CN102336814B - Polypeptide sequence for preparing anti-liver-neoplasm marker CK-19 antibody, polyclonal antibody and application thereof - Google Patents

Abstract

The invention discloses a polypeptide sequence for preparing an anti-liver-neoplasm marker CK-19 antibody, a polyclonal antibody and application thereof. The amino acid sequence of the polypeptide sequence is shown as SEQNO.1. In the invention, a segment of amino acid sequence of a CK-19 protein serving as an immunogen is subjected to polypeptide synthesis to obtain an immune animal preparation antibody. The invention relates to human CK-19 protein amino acid characteristic analysis, polypeptide design and synthesis, coupling of a polypeptide with a carrier protein KLH, coupling product purification, conjugate immune New Zealand rabbits, antiserum ELISA (Enzyme-Linked Immuno Sorbent Assay) detection, collection and purification, a purified antibody ELISA and western blotting identification. The polyclonal antibody can be used for specifically identifying and detecting human CK-19 proteins, and can be used for assisting in liver cancer diagnosis, and clinical determination of prognosis and treating scheme selection.

Description

A kind of peptide sequence, polyclonal antibody and application that is used to prepare anti-liver neoplasm mark CK-19 antibody

Technical field

The present invention relates to detect the rabbit polyclonal antibody that liver cancer marker CK-19 uses, specifically, relate to the preparation and the application thereof of the polypeptide antibody of a kind of anti-liver neoplasm mark CK-19.

Background technology

Cytokeratin (Keratin typeIcytoskeletal) is the intermediate filament of cell paste, according to its molecular weight different with iso-electric point be divided into 20 kinds dissimilar.Express CK8, CK18, CK-19 in the embryonic liver cell, along with the differentiation of liver, CK-19 does not express at normal liver cell, only expresses in bile duct.CK-19 has specific stain to epithelial duct and cholangiocellular carcinoma (ICC), and positive rate reaches 77%～100%, is one of best immunohistochemical methods mark of current diagnosis ICC.When canceration took place liver cell, some cancer cells was expressed CK-19 again, and part is the incomplete CK-19 of molecular structure, and the differentiation degree of expression degree and cell is negative correlation.When necrosis appearred in cell, CK-19 was released in the serum to dissolve segmental form, can detect in blood, and detecting index is cytokeratin antigen on the low side.Reports such as Nagai, in 70 routine liver cancer patients, 33 routine peripheral blood CK-19 raise, and detect at the same time among the patient of CK-19 and AFP, have 12.3% patient CK-19 to raise and AFP is normal.The combined detection has certain complementarity to diagnosis.DingSJ etc. find CK-19 high expression level in high metastatic capacity cell strain, and along with the rising expression amount of metastatic capacity is also in rising trend.Li Yan etc. have further explored the relation of CK-19 and liver cancer patient clinicopathological parameters, and it is low with the HCC differentiation degree to show that serum CK-19 raises, and involvement of blood vessel power is strong, and clinical disease period has certain relation evening.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art, but the polyclonal antibody of a kind of CK-19 of specific recognition human liver cancer cell expression is provided.

To achieve these goals, the present invention adopts following technical scheme:

A kind of peptide sequence that is used to prepare anti-liver neoplasm mark CK-19 antibody, its aminoacid sequence is shown in SEQNO.1.

For ease of with carrier protein couplet, connect a halfcystine at the C of aforementioned polypeptides sequence end.

The present invention has selected CK-19 albumen n end 14 peptides as candidate's polypeptide, and carries out the synthetic preparation of polypeptide immunogen with manual method.

Aforementioned polypeptides epi sequence animal can obtain polyclonal antibody, and described polyclonal antibody can be used to prepare the diagnosing cancer of liver medicine.

The present invention is with above-mentioned immunogen immune new zealand rabbit, but obtains the polyclonal antibody of the CK-19 that the specific recognition human liver cancer cell expresses.

Compared with prior art, the present invention has following beneficial effect:

The present invention analyzes the secondary structure of CK-19 Argine Monohydrochloride sequence, immunogenicity, hydrophilic and hydrophobic, surperficial accessibility etc., determines that one section suitable peptide sequence carries out synthetic; With synthetic polypeptide and maleoyl imido activatory carrier mcKLH coupling, this coupled product is carried out immune new zealand rabbit behind the desalting column purifying; Rabbit anteserum through repeatedly immunity is tired with ELISA method antagonist and is detected, tires to reach collection immunize rabbit serum after the ideal value, and with ProteinG protein purification column purification antibody; Antibody behind the purifying is carried out evaluations such as ELISA, westernblot.But qualification result shows this polyclonal antibody specific recognition CK-19 albumen, can be used for detecting the CK-19 albumen that liver cancer cell is expressed, and for diagnosing cancer of liver is offered help, and can instruct its clinical prognosis to judge and the selection of treatment plan.

Description of drawings

Fig. 1 be anti-CK-19 polyclonal antibody through proteinG gel column purifying after SDS-PAGE electrophoresis (12%) detected result.M: albumen Marker:Lane1: go up the sample peak; Lane2: stream is worn the