CN109678945A

CN109678945A - A kind of preparation method of serum amyloid A protein epitope, its prediction and verification method and monoclonal antibody

Info

Publication number: CN109678945A
Application number: CN201811560976.9A
Authority: CN
Inventors: 顾悦; 周俊花; 徐长银
Original assignee: Dia Lembo (zhangjiagang) Biotechnology Co Ltd
Current assignee: Dia Lembo (zhangjiagang) Biotechnology Co Ltd
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2019-04-26

Abstract

The present invention relates to a kind of prediction of serum amyloid A protein epitope and verification method and the preparation methods of monoclonal antibody, prediction is surface accessibility, elastomeric sequences region, β-bend, antigenicity, hydrophily, linear epitope, the long segment epitope for predicting serum amyloid A protein antigen with verification method, obtains multiple epitope sequences respectively；It removes the duplicate epitope in multiple epitope sequences and includes the sequence in other longer epitopes, obtain multiple nonredundancies prediction epitopes；ELISA is used whether to verify multiple nonredundancy prediction epitopes for the identification epitope of antibody.The present invention overcomes the low problems of single method predictablity rate；And by experimental verification, solve the problems, such as that SAA epitope is unknown；Monoclonal antibody is prepared using the antigen epitope polypeptide of synthesis, SAA is overcome and is difficult to purify the difficult problem of acquisition, reduce cost by one times or more.

Description

A kind of serum amyloid A protein epitope, its prediction and verification method and monoclonal The preparation method of antibody

Technical field

The invention belongs to biotechnologys to prepare in-vitro diagnosis raw material field, belong to in-vitro diagnosis industry the detection kit The raw material of preparation, and in particular to a kind of serum amyloid A protein epitope, its prediction and verification method and monoclonal antibody Preparation method.

Background technique

Humoral immunity or antibody generation are mainly mediated by B cell, and B cell identifies that antigen passes through membrane-bound antibody or B cell Receptor (B cell receptor, BCR) identifies antigen, in conjunction with antigenic site be B cell epitope, final induction is anti- The editor of body gene and the secretion of antibody, the antibodies bind antigen of secretion is to inactivate or eliminate antigen.

Immunoinformatics are that the Informatics Method of immune problem is applied and solved in immunology using computer approach, are One branch of bioinformatics.Two focus targets of Immunoinformatics are exactly the prediction of B cell epitope and t cell epitope, Very important prediction reference result is provided for practicable achievements conversion.Derived from the fast of high throughput sequencing technologies in recent years Exhibition is hailed, biological information researcher develops panimmunity Bioinformatic tool, and still, this tool is all independent for height The big data analysis that a certain specific feature or feature of flux data carry out, lacks the epitope analysis to specific antigen, more at present The practical applications such as screening verification and Antibody preparation are carried out to the epitope analyzed it is important that lacking.Serum amyloid protein at present The epitope (or B cell epitope) of A (Serum amyloid A, SAA) is tested without Immunoinformatics prediction and experiment screening Card.

Homeostasis is all necessary, and acute-phase response (acute- for all biosystems including people Phase response, APR) it is one of the mechanism that stable state is rebuild under the conditions of the original homeostasis of one kind is destroyed.Acute When phase reaction in serum variation most significantly serum amyloid A protein (Serum amyloid A, SAA) and c reactive protein (C-reactive protein,CRP)。

SAA is made of 104 amino acid, and after body is infected, SAA can increase rapidly about 1000 times in 4-6h, It can be rapidly reduced to normal level again after removing pathogen, be that emerging reflection organism infection situation and inflammation restore sensitive Index.SAA has its apparent advantage compared with current clinical most widely used CRP: (1) SAA raising sees virus, branch Substance, bacterium infection, and sensibility is higher than CRP；(2) in bacterial infection disease, SAA ratio CRP rising is early, amplitude is big, spirit Sensitivity is high, and especially in acute bacterial infection early stage, SAA testing result is more significant；(3) SAA is in disease of viral infection, SAA is significantly increased, and CRP is not increased, therefore SAA can be used as the sensitive indicator of judging viral infection.In addition, SAA level also with Kinds of tumors, obesity and its metabolism complication, secondary amyloidosis, coronary heart disease, graft-rejection, atherosclerosis etc. Many diseases are closely related, and numerous valuable reference informations can be provided for clinical diagnosis.

SAA poorly water-soluble is always the important obstruction for hindering its functional study and antigen purification, also exactly therefore, people SAA Acquisition with recombination SAA (rSAA) is the significant difficulty in SAA diagnosis antibody research and development preparation process.

SAA is a kind of lipophilic albumen, and more than 95% together with the contaminated with lipid such as high density lipoprotein level bletilla cholesterol, Also possess interaction and combination with various kinds of cell receptor, mucopolysaccharide, cystatin C etc., free SAA is seldom and easily by cell Phagocytosis is degraded to tissue amyloid A (AA).The combination of SAA and HDL etc. is but also at the epitope sequence of only part Under surface accessibility state in clinical diagnosis, and fixed SAA epitope (or B cell epitope) is had no so far Report.

