CN109734790B

CN109734790B - Human Agrin antigen, human Agrin antibody detection kit, preparation method and application thereof

Info

Publication number: CN109734790B
Application number: CN201910043457.3A
Authority: CN
Inventors: 张崇珍; 王颖; 郝洪军
Original assignee: Wuhan Easydiagnosis Biomedicine Co ltd
Current assignee: Wuhan Easydiagnosis Biomedicine Co ltd
Priority date: 2019-01-17
Filing date: 2019-01-17
Publication date: 2022-05-24
Anticipated expiration: 2039-01-17
Also published as: CN109734790A

Abstract

The invention provides a human Agrin antigen, an ELISA detection kit, a preparation method and application thereof. The human Agrin antigen comprises amino acid sequences shown by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, seven detection kits respectively containing the seven human Agrin antigens and 1 detection kit simultaneously containing the seven human Agrin antigens are also provided. The human Agrin detection kit is used for detecting the Agrin antibody in human serum, has strong specificity, high reaction sensitivity, high flux and low cost, can diagnose myasthenia gravis, and is suitable for large-scale popularization and application.

Description

Human Agrin antigen, human Agrin antibody detection kit, preparation method and application thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a human Agrin antigen, a human Agrin antibody detection kit, a preparation method and application thereof.

Background

Myasthenia Gravis (MG), a common muscle nerve junction (NMJ) disease, is a common disease of the muscular nerve junction. In most patients, MG may result from an autoimmune response to acetylcholine receptors (AChRs), and autoantibodies to AChRs can be detected in 80% to 85% of MG patients. However, AChR antibody was not detected in about 20% of MG patients. There is evidence that these "seronegative" patients can produce protein antibodies that are critical for NMJ formation or maintenance. The aggrecan, Agrin, released from motor neurons, binds to low density lipoprotein receptor-related protein 4(LRP4), activating the receptor tyrosine kinase, MuSK, to direct NMJ formation. Approximately 40% -70% of seronegative patients have MuSK autoantibodies. The remaining 6% -12% of MG patients had a double seronegative response to AChR and MuSK antibodies. Agrin is a heparan sulfate proteoglycan released from motor nerve terminals. Which modulate the formation, maintenance and regeneration of neuromuscular junctions and interfere with collectin function, resulting in neuromuscular transmission deficits antibodies to collectin in a minority of MG patients can be detected with or without antibodies to AChR, MuSK or LRP 4. The aggrecan antibodies were only detected in MG patients, suggesting that these antibodies are specific for MG. At present, no report related to a human Agrin antibody detection kit exists.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a human Agrin antigen and a preparation method thereof, a human Agrin antibody detection kit and a preparation method and application thereof, and the detection kit can specifically detect LRP4 antibody in human serum, has diagnostic significance on severe myasthenia, is sensitive in reaction, low in cost and suitable for large-scale popularization and application.

The invention is realized by the following steps:

one of the purposes of the invention is a human Agrin antigen, which comprises any one of the following fragments or seven fragments:

fragment 1: agrin-40, the amino acid sequence is shown as SEQ ID NO. 1;

fragment 2: agrin-411, the amino acid sequence is shown as SEQ ID NO. 2;

fragment 3: the amino acid sequence of the Agrin-701 is shown as SEQ ID NO. 3;

fragment 4: agrin-1103, the amino acid sequence is shown in SEQ ID NO. 4;

fragment 5: agrin-1281, the amino acid sequence is shown in SEQ ID NO. 5;

fragment 6: agrin-1635, the amino acid sequence is shown in SEQ ID NO. 6;

fragment 7: the amino acid sequence of the Agrin-1864 is shown in SEQ ID NO. 7.

The invention also aims to provide a preparation method of the human Agrin antigen, which comprises the following steps:

step 1, carrying out gene synthesis on DNA sequences of 7 segments of Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864 respectively, designing primers, carrying out PCR amplification, and then respectively connecting the primers into expression vectors to construct recombinant expression plasmids;

step 2, transforming the constructed recombinant plasmid into an expression bacterium to construct a recombinant expression engineering bacterium;

and 3, inducing expression and purification of the human Agrin antigen.

Specifically, the primer pairs of the 7 fragments in step 1 are respectively:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Specifically, the reaction system for PCR amplification in step 1 is: h₂O38.7 ul; buffer 5 ul; dNTP 3 ul; 1ul of upper primer; 1ul of lower primer; 1ul of DNA; taq E0.3 ul; the amplification procedure was: denaturation at 94 deg.C for 5 min; 94 degrees of denaturation 45sec, 57 degrees of 150sec, 72 degrees of 90sec, 32 cycles; the extension is 72 degrees for 10 min.

Specifically, the specific steps of inducing expression in step 3 are as follows: respectively inoculating 7 recombinant bacteria into LB culture solution, shaking to OD600 to 0.6-0.8, adding IPTG with the concentration of 24mg/ml according to the ratio of 1:1000, and inducing for 4-6 hours.

Specifically, the purification conditions after the induction of expression in step 3 are as follows: loading buffer solution: 0.5M NaCl, 20mM Na₂HPO₃10mM imidazole; binding buffer: 0.5M NaCl, 20mM Na₂HPO₃20mM imidazole; elution buffer: 0.5M NaCl, 20mM Na₂HPO₃500mM imidazole.

The invention also aims to provide an expression vector of human Agrin antigen, wherein the number of the expression vectors is 7, and the nucleotide sequences of expression regions of the 7 expression vectors are respectively as follows: as shown in SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO. 28.

The fourth purpose of the invention is to provide 7 engineering bacteria for expressing the human Agrin antigens, wherein the 7 engineering bacteria respectively comprise expression vectors of the 7 human Agrin antigens.

The fifth purpose of the invention is to provide a human Agrin antibody detection kit, wherein the ELISA detection kit comprises:

(A) the ELISA plate is coated with the human Agrin antigen; the human Agrin antigen comprises any one or seven of Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864;

(B) standard negative sera: serum negative for Agrin antibody;

(C) standard positive sera: serum positive for Agrin antibody;

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

(E) sample diluent, coating buffer solution, confining liquid, ELISA plate washing liquid, antibody diluent, developing liquid and stop solution.

Wherein the coating concentrations of the human Agrin antigens, namely Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864 are respectively 200ng/ml, 250ng/ml, 300ng/ml, 150ng/ml and 150 ng/ml.

The sixth purpose of the invention is to provide a detection method of a human Agrin antibody, which comprises the following steps:

s1, preparing and detecting an ELISA plate: diluting the human Agrin antigen of claim 1 by using a coating buffer solution, adding the diluted human Agrin antigen into an enzyme label plate for adsorption, drying the coating in the air, using a washing solution, and sealing the coating by using a sealing solution; the human Agrin antigen comprises any one or seven of Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864;

s2, taking the serum to be detected, the negative serum and the positive serum as primary antibodies respectively, and adding the primary antibodies into an ELISA plate hole for incubation;

s3, incubation of enzyme-labeled secondary antibody: adding an enzyme-linked immunosorbent assay (ELISA) second antibody marked by horse radish peroxidase into an ELISA plate, washing with a washing solution, and spin-drying;

s4, adding a color development solution, incubating at room temperature in a dark place, and adding a stop solution to stop the reaction; OD values were determined on a microplate reader at a wavelength of 450 nm.

The seventh purpose of the invention is to provide the application of the detection kit in diagnosing Myasthenia Gravis (MG) diseases.

The invention has the beneficial effects that:

the invention provides a human Agrin antigen, a human Agrin antibody detection kit and a detection method, wherein the human Agrin antigen (Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864) is prepared firstly, and then the human Agrin antigen is used as a coating antigen to carry out indirect ELISA detection on sample serum.

Drawings

FIG. 1 is an enzyme-cleaved diagram of a constructed recombinant plasmid provided in example 1 of the present invention; wherein (A) is an enzyme cutting diagram of recombinant plasmid containing Agrin-40; (B) is an enzyme map of a recombinant plasmid containing Agrin-411; (C) is an enzyme map of a recombinant plasmid containing Agrin-701; (D) is an enzyme cutting picture of a recombinant plasmid containing Agrin-1103; (E) is an enzyme cutting chart of a recombinant plasmid containing the Agrin-1281; (F) is an enzyme cutting picture of a recombinant plasmid containing the Agrin-1635; (G) is an enzyme cutting picture of a recombinant plasmid containing Agrin-1864; wherein Lane 1 is the plasmid without restriction enzyme, Lane 2 is the band cut by the corresponding endonuclease; (F) a Marker strip chart, wherein the Marker is 1Kb ladder;

FIG. 2 is an electrophoretogram of purified products of fragments of Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864, according to example 2 of the present invention.

Detailed Description

Example 1 construction of recombinant expression plasmids and engineering bacteria

1. Agrin is a heparan sulfate proteoglycan composed of multiple domains including 9 protein kinase inhibitor domains, 4 epidermal growth factor-like domains and 1 adhesion molecule G homeodomain. The mature Agrin protein consists of 2038 amino acids. The amino acid sequence of the Agrin protein is analyzed through hydrophilicity, surface accessibility and the like, and the fragments of 7 regions of the mature Agrin protein are selected to be obtained respectively by combining the space conformation and the modification characteristics of each structural domain. The inventor obtains seven human Agrin antigens, namely Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864 through a series of researches.

2. PCR amplification of human Agrin antigen gene

1. The DNA sequences of the 7 fragments were subjected to gene synthesis, and PCR amplification was performed using primers (with restriction sites) designed as shown in Table 1, with restriction endonuclease sites underlined.

TABLE 1

2. The PCR amplification system was used, and the temperature cycle parameters are shown in Table 2: 94 ℃, 5min → (94 ℃,45S, → 57 ℃,150S, → 72 ℃,90S) × 32 → 72 ℃,10 min. The amplified product is used for subsequent enzyme digestion enzyme connection.

TABLE 2

4. After PCR amplification, agarose gel electrophoresis is carried out to recover an amplification band, enzyme digestion and enzyme ligation are carried out, and 7 DNA fragments are respectively connected into a PET-28a expression vector to construct recombinant expression plasmids. The recombinant plasmid colloidases cleavage map is shown in the attached figure 1 of the specification.

5. The constructed recombinant plasmid is transformed into BL21(DE3) bacteria to construct a recombinant bacterium, and the result of the sequencing of the recombinant bacterium verifies that the construction of the recombinant plasmid in the embodiment is successful.

EXAMPLE 2 expression and purification of antigen

1. The constructed recombinant protein expression engineering bacteria are used for carrying out induction expression experiments. The 7 recombinant bacteria were inoculated into 600ml of LB medium (ingredients: 10g sodium chloride/liter, 10g peptone/liter and 5g yeast extract/liter), shaken at 37 ℃ and 200RPM until OD600 reached 0.6-0.8, and then induced for 4 hours by adding IPTG (isopropyl thiogalactoside) at a concentration of 24mg/ml at a ratio of 1: 1000. Centrifuging, collecting bacteria, and preparing for purification.

2. The packing material selected for purification was GE Ni Sepharose (cat number 17-0729-10) and the following solutions were prepared separately according to the specifications:

loading buffer A: 0.5M NaCl +20mM Na₂HPO₃+10mM imidazole.

Binding buffer B: 0.5M NaCl +20mM Na₂HPO₃+20mM imidazole

Elution buffer C: 0.5M NaCl, 20mM Na₂HPO₃500mM imidazole;

and the solution used for purification of inclusion bodies:

loading buffer for purification of inclusion body antigen a: 0.5M NaCl, 20mM Na₂HPO₃20mM imidazole, 8M urea;

binding buffer b: 0.5M NaCl, 20mM Na₂HPO₃20mM imidazole, 8M urea;

elution buffer c: 0.5M NaCl, 20mM Na₂HPO₃300mM imidazole, 8M urea.

3. The 7 centrifugally collected bacteria were dispersed uniformly with the loading buffer A, sonicated (250W for over 3s, 3s apart, 20min throughout), and centrifuged for the first time (12000RPM, 15min, 4 ℃ C.) to obtain a supernatant containing the desired antigen at a higher concentration. The inclusion body antigen is prepared by uniformly re-dispersing the precipitate obtained by the first centrifugation by using a loading buffer solution a, performing ultrasonic treatment (250W, 3s of ultra, 3s of interval, 20min of the whole process) and re-centrifuging (12000RPM, 15min, 4 ℃), and discarding the precipitate to obtain a solution containing the target antigen with higher concentration. And (3) respectively carrying out loading, washing and elution on the packed column by using the solution of the 7 target proteins to respectively obtain the 7 target proteins. The purified inclusion body antigen needs to be renatured, renaturation is continuously dialyzed by adopting renaturation buffer solutions with the same volume with the inclusion body antigen to be renatured and different urea concentrations (4.5M, 3.5M, 2.5M, 1.5M, 0.5M and 0M), and each renaturation buffer solution is dialyzed for 4 hours.

Performing SDS-PAGE electrophoresis on a 12% concentration gel by using a soluble antigen (Agrin-609) and renatured inclusion body antigens (Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864), and detecting the purity of seven target proteins as follows: 93.5%, 94.9%, 93.4%, 94.5%, 94.1%, 94.8%, and 95.1%. See figure 2 of the specification. Each antigen was stored for later use after concentration determination.

Example 3 human Agrin antibody detection kit and method of use

1. Coated enzyme label plate

(1) Coating liquid: NaCl 8.5g, Na₂HPO₄·12H₂O 30.8g，KH₂PO₄2.2g, plus ddH₂O to 1000ml, adjust pH to 7.4.

(2) Coating lotion: NaCl 8.0g, KH₂PO₄0.24g、Na₂HPO₄·12H₂O2.9 g, KCl 0.2g, TWEEN200.5ml, to ddH₂O to 1000ml, adjusted to pH 7.4.

(3) The coating method comprises the following steps: 7 fragments of the Agrin antigen were coated in wells of an microplate with 0.1M PBS (pH7.4) coating buffer at 4 ℃ overnight, wherein the coating concentrations of Agrin-25, Agrin-344, Agrin-530, Agrin-824, and Agrin-1048 were 200ng/ml, 250ng/ml, 150ng/ml, and 250ng/ml, respectively. Adding 100 mu L of the enzyme label plate into each hole of the 96-hole enzyme label plate, and adsorbing for 24 hours at the temperature of 2-8 ℃. The coating solution was emptied and the plates were washed 3 times with wash solution.

2. Sealing of

(1) Sealing liquid for coating: na (Na)₂HPO₄·12H₂O 3.582g，NaH₂PO₄·2H₂O1.561 g, NaCl 9.0g, BSA20g, xylose 10g, pH adjusted to 7.2, volume to 1000 ml.

(2) And (3) sealing operation: the bags were air-dried with the wash solution, blocked with 1.5% BSA blocking solution at 37 ℃ for 2 hours, or left overnight at 2-8 ℃. Removing the sealing liquid, naturally drying, and sealing.

3. Incubation of Positive and negative serum (Primary antibody incubation)

Diluting the serum to be detected by 10-100 times, respectively adding the diluted serum into a batten of 7 antigens (each antigen has optimized specific serum dilution), adding a positive control and a negative control, incubating at 37 ℃ for 1 hour, and washing the plate by using a washing solution, wherein the formula of the washing solution is as follows:

washing solution (0.15M): NaCl 8.0g, KH₂PO₄0.24g、Na₂HPO₄·12H₂O2.9 g, KCl 0.2g, TWEEN200.5ml, to ddH₂O to 1000ml, adjusted to pH 7.4.

The operation is as follows: the serum to be detected is diluted by PBS as serum diluent according to the proportion of 1:400 times, and added into the hole of the coating plate with 100 mu L/hole. The standard positive serum or the standard negative serum is directly sucked and added into a coated plate hole, and the volume is 100 mu L/hole. The plate was placed on an ELISA plate at 37 ℃ for 30 min.

4. Incubation of enzyme-labeled Secondary antibodies

Adding horseradish peroxidase-labeled secondary antibodies (anti-human IGG, IGM and IGA) with a certain concentration, adding 100 mu L/hole into the wells of the enzyme label plate, placing at 37 ℃ for 15min, and washing the plate.

5. Color development

The substrate solution A50. mu.L and the substrate solution B50. mu.L were added thereto, and the mixture was gently shaken and reacted at 37 ℃ for 15 min.

(1) Substrate solution a: 13.6g of sodium acetate, 1.6g of citric acid, 0.3ml of 30% hydrogen peroxide and distilled water added to 500ml

(2) Substrate solution B: 0.2g of TMB is dissolved in 20ml of DMSO, 0.2g of disodium ethylenediamine tetraacetic acid, 0.95g of citric acid, 50ml of glycerol and distilled water are added to 500 ml.

6. And (4) terminating:

stopping liquid: 2mol/L H₂SO₄。

After the color development was completed, 50ul of stop solution was added to each well to terminate the reaction.

7. Reading a plate:

OD450 values were measured with a microplate reader.

The serum was tested on 7 antigen panels, and when the OD450 value of any panel reached a positive value, the serum was judged to be positive for Agrin and positive for the antigen fragment. Antibodies to Agrin and the Agrin fragments are present in the serum individual. The 7 detection kits prepared from the 7 antigens can be used separately or in combination.

The human Agrin antibody detection kit provided by the invention can also be used for preparing products in various forms such as immune colloidal gold detection test strips, immunofluorescence, immunoturbidimetry, chemiluminescence and the like.

Example 4 application of human Agrin antibody detection kit

Application of

1. 613 sera from patients with autoimmune diseases of the nervous system, such as CIDP, Guillain-Barre GBS and myasthenia gravis MG, were tested in example 3, wherein the sera from CIDP patients with chronic inflammatory demyelinating polyneuritis were tested clinically in hospitals to be CIDP antibody positive sera, and 300 normal human sera were tested in the same batch.

2. The experimental results are shown in the following table (only 60 samples are listed), and the detection result shows that the Agrin-1635 antibody detection kit is provided. The positive rate of the serum of MG patients reaches 25%, the positive rate of the serum of CIDP positive patients reaches 10%, the positive rate of the serum of normal patients reaches 5%, and the critical value is 0.49.

TABLE 3 sample data test results

4. In addition, the detection of Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864 also shows that the positive proportion of the serum in MG patients is more than 3 times of that of normal people. Results single-factor analysis of variance was performed using SPSS statistical analysis software 17.0 and multiple comparison tests were performed using Student-Newman-Keuls. The results are given in the following table:

TABLE 4 statistical analysis of test results Table

Note: the data were analyzed by multiple comparisons using the Student-Newman-Keuls test method of the One-WayANOVA program of SPSS17.0 statistical analysis software, with significant differences between groups with different letter shoulder numbers (a, b, c) in the same column (P < 0.05).

Technical indexes of two and 7 ELISA detection kits

1. Critical value: the method for determining the validity of the test, the method for calculating the CUT-OFF value (CUT OFF), and the method for determining the negative or positive of the sample were determined by examining the serum and the serum of the normal human patient according to example 3.

(1)Agrin-40：

And (3) testing validity judgment: the average value of the positive control holes is more than or equal to 0.53; the average value of the negative control is less than or equal to 0.28;

cutoff value-negative control well mean + 0.13-0.41;

and (4) negative judgment: the sample with OD value less than the critical value (0.41) is negative to the Agrin-40 antibody;

and (3) positive judgment: the sample with the OD value larger than or equal to the critical value (0.41) is positive for the Agrin-40 antibody.

(2)Agrin-411：

And (3) testing validity judgment: the average value of the positive control holes is more than or equal to 0.53; the average value of the negative control is less than or equal to 0.29;

cutoff value + mean of negative control wells + 0.14-0.43;

and (4) negative judgment: the sample with OD value less than the critical value (0.43) is negative to the Agrin-411 antibody;

and (3) positive judgment: the sample with the OD value larger than or equal to the critical value (0.43) is positive for the Agrin-411 antibody.

(3)Agrin-701：

cutoff value + mean of negative control wells + 0.14-0.43;

and (4) negative judgment: the sample with OD value less than the critical value (0.43) is negative to the Agrin-701 antibody;

and (3) positive judgment: the sample with the OD value larger than or equal to the critical value (0.43) is positive for the Agrin-701 antibody.

(4)Agrin-1103：

And (3) testing validity judgment: the average value of the positive control holes is more than or equal to 0.53; the average value of negative control is less than or equal to 0.25;

cutoff value is the average of negative control wells + 0.14;

and (4) negative judgment: the sample with OD value less than the critical value (0.39) is negative to the Agrin-1103 antibody;

and (3) positive judgment: the sample with the OD value larger than or equal to the critical value (0.39) is positive for the Agrin-1103 antibody.

(5)Agrin-1281：

And (3) testing validity judgment: the average value of the positive control holes is more than or equal to 0.53; the average value of the negative control is less than or equal to 0.26;

threshold value +0.14 to 0.40 for the mean negative control wells;

and (4) negative judgment: the sample with OD value less than the critical value (0.40) is negative to the Agrin-1281 antibody;

and (3) positive judgment: the sample with the OD value larger than or equal to the critical value is positive by the (0.40) Agrin-1281 antibody.

(6)Agrin-1635：

And (3) testing validity judgment: the average value of the positive control holes is more than or equal to 0.53; the average value of the negative control is less than or equal to 0.36;

cutoff value negative control wells mean +0.13 to 0.49;

and (4) negative judgment: the sample with OD value less than the critical value (0.49) is negative to the Agrin-1635 antibody;

and (3) positive judgment: the sample with OD value larger than or equal to the critical value (0.49) is positive for the Agrin-1635 antibody.

(7)Agrin-1864：

And (3) test effectiveness judgment: the average value of the positive control holes is more than or equal to 0.53; the average value of negative control is less than or equal to 0.21;

cutoff value + mean of negative control wells + 0.13-0.34;

and (4) negative judgment: the sample with OD value less than the critical value (0.34) is negative to the Agrin-1864 antibody;

and (3) positive judgment: the sample with OD value larger than or equal to the critical value (0.34) is positive for the Agrin-1864 antibody.

2. Specificity: except for positive serum, other test samples were negative. These data indicate that there is no cross-reaction between the kits provided by the invention and other serum antibodies.

3. Sensitivity: a12800 fold dilution of positive serum was detected (i.e., 1. mu.L of serum was added to 12800. mu.L of sample dilution, 100. mu.L of which was added to the sample well).

4. Stability: the kit is placed in a kit stored at 37 ℃ for not less than 2 days and 4 ℃ for synchronously detecting 20 samples, and the coincidence rate is 100%.

5. Precision: taking 7 serum samples with gradient concentrations, diluting respectively, and determining for 10 times in the same batch respectively, wherein the variation coefficients in the batch are all lower than 4%; 7 parts of serum are measured for 10 times every other day, and the variation is lower than 5%; meets the precision requirement of the kit.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Wuhanming Germany Biotechnology GmbH

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165 170 175

His Gln Ile Ser Leu His Val Ala Ser Ala Gly Pro Cys Glu Thr Cys

180 185 190

Gly Asp Ala Val Cys Ala Phe Gly Ala Val Cys Ser Ala Gly Gln Cys

195 200 205

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

210 215 220

Asp Gly Val Thr Tyr Gly Ser Ala Cys Glu Leu Arg Glu Ala Ala Cys

225 230 235 240

Leu Gln Gln Thr Gln Ile Glu Glu Ala Arg Ala Gly Pro Cys Glu Gln

245 250 255

Ala Glu Cys Gly Ser Gly Gly Ser Gly Ser Gly Glu Asp Gly Asp Cys

260 265 270

Glu Gln Glu Leu Cys Arg Gln Arg Gly Gly Ile Trp Asp Glu Asp Ser

275 280 285

Glu Asp

290

<210> 3

<211> 400

<212> PRT

<213> human Agrin (Agrin-701)

<400> 3

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> human 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> human 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> human 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> human 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> human 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> human 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> human 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> human 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> human 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> human 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> human 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

1. A preparation method of a human Agrin antigen is characterized in that the human Agrin antigen consists of any one or seven segments as follows:

fragment 1: agrin-40, the amino acid sequence is shown as SEQ ID NO. 1;

fragment 2: agrin-411, the amino acid sequence is shown as SEQ ID NO. 2;

fragment 3: the amino acid sequence of the Agrin-701 is shown as SEQ ID NO. 3;

fragment 4: agrin-1103, the amino acid sequence is shown in SEQ ID NO. 4;

fragment 5: agrin-1281, wherein the amino acid sequence is shown as SEQ ID NO. 5;

fragment 6: agrin-1635, the amino acid sequence is shown in SEQ ID NO. 6;

fragment 7: agrin-1864, the amino acid sequence is shown in SEQ ID NO. 7;

the preparation method comprises the following steps:

step 1, carrying out gene synthesis on DNA sequences of 7 segments including Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864 respectively, designing primers, carrying out PCR amplification, and then respectively connecting the primers into expression vectors to construct 7 recombinant expression plasmids;

step 2, respectively transforming the constructed recombinant plasmids into expression bacteria, and constructing 7 recombinant expression engineering bacteria;

3, performing induced expression and purification on antigen fragments of the Agrin-40, the Agrin-411, the Agrin-701, the Agrin-1103, the Agrin-1281, the Agrin-1635 and the Agrin-1864;

the specific steps of inducing expression in the step 3 are as follows: respectively inoculating 7 recombinant bacteria into LB culture solution, shaking to OD600 to 0.6-0.8, adding IPTG with the concentration of 24mg/ml according to the ratio of 1:1000, and inducing for 4-6 hours.

2. The method according to claim 1, wherein the primer pairs for the 7 fragments in step 1 are:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

3. The method according to claim 1, wherein the reaction system for PCR amplification in step 1 is: h₂O38.7 ul; buffer 5 ul; dNTP 3 ul; 1ul of upper primer; 1ul of lower primer; 1ul of DNA; taq E0.3 ul; the amplification procedure was: denaturation at 94 deg.C for 5 min; 94 degrees of denaturation 45sec, 57 degrees of 150sec, 72 degrees of 90sec, 32 cycles; the extension is 72 degrees for 10 min.

4. The method according to claim 1, wherein the reaction mixture,

the purification conditions after the induction expression in the step 3 are as follows: loading buffer solution: 0.5M NaCl, 20mM Na₂HPO₃10mM imidazole; or loading buffer: 8M Urea, 0.5M NaCl, 20mM Na₂HPO₃10mM imidazole; binding buffer: 0.5M NaCl, 20mM Na₂HPO₃20mM imidazole; elution buffer: 0.5M NaCl, 20mM Na₂HPO₃500mM imidazole.

5. The expression vector of the human Agrin antigen prepared by the preparation method of claim 1, wherein the number of the expression vectors is 7, and the nucleotide sequences of the expression regions of the 7 expression vectors are respectively as follows: as shown in SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO. 28.

6. A human Agrin antibody detection kit, characterized in that, the detection kit includes:

(A) an ELISA microplate coated with the human Agrin antigen of claim 1; the human Agrin antigen comprises any one or seven of Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635 and Agrin-1864;

(B) standard negative sera: serum negative for Agrin antibody;

(C) standard positive sera: serum positive for Agrin antibody;

7. The human Agrin antibody detection kit of claim 6, wherein the human Agrin antigen has a coating concentration of Agrin-40, Agrin-411, Agrin-701, Agrin-1103, Agrin-1281, Agrin-1635, and Agrin-1864 of 200ng/ml, 250ng/ml, 300ng/ml, 150ng/ml, and 150ng/ml, respectively.

8. Use of the test kit according to claim 6 for the preparation of a test kit for the diagnosis of myasthenia gravis.