aaacggccgc 300

aagggtgccc tgcgtgtggg cgacggcccc cgtgtgttgg gggagtcccc gaaatcccgc 360

aaggttccgc acaccgtcct caacctgaag gagccgctct acgtaggggg cgctcccgac 420

ttcagcaagc tggcccgtgc tgctgccgtg tcctctggct tcgacggtgc catccagctg 480

gtctccctcg gaggccgcca gctgctgacc ccggagcacg tgctgcggca ggtggacgtc 540

acgtcctttg caggtcaccc ctgctga 567

<210> 28

<211> 618

<212> DNA

<213>people Agrin (Agrin-1864)

<400> 28

tcagcggggg acgtggatac cttggccttt gacgggcgga cctttgtcga gtacctcaac 60

gctgtgaccg agagcgaact ggccaatgag atccccgtcc ccgaaactct ggattccggg 120

gcccttcaca gcgagaaggc actgcagagc aaccactttg aactgagcct gcgcactgag 180

gccacgcagg ggctggtgct ctggagtggc aaggccacgg agcgggcaga ctatgtggca 240

ctggccattg tggacgggca cctgcaactg agctacaacc tgggctccca gcccgtggtg 300

ctgcgttcca ccgtgcccgt caacaccaac cgctggttgc gggtcgtggc acatagggag 360

cagagggaag gttccctgca ggtgggcaat gaggcccctg tgaccggctc ctccccgctg 420

ggcgccacgc agctggacac tgatggagcc ctgtggcttg ggggcctgcc ggagctgccc 480

gtgggcccag cactgcccaa ggcctacggc acaggctttg tgggctgctt gcgggacgtg 540

gtggtgggcc ggcacccgct gcacctgctg gaggacgccg tcaccaagcc agagctgcgg 600

ccctgcccca ccccatga 618

Claims (10)

1. a kind of people Agrin antigen, which is characterized in that include any of the following segment or seven kinds of segments:

Segment 1:Agrin-40, amino acid sequence is as shown in SEQ ID NO.1；

Segment 2:Agrin-411, amino acid sequence is as shown in SEQ ID NO.2；

Segment 3:Agrin-701, amino acid sequence is as shown in SEQ ID NO.3；

Segment 4:Agrin-1103, amino acid sequence is as shown in SEQ ID NO.4；

Segment 5:Agrin-1281, amino acid sequence is as shown in SEQ ID NO.5；

Segment 6:Agrin-1635, amino acid sequence is as shown in SEQ ID NO.6；

Segment 7:Agrin-1864, amino acid sequence is as shown in SEQ ID NO.7.

2. a kind of preparation method of people Agrin antigen described in claim 1, which comprises the steps of:

Step 1, by Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and The DNA sequence dna of Agrin-1864 totally 7 segments carries out gene chemical synthesis respectively, after design primer PCR amplification, is separately connected into expression Carrier constructs 7 recombinant expression plasmids；

Step 2 converts the recombinant plasmid built respectively into expression bacterium, constructs 7 recombinant expression engineering bacterias；

Step 3, Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and The inducing expression and purifying of the antigen fragment of Agrin-1864.

3. preparation method as claimed in claim 2, which is characterized in that the primer pair difference of 7 segments described in the step 1 Are as follows:

Agrin-40-P1: nucleotide sequence is as shown in SEQ ID NO.8；

Agrin-40-P2: nucleotide sequence is as shown in SEQ ID NO.9；

Agrin-411-P1: nucleotide sequence is as shown in SEQ ID NO.10；

Agrin-411-P2: nucleotide sequence is as shown in SEQ ID NO.11；

Agrin-701-P1: nucleotide sequence is as shown in SEQ ID NO.12；

Agrin-701-P2: nucleotide sequence is as shown in SEQ ID NO.13；

Agrin-1103-P1: nucleotide sequence is as shown in SEQ ID NO.14；

Agrin-1103-P2: nucleotide sequence is as shown in SEQ ID NO.15；

Agrin-1281-P1: nucleotide sequence is as shown in SEQ ID NO.16；

Agrin-1281-P2: nucleotide sequence is as shown in SEQ ID NO.17；

Agrin-1635-P1: nucleotide sequence is as shown in SEQ ID NO.18；

Agrin-1635-P2: nucleotide sequence is as shown in SEQ ID NO.19；

Agrin-1864-P1: nucleotide sequence is as shown in SEQ ID NO.20；

Agrin-1864-P2: nucleotide sequence is as shown in SEQ ID NO.21.

4. preparation method as claimed in claim 2, which is characterized in that the reaction system of PCR amplification in the step 1 are as follows: H₂O 38.7ul；Buffer 5ul；dNTP 3ul；Upper primer 1ul；Lower primer 1ul；DNA 1ul；Taq E 0.3ul；Amplification program Are as follows: 94 degree of denaturation 5min；94 degree of denaturation 45sec, 57 degree of 150sec, 72 degree of 90sec, 32 circulations；72 degree of extension 10min.

5. preparation method as claimed in claim 2, which is characterized in that the specific steps of inducing expression in the step 3 are as follows: will 7 recombinant bacteriums are inoculated in respectively when shaking bacterium to OD600 to 0.6-0.8 in LB culture solution, and 24mg/ml concentration is added by 1:1000 IPTG is induced 4-6 hours；

Purification condition in the step 3 after inducing expression is: sample-loading buffer: 0.5M NaCl, 20mM Na₂HPO₃, 10mM miaow Azoles；Or sample-loading buffer: 8M urea, 0.5M NaCl, 20mM Na₂HPO₃, 10mM imidazoles；Combination buffer: 0.5M NaCl, 20mM Na₂HPO₃, 20mM imidazoles；Elution buffer: 0.5M NaCl, 20mM Na₂HPO₃, 500mM imidazoles.

6. a kind of expression vector of people Agrin antigen, which is characterized in that the expression vector is 7,7 expression vectors The nucleotide sequence in expression area be respectively as follows: such as SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, shown in SEQ ID NO.28.

7. a kind of people Agrin antibody assay kit, which is characterized in that the detection kit includes:

(A) it is coated with the ELISA ELISA Plate of people Agrin antigen described in claim 1；The people Agrin antigen includes In Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864 Any one or seven kinds；

(B) standard female serum: the serum of Agrin negative antibody；

(C) standard positive serum: the serum of Agrin antibody positive；

(D) ELIAS secondary antibody of horseradish peroxidase-labeled: anti-human IGG, IGM and IGA；

(E) sample diluting liquid, coating buffer, confining liquid, ELISA ELISA Plate cleaning solution, antibody diluent, developing solution and termination Liquid.

8. people Agrin antibody assay kit as claimed in claim 7, which is characterized in that in the people Agrin antigen The packet of Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864 It is respectively 200ng/ml, 200ng/ml, 250ng/ml, 250ng/ml, 300ng/ml, 150ng/ml and 150ng/ml by concentration.

9. a kind of detection method of people Agrin antibody, which is characterized in that the detection method comprises the following steps:

S1, preparation detection ELISA ELISA Plate: coating buffer will be added to enzyme after people Agrin antigen diluent described in claim 1 It is adsorbed in target, the dry coating washing lotion of sky adds coating to be closed with confining liquid；The people Agrin antigen include Agrin-40, Any one in Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864 Or seven kinds；

Respectively as primary antibody, sample-adding is incubated for into ELISA ELISA Plate hole for S2, serum to be checked, negative serum, positive serum；

The incubation of S3, ELIAS secondary antibody: ELISA ELISA Plate is added in the ELIAS secondary antibody of horseradish peroxidase-labeled, and cleaning solution washs, Drying；

S4, developing solution is added, room temperature is protected from light incubation, and terminate liquid is added and terminates reaction；OD is measured under 450nm wavelength in microplate reader Value.

10. application of the detection kit as claimed in claim 7 in diagnosing myasthenia myasthenia disease.