In conclusion the prior art is primarily present following problem:

1, current Immunoinformatics Method and kit for is general pre- mainly for the B cell antigen epi-position of high-throughput data It surveys, predictablity rate is low, and lacks the experiment screening verifying to epitope, also lacks and has verified that prepared by epitope to application The application of antibody.

2, have no so far SAA epitope research report, SAA epitope Immunoinformatics analysis and it is subsequent Experiment screening verifying is also at space state.

3, SAA is the sensitive indexes of a kind of emerging reflection organism infection situation and inflammation recovery, but existing market is It is not high, specific bad there is anti-SAA antibody sensitivity, and is mostly scientific research antibody, it can be anti-using the SAA of in-vitro diagnosis Body is deficient, and paratope identification epitope is unknown.

4, SAA half-life period is about 1 day in blood of human body, and more than 95% and the lipids knot such as high density lipoprotein level bletilla cholesterol It is combined, also possesses interaction and combination with various kinds of cell receptor, mucopolysaccharide, cystatin C etc., in addition blood dissociates SAA Easily by protease fast degradation, so purification of human blood SAA difficulty is huge.

5, recombination SAA (rSAA) is lipophilic since SAA has and purification difficult, expensive, and recombinates SAA and generally present The forms such as six aggressiveness, the tetramer, eight aggressiveness, lack blood of human body SAA native conformation, and antigenicity is poor.

Summary of the invention

Technical problem to be solved by the invention is to provide a kind of clearly effective serum amyloid A protein epitope, The prediction of the epitope and verification method, and using the method for epitope preparation monoclonal antibody, the preparation method It is at low cost.

To achieve the above object, the present invention adopts the following technical scheme that:

The first purpose of this invention is to provide a kind of serum amyloid A protein epitope, the epitope Including the sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4.

Second object of the present invention is to provide prediction and the authentication of a kind of serum amyloid A protein epitope Method includes the following steps:

(1) the surface accessibility for predicting serum amyloid A protein antigen, obtains multiple first epitope sequences；

(2) the elastomeric sequences region for predicting serum amyloid A protein antigen, obtains multiple second epitope sequences；

(3) β-bend for predicting serum amyloid A protein antigen, obtains multiple third epitope sequences；

(4) antigenicity for predicting serum amyloid A protein antigen, obtains multiple 4th epitope sequences；

(5) hydrophily for predicting serum amyloid A protein antigen, obtains multiple 5th epitope sequences；

(6) linear epitope for predicting serum amyloid A protein antigen, obtains multiple 6th epitope sequences；

(7) the long segment epitope for predicting serum amyloid A protein antigen, obtains multiple 7th epitope sequences；

(8) removal first epitope sequences, second epitope sequences, the third epitope sequences, described The 4th epitope sequences, the 5th epitope sequences, the 6th epitope sequences, the weight in the 7th epitope sequences Multiple epitope and include sequence in other longer epitopes, obtains multiple nonredundancies prediction epitopes；

(9) whether the multiple nonredundancies prediction epitope for using ELISA verifying described is for the identification epitope of antibody.

Preferably, step (1) is predicted using Emini Surface Accessibility Prediction.

Preferably, step (2) is predicted using Karplus&Schulz Flexibility Prediction.

Preferably, step (3) is predicted using Chou&Fasman Beta-Turn Prediction.

Preferably, step (4) is predicted using Kolaskar&Tongaonkar Antigenicity.

Preferably, step (5) is predicted using Parker Hydrophilicity Prediction.

Preferably, step (6) is predicted using Bepipred Linear Epitope Prediction 2.0.

Preferably, step (7) is predicted using ABCpred Prediction.

Preferably, first epitope sequences are the sequence of threshold value >=1.

Preferably, second epitope sequences are the sequence of threshold value >=0.999.

It is further preferred that second epitope sequences are ten high epitope sequences of score.

Preferably, the third epitope sequences are the sequence of threshold value >=1.041.

It is further preferred that the third epitope sequences are ten high epitope sequences of score.

Preferably, the 4th epitope sequences are the sequence of threshold value >=0.976.

Preferably, the 5th epitope sequences are the sequence of threshold value >=2.410.

It is further preferred that the 5th epitope sequences are ten high epitope sequences of score.

Preferably, the 6th epitope sequences are the sequence of threshold value >=0.500.

Preferably, the 7th epitope sequences are the sequence of threshold value >=0.51.

It is further preferred that the length of the 7th epitope sequences is 16.

Preferably, the specific embodiment of step (9) includes the following steps successively carried out:

(a) multiple nonredundancies are synthesized using solid-phase synthesis and predicts epitope polypeptide；

(b) multiple nonredundancy prediction epitope polypeptides are coupled with bovine serum albumin respectively, then recycling is even Be associated with the bovine serum albumin of the nonredundancy prediction epitope polypeptide, wherein control the nonredundancy prediction epitope polypeptide with The molar ratio of the bovine serum albumin is 90~110:1, and the coupling agent used is glutaraldehyde；

(c) step (b) coupling obtained there is the bovine serum albumin of the nonredundancy prediction epitope polypeptide be dissolved in pH 7~7.5 PBS buffer, is configured to the solution to be measured of 0.8~1.2 μ g/ml, and blank control is and the solution to be measured Then the solution to be measured and the bovine serum albumen solution are added separately to by the bovine serum albumen solution of same concentrations In the hole of ELISA plate, 36~38 DEG C of 1~3h of processing are subsequently placed in 2~6 DEG C of coatings overnight；

(d) it is repeatedly washed with the Tween20-PBS of 0.04~0.06wt%；

(e) skimmed milk power-PBS for adding 4~6wt%, under air-proof condition, 36~38 DEG C of 1~3h of closing；

(f) it is repeatedly washed with the Tween20-PBS of 0.04~0.06wt%；

(g) every hole addition diluted mouse anti-human serum amyloid A antibody diluent of PBS, 36~38 DEG C of incubations 1~ 1.5h；

(h) it is repeatedly washed with the Tween20-PBS of 0.04~0.06wt%；

(i) the sheep anti-mouse antibody dilution of the diluted HRP label of PBS, 36~38 DEG C of 40~60min of incubation are added in every hole；

(j) it is repeatedly washed with the Tween20-PBS of 0.04~0.06wt%；

(k) TMB developing solution is added, develop the color 10~30min, and colour developing terminate liquid is then added, and 450nm measures the suction of corresponding aperture Light value；

(l) it is the serum amyloid A protein antigen that ELISA testing result, which is positive nonredundancy prediction epitope polypeptide, Epitope.

Third object of the present invention is to provide a kind of preparation side of mouse anti-human serum amyloid A monoclonal antibody Method includes the following steps:

(1) it is respectively synthesized the sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 Polypeptide；

(2) polypeptide that step (1) synthesizes is coupled with carrier protein respectively, after purification, 0.8 is diluted to PBS~ Then the polypeptide solution is mixed to get mixed liquor by the polypeptide solution of 1.2 mg/ml in equal volume；

(3) mouse is immunized with the mixed liquor；

(4) immune spleen cell is produced within three days after mouse final immunization；

(5) after myeloma cell and the immune spleen cell being carried out fusion culture, screening secretion antiserum amyloid The positive hybridoma cell of albumin A antibody；

(6) further cultivating the positive hybridoma cell being capable of stably excreting antiserum amyloid protein with screening The cell strain of A antibody；

(7) after the cell strain described in mouse inoculation, the ascites of mouse is collected, the ascites is centrifuged, is filtered, Affinity chromatography collects eluent, neutralizes to obtain the mouse anti-human serum amyloid A monoclonal for the eluent and resists Body.

Preferably, in step (2), the carrier protein is keyhole limpet hemocyanin, the polypeptide and the carrier Albumen is according to molecular number than being added for the amount of 90~110:1.

Preferably, the specific embodiment of the preparation method are as follows:

(1) it is respectively synthesized with solid-phase synthesis such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID The polypeptide of sequence shown in NO.4；

(2) polypeptide is coupled respectively to obtain polypeptide idol according to molecular number ratio 100:1 with keyhole limpet hemocyanin Co-product；After purification by the polypeptide coupled product, the polypeptide solution of 1mg/ml is diluted to PBS, it then will be described more Peptide solution is mixed to get mixed liquor in equal volume；

(3) mixed liquor is mixed in equal volume with Freund's complete adjuvant, obtains oil emulsion；By the oily cream Liquid injects BALB/c mouse back site with the dose subcutaneous of 0.1mL, after 14 days immune for the first time, with the mixed liquor with Abdominal cavity enhancing is immune after incomplete Freund's adjuvant mixing, and enhancing is immunized to after three needles, adopts tail blood and carries out bioactivity, potency reaches Fusion requires, and merges first 3 days, immune with the intraperitoneal injection impact of same dose antigen；

(4) for murine myeloma cell Sp2/0 after 8-anaguanine screens, it is outstanding that cell is made in culture to logarithmic growth phase Liquid, centrifugation abandon supernatant, are repeatedly resuspended with RPMI1640 basic culture solution, obtain myeloma cell；

(5) by three days after the mouse final immunization of step (3), spleen is aseptically taken out, is placed in plate, used RPMI1640 basic culture solution rinses, and then grinds filtering, cell suspension is made, and is centrifuged, and abandons supernatant, the culture of the basis RPMI1640 Liquid is repeatedly resuspended, and obtains immune spleen cell；

(6) myeloma cell and the immune spleen cell are mixed in 1:10 ratio, with the basis RPMI1640 Culture solution washing, 1200rpm are centrifuged 5 minutes, abandon supernatant, cell is mixed, the PEG-2000 of 1mL 50wt% is slowly added to Fusion, the RPMI1640 basic culture solution that 15mL is added after fusion 1 minute terminate cell fusion, and 1000rpm is centrifuged 5 minutes, is abandoned Supernatant is gently resuspended with the RPMI1640 screening and culturing liquid of 50mL, is divided equally in 96 orifice plate of muti-piece, 50 holes μ L/, 37 DEG C, 5%CO₂ Culture cultivates 6 days, changes HAT culture solution, continue to cultivate and change the liquid once；

(7) it is coated with ELISA plate with the mixed liquor, the positive of screening secretion antiserum amyloid A antibody is miscellaneous Oncocyte clone is handed over, RPMI1640 complete culture solution is positive thin with ratio >=2.1 of measured value and control value as negative control Hilum；

(8) cell in the positive cell hole is used into limiting dilution assay gram with 1 cells/well on 96 well culture plates It is grand, after expanding culture, frozen with the culture solution containing 10wt%DMSO, cell density 10⁶It is anti-to obtain energy stably excreting by a/mL The cell strain of serum amyloid A protein antibody；

(9) 6-8 weeks healthy and strong BALB/c mouse, the norphytane of every mouse peritoneal injection 0.5mL are selected；Abdominal cavity is infused after 10 days Penetrate 1 × 10⁶The hybridoma that a step (8) screens, ascites to be generated and mouse frequency domain before death, put to death mouse, receive Collect ascites, centrifuging and taking supernatant is filtered with filter paper after the PBS dilution of 3 times of volumes, by resulting filtrate under the flow velocity of 1mL/min It is added to the protein g affinity chromatography column balanced with PBS, is then washed with PBS with the flow velocity of 1mL/min and is not inhaled by Protein G Attached substance is eluted and is recycled until the absorption value at OD280nm reaches baseline, then with the glycine elution liquid of 0.1M Eluent, the eluent are neutralized with the Tris of pH8.8,0.1M, and it is anti-to obtain mouse anti-human serum amyloid A monoclonal Body.

Due to the implementation of above technical scheme, the invention has the following advantages over the prior art:

1, SAA epitope is predicted using panimmunity Informatics Method for the first time, overcomes single method predictablity rate Low problem.

2, epitope is verified using experiment, solves the problems, such as that SAA epitope is unknown.

3, monoclonal antibody is prepared using the antigen epitope polypeptide of synthesis, SAA is overcome and is difficult to purify the difficulty of acquisition, Solves the difficulty of antigen preparation, cost reduces by one times or more.

Detailed description of the invention

Fig. 1 is the result figure that Emini Surface Accessibility Prediction predicts surface accessibility；

Fig. 2 is the result that Karplus&Schulz Flexibility Prediction predicts SAA elastomeric sequences region Figure；

Fig. 3 is the result figure that Chou&Fasman Beta-Turn Prediction predicts β-bend；

Fig. 4 is the result figure of Kolaskar&Tongaonkar Antigenicity antigenicity prediction；

Fig. 5 is the result figure of Parker Hydrophilicity Prediction hydrophily prediction；

Fig. 6 is the result figure of 2.0 Antigen Epitope Prediction of Bepipred Linear Epitope Prediction；

Fig. 7 is that all prediction epitopes are overlapped display figure on SAA.

Fig. 8 is the colour developing result figure of the anti-human SAA monoclonal antibody of mouse and the commercially available anti-human SAA antibody of two kinds of mouse.

Specific embodiment

Below will by specific embodiment, the present invention is further explained, but the protection scope being not intended to restrict the invention. Those skilled in the art can be made improvements to preparation method and using instrument within the scope of the claims, these improvement also should be regarded as Protection scope of the present invention.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

One, SAA Epitope prediction

1, SAA sequence information is obtained by UniProtKB database.

Protein sequence:

RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPGGVWAAEAIS DARENIQR FFGHGAEDSLADQAANEWGRSGKDPNHFRPAGLPEKY(SEQ ID NO.5)。

(remarks reference information: SAA precursor protein overall length is 122 amino acid, and 18 amino acid of N section are signal peptide, in vivo Albumen excision in synthesis secretion process forms the mature form of 104 amino acid as above).

2, Emini Surface Accessibility Prediction predicts surface accessibility

As a result such as Fig. 1 (threshold value 1), predict that epitope sequences are shown in Table 1.

Table 1

Serial number	Start	Terminate	Polypeptide	Length
					1	20	27	AYSDMREA	8
2	43	48	DAAKRG	6
					3	60	65	DARENI	6
4	87	95	RSGKDPNHF	9

3, Karplus&Schulz Flexibility Prediction predicts SAA elastomeric sequences region

As a result such as Fig. 2 (threshold value 0.999), prediction epitope result such as table 2 (obtaining point highest preceding ten epitopes).

Table 2

Position	Residue	Start	Terminate	Polypeptide	Score
						89	G	86	92	GRSGKDP	1.1
88	S	85	91	WGRSGKD	1.09
						90	K	87	93	RSGKDPN	1.087
48	G	45	51	AKRGPGG	1.08
						49	P	46	52	KRGPGGV	1.078
87	R	84	90	EWGRSGK	1.066
						91	D	88	94	SGKDPNH	1.062
47	R	44	50	AAKRGPG	1.057
						50	G	47	53	RGPGGVW	1.052
62	R	59	65	SDARENI	1.047

4, Chou&Fasman Beta-Turn Prediction predicts β-bend

As a result such as Fig. 3 (threshold value 1.041), predict that epitope the results are shown in Table 3 (obtaining point highest preceding ten epitopes).

Table 3

5, Kolaskar&Tongaonkar Antigenicity antigenicity is predicted

As a result as (threshold value: 0.976), prediction epitope is shown in Table 4 to Fig. 4.

Table 4

Serial number	Start	Terminate	Polypeptide	Length
					1	4	10	FSFLGEA	7
2	32	39	SDKYFHAR	8
					3	49	60	PGGVWAAEAISD	12
4	66	71	QRFFGH	6
					5	74	80	EDSLADQ	7

6, Parker Hydrophilicity Prediction hydrophily is predicted

As a result as (threshold value: 2.410), prediction epitope the results are shown in Table 5 (obtaining point highest preceding ten epitopes) to Fig. 5.

Table 5

7,2.0 Antigen Epitope Prediction of Bepipred Linear Epitope Prediction

As a result as (threshold value: 0.500), prediction epitope is shown in Table 6 to Fig. 6.

Table 6

Serial number	Start	Terminate	Polypeptide	Length
					1	57	63	AISDARE	7
2	77	89	LADQAANEWGRSG	13

8, ABCpred Prediction long segment Antigen Epitope Prediction (threshold value: 0.51,16) prediction table bit length is set as, in advance It surveys epitope and is shown in Table 7.

Table 7

9, all prediction epitopes are shown in Table 8.

Table 8

All prediction epitopes are overlapped display Fig. 7 on SAA.

Remove duplicate epitope and include other predictions longer epitope in sequence, obtain following nonredundancy prediction table Position, is shown in Table 9.

Table 9

Number	Initiation site	End locus	Polypeptide	Length
					E1	4	10	FSFLGEA	7
E2	7	22	LGEAFDGARDMWRAYS	16
					E3	15	30	RDMWRAYSDMREANYI	16
E5	21	36	YSDMREANYIGSDKYF	16
					E7	27	42	ANYIGSDKYFHARGNY	16
E10	39	54	RGNYDAAKRGPGGVWA	16
					E22	49	60	PGGVWAAEAISD	12
E23	50	65	GGVWAAEAISDARENI	16
					E27	60	75	DARENIQRFFGHGAED	16
E29	69	84	FGHGAEDSLADQAANE	16
					E34	77	92	LADQAANEWGRSGKDP	16
E43	86	101	GRSGKDPNHFRPAGLP	16

Two, ELISA verifies whether nonredundancy prediction epitope is antibody identification meter position

1, nonredundancy predict Peptide systhesis using solid-phase synthesis synthesize above-mentioned nonredundancy prediction polypeptide E1, E2, E3, E5, E7、E10、E22、E23、E27、E29、E34、E43。

2, the nonredundancy of synthesis prediction polypeptide is coupled by coupled antigen carrier with bovine serum albumin BSA, polypeptide and BSA molar ratio is 100:1, and coupling agent is glutaraldehyde, and recycling coupling has the BSA of corresponding polypeptide after coupling.

3, the BSA of coupled peptide is dissolved in PBS buffer (pH 7.4) by coating, is configured to 1 μ g/ml solution, blank Control is 1 μ g/ml BSA solution, is added in ELISA plate according to 100 holes μ l/, and every group setting 3 are parallel, 37 DEG C, 2h, It is subsequently placed in 4 DEG C of coatings overnight.

4, washing discards coating buffer, is washed with 0.05%Tween20-PBS, every 200 μ l of hole, stands 5min, discards Washing lotion washes repeatedly step 3 time.

5, closing addition 200 μ l, 5% skimmed milk power (PBS), sealing, closes 2h by 37 DEG C.

6, washing discards confining liquid, is washed with 0.05%Tween20-PBS, every 200 μ l of hole, stands 5min, discards Washing lotion washes repeatedly step 3 time.

7, primary antibody is incubated for every hole and the anti-human 100 μ l of SAA antibody diluent of the diluted mouse of PBS is added.37 DEG C of incubation 1h.

8, washing discards primary antibody Incubating Solution, is washed with 0.05%Tween20-PBS, every 200 μ l of hole, and 5min is stood, Washing lotion is discarded, step 3 time is washed repeatedly.

9, secondary antibody is incubated for the 100 μ l of sheep anti-mouse antibody dilution that the diluted HRP label of PBS is added in every hole.37 DEG C of incubations 45min。

10, washing discards secondary antibody Incubating Solution, is washed with 0.05%Tween20-PBS, every 200 μ l of hole, stands 5min discards washing lotion, washes repeatedly step 5 time

11,100 μ l TMB developing solutions are added in colour developing, and develop the color 20min, and 100 μ l colour developing terminate liquid, 450 nm are then added Measure the light absorption value of corresponding aperture.

12, as a result ELISA testing result is shown in Table 10, the results showed that E5 (SEQ ID NO.1: YSDMREANYIGSDKYF), E7 (SEQ ID NO.2:ANYIGSDKYFHARGNY), E34 (SEQ ID NO.3: LADQAANEWGRSGKDP), E43 (SEQ ID NO.4:GRSGKDPNHFRPAGLP) is coupled BSA test positive.

Table 10

Three, the anti-human SAA monoclonal antibody of mouse is prepared using polypeptide E5, E7, E34, E43

1, polypeptide coupling carrier albumen is by polypeptide E5, E7, E34, E43 respectively with keyhole limpet hemocyanin (KLH) according to molecule Number is coupled to obtain polypeptide and KLH coupled product E5-KLH, E7-KLH, E34-KLH, E43-KLH than 100:1.After being coupled Afterwards after product purification, it is diluted to 1mg/ml with PBS, then mixes E5-KLH, E7-KLH, E34-KLH, E43-KLH in equal volume Obtain mixed liquor mix-KLH.

2, mouse immune antigen is immune by mixKLH and Freund's complete adjuvant (appearance: amber cell suspending liquid；Component: Paraffin Oil 85%, Mannide Monooleate 15%, Mycobacterium smegmatis 1mg/mL) etc. bodies Product mixing, obtains oil emulsion.The lotion is injected into BALB/c mouse back site with the dose subcutaneous of 0.1mL, is exempted from for the first time Abdominal cavity enhances immune (equivalent amount of antigen is mixed with incomplete Freund's adjuvant) after epidemic disease 14 days, enhances and is immunized to after three needles, adopt tail blood into Row bioactivity, potency reach fusion and require.First 3 days of fusion, it is immune with the intraperitoneal injection impact of same dose antigen.

3, myeloma cell prepares murine myeloma cell Sp2/0 after 8-anaguanine screens, culture to logarithmic growth Phase takes two big bottles that cell suspension is made, and supernatant is abandoned in centrifugation, is resuspended with RPMI1640 basic culture solution, such as a bit in triplicate, obtains To myeloma cell, count.

4, B cell is prepared after mouse final immunization three days, is aseptically taken out spleen, is placed in plate, RPMI1640 basic culture solution rinses once, is put on the nylon wire of small beaker and grinds filtering, cell suspension is made.Centrifugation is abandoned Supernatant, RPMI1640 basic culture solution are resuspended, and so in triplicate, obtain immune spleen cell, count.

5, cell fusion and filtering hybridoma are mixed myeloma cell and immune spleen cell in 1:10 ratio, It is washed 1 time, 1200rpm, is centrifuged 5 minutes with RPMI1640 basic culture solution in 50mL sterile centrifugation tube.Supernatant is abandoned, cell is mixed It is even, it is slowly added to the PEG-2000 fusion of 1mL50%, the RPMI1640 basic culture solution that 15mL is added after fusion 1 minute terminates Cell fusion.1000rpm is centrifuged 5 minutes.Supernatant is abandoned, is gently resuspended, is divided equally in 10 with the RPMI1640 screening and culturing liquid of 50mL 96 orifice plate of block, 50 holes μ L/, 37 DEG C, 5%CO₂Culture.Culture 6 days, changing HAT culture solution, (RPMI1640 containing HAT is cultivated completely Liquid), continue to cultivate and change the liquid once.

6, positive colony screens mix-KLH albumen and is coated with ELISA plate, and the positive hybridoma of anti-SAA antibody is secreted in screening Cell clone.RPMI1640 complete culture solution is as negative control, with ratio >=2.1 (i.e. P/N >=2/1) of measured value and control value For positive cell hole.

7, hybridoma subclone screening secretory antibody positive cell hole is used on 96 well culture plates with 1 cells/well Limiting dilution assay clone, method on positive Kong Yi of screening continuously is cloned three times, after expanding culture, is frozen with the culture solution containing 10%DMSO It deposits, cell density 10⁶A/mL.The final cell strain for screening 5 plants of energy stably excreting antibody altogether: SAA-L1, SAA-L2, SAA-L3、SAA-L4、SAA-L5。

8,6-8 weeks healthy and strong BALB/c mouse is selected in monoclonal antibody preparation, and the drop of every mouse peritoneal injection 0.5mL is planted Alkane；1 × 10 is injected intraperitoneally after 10 days⁶A hybridoma.It can produce ascites after inoculating cell 7-10 days, close observation animal Health status and sign of ascites as, it is as more as possible to ascites, and before mouse is frequency domain dead, put to death mouse, inhaled ascites with dropper Enter in test tube, a general mouse can obtain 5-10mL ascites.Ascites is collected, centrifuging and taking supernatant is put in -20 DEG C of refrigerators and saves.It takes Ascites supernatant is filtered with filter paper after the PBS dilution of 3 times of volumes.Resulting filtrate is added to one under the flow velocity of 1mL/min The protein g affinity chromatography column balanced with PBS.Then it is straight the substance not adsorbed by Protein G to be washed with the flow velocity of 1mL/min with PBS Until the absorption value at OD280nm reaches baseline.It is eluted again with the glycine elution liquid (pH2.5) of 0.1M and recycles this Antibody.The solution recycled is neutralized with 0.1M Tris (pH8.8), by ultrafiltration that antibody concentration adjusting is suitable dense to one Degree.The antibody of five strain of hybridoma purifying is subjected to equal proportion and is mixed to get the anti-human SAA monoclonal antibody of mouse, -20 DEG C of packing freeze.

9, monoclonal antibody evaluation carries out non-denaturing polyacrylamide gel, 0.5%BSA- with diagnosis with SAA standard items PBS closes 30min, the anti-human SAA monoclonal antibody of quality mouse such as addition and the commercially available anti-human SAA antibody of two kinds of mouse, and 4 DEG C of overnight incubations are used 0.05%Tween20-PBS carries out washing 3min, washs 3 times, and equivalent AP label sheep anti mouse secondary antibody is added later in 0.5% 45min is incubated in BSA-PBS, 0.05%Tween20-PBS, which is shown after carrying out washing 3 times with AP, also to develop the color, as a result see Fig. 8, In, band 1: the anti-human SAA monoclonal antibody of mouse；Band 2 and 3: two kind of commercially available SAA monoclonal antibody.

10, SAA monoclonal antibody of the present invention, which is evaluated, using the anti-human SAA monoclonal antibody cost advantage of polypeptide preparation mouse prepares For method compared to previous monoclonal preparation method the difference is that the difference of antigen, antigen needed for the present invention has obvious price excellent Gesture: synthesis polypeptide price is about 100 yuan every milligram, and recombinates SAA price not less than 2000 yuan every milligram, and human plasma purifies SAA is then without market products.

The above embodiments merely illustrate the technical concept and features of the present invention, and its object is to allow person skilled in the art Scholar cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.It is all according to the present invention Equivalent change or modification made by Spirit Essence, should be covered by the protection scope of the present invention.

Sequence table

<110>Di Yalaibo (Zhangjiagang) Biotechnology Co., Ltd

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Claims

1. a kind of serum amyloid A protein epitope, it is characterised in that: the epitope include as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, sequence shown in SEQ ID NO.4.

2. prediction and the verification method of a kind of serum amyloid A protein epitope, characterized by the following steps:

(8) removal first epitope sequences, second epitope sequences, the third epitope sequences, described the It is four epitope sequences, the 5th epitope sequences, the 6th epitope sequences, duplicate in the 7th epitope sequences Epitope and include sequence in other longer epitopes, obtains multiple nonredundancies prediction epitopes；

3. prediction and the verification method of serum amyloid A protein epitope according to claim 2, it is characterised in that: Step (1) is predicted using Emini Surface Accessibility Prediction；Step (2) uses Karplus& Schulz Flexibility Prediction is predicted；Step (3) uses Chou&Fasman Beta-Turn Prediction is predicted；Step (4) is predicted using Kolaskar&Tongaonkar Antigenicity；Step (5) it is predicted using Parker Hydrophilicity Prediction；Step (6) uses Bepipred Linear Epitope Prediction 2.0 is predicted；Step (7) is predicted using ABCpred Prediction.

4. the prediction of serum amyloid A protein epitope according to claim 2 or 3 and verification method, feature exist In: first epitope sequences are the sequence of threshold value >=1；Second epitope sequences are the sequence of threshold value >=0.999；Institute The third epitope sequences stated are the sequence of threshold value >=1.041；4th epitope sequences are the sequence of threshold value >=0.976；Institute The 5th epitope sequences stated are the sequence of threshold value >=2.410；6th epitope sequences are the sequence of threshold value >=0.500；Institute The 7th epitope sequences stated are the sequence of threshold value >=0.51.

5. prediction and the verification method of serum amyloid A protein epitope according to claim 4, it is characterised in that: Second epitope sequences are ten high epitope sequences of score；The third epitope sequences are ten high epitopes of score Sequence；5th epitope sequences are ten high epitope sequences of score；The length of 7th epitope sequences is 16.

6. prediction and the verification method of serum amyloid A protein epitope according to claim 2, it is characterised in that: The specific embodiment of step (9) includes the following steps successively carried out:

(b) multiple nonredundancy prediction epitope polypeptides are coupled with bovine serum albumin respectively, then recycling coupling has The bovine serum albumin of the described nonredundancy prediction epitope polypeptide, wherein control the nonredundancy prediction epitope polypeptide with it is described Bovine serum albumin molar ratio be 90~110:1, the coupling agent used is glutaraldehyde；

(c) will step (b) coupling obtained have the bovine serum albumin of the nonredundancy prediction epitope polypeptide be dissolved in pH 7~ 7.5 PBS buffer, is configured to the solution to be measured of 0.8~1.2 μ g/ml, and blank control is identical as the solution to be measured Then the solution to be measured and the bovine serum albumen solution are added separately to by the bovine serum albumen solution of concentration In the hole of ELISA plate, 36~38 DEG C of 1~3h of processing are subsequently placed in 2~6 DEG C of coatings overnight；

(d) it is repeatedly washed with the Tween20-PBS of 0.04~0.06wt%；

(f) it is repeatedly washed with the Tween20-PBS of 0.04~0.06wt%；

(g) the diluted mouse anti-human serum amyloid A antibody diluent of PBS, 36~38 DEG C of 1~1.5h of incubation are added in every hole；

(h) it is repeatedly washed with the Tween20-PBS of 0.04~0.06wt%；

(j) it is repeatedly washed with the Tween20-PBS of 0.04~0.06wt%；

(k) TMB developing solution is added, develop the color 10~30min, and colour developing terminate liquid is then added, and 450nm measures the extinction of corresponding aperture Value；

(l) it is the serum amyloid A protein antigen table that ELISA testing result, which is positive nonredundancy prediction epitope polypeptide, Position.

7. a kind of preparation method of mouse anti-human serum amyloid A monoclonal antibody, characterized by the following steps:

(1) the more of the sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 are respectively synthesized Peptide；

(2) polypeptide that step (1) synthesizes is coupled with carrier protein respectively, after purification, 0.8 is diluted to PBS~ Then the polypeptide solution is mixed to get mixed liquor by the polypeptide solution of 1.2mg/ml in equal volume；

(3) mouse is immunized with the mixed liquor；

(5) after myeloma cell and the immune spleen cell being carried out fusion culture, screening secretion antiserum amyloid A The positive hybridoma cell of antibody；

(6) by the positive hybridoma cell further cultivate with screen can stably excreting antiserum amyloid A resist The cell strain of body；

(7) after the cell strain described in mouse inoculation, the ascites of mouse is collected, the ascites is centrifuged, is filtered, is affine Chromatography collects eluent, and the eluent is neutralized to obtain the mouse anti-human serum amyloid A monoclonal antibody.

8. the preparation method of mouse anti-human serum amyloid A monoclonal antibody according to claim 7, feature exist In: in step (2), the carrier protein is keyhole limpet hemocyanin, and the polypeptide and the carrier protein are according to molecule Number is than being that the amount of 90~110:1 is added.

9. the preparation method of mouse anti-human serum amyloid A monoclonal antibody according to claim 7, feature exist In: the specific embodiment of the preparation method are as follows:

(1) it is respectively synthesized with solid-phase synthesis such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 The polypeptide of shown sequence；

(2) polypeptide is coupled respectively to obtain polypeptide coupling production according to molecular number ratio 100:1 with keyhole limpet hemocyanin Object；After purification by the polypeptide coupled product, the polypeptide solution of 1mg/ml is diluted to PBS, it is then that the polypeptide is molten Liquid is mixed to get mixed liquor in equal volume；

(3) mixed liquor is mixed in equal volume with Freund's complete adjuvant, obtains oil emulsion；By the oil emulsion with The dose subcutaneous of 0.1mL injects BALB/c mouse back site, after 14 days immune for the first time, not with the mixed liquor and Freund Abdominal cavity enhancing is immune after Freund's complete adjuvant mixing, and enhancing is immunized to after three needles, adopts tail blood and carries out bioactivity, potency reaches fusion and wants It asks, merges first 3 days, it is immune with the intraperitoneal injection impact of same dose antigen；

(4) for murine myeloma cell Sp2/0 after 8-anaguanine screens, cell suspension is made in culture to logarithmic growth phase, from The heart abandons supernatant, is repeatedly resuspended with RPMI1640 basic culture solution, obtains myeloma cell；

(6) myeloma cell and the immune spleen cell are mixed in 1:10 ratio, is cultivated with the basis RPMI1640 Liquid washing, 1200rpm are centrifuged 5 minutes, abandon supernatant, cell is mixed, and are slowly added to the PEG-2000 fusion of 1mL 50wt%, The RPMI1640 basic culture solution that 15mL is added after fusion 1 minute terminates cell fusion, and 1000rpm is centrifuged 5 minutes, abandons supernatant, It is gently resuspended, is divided equally in 96 orifice plate of muti-piece, 50 holes μ L/, 37 DEG C, 5%CO with the RPMI1640 screening and culturing liquid of 50mL₂Culture, Culture 6 days, changes HAT culture solution, continues to cultivate and change the liquid once；

(7) ELISA plate, the positive hybridoma of screening secretion antiserum amyloid A antibody are coated with the mixed liquor Cell clone, for RPMI1640 complete culture solution as negative control, ratio >=2.1 with measured value and control value are positive cell Hole；

(8) cell in the positive cell hole is cloned on 96 well culture plates with limiting dilution assay with 1 cells/well, After expanding culture, frozen with the culture solution containing 10wt%DMSO, cell density 10⁶A/mL, obtaining can stably excreting antiserum The cell strain of amyloid A antibody；

(9) 6-8 weeks healthy and strong BALB/c mouse, the norphytane of every mouse peritoneal injection 0.5mL are selected；1 is injected intraperitoneally after 10 days ×10⁶The hybridoma that a step (8) screens, ascites to be generated and mouse frequency domain before death, put to death mouse, collect abdomen Water, centrifuging and taking supernatant are filtered with filter paper after the PBS dilution of 3 times of volumes, resulting filtrate are added under the flow velocity of 1mL/min Then one protein g affinity chromatography column balanced with PBS is washed not with the flow velocity of 1mL/min by Protein G absorption with PBS Substance is until the absorption value at OD280nm reaches baseline, then is eluted with the glycine elution liquid of 0.1M and recycle elution Liquid, the eluent are neutralized with the Tris of pH8.8,0.1M, obtain mouse anti-human serum amyloid A monoclonal antibody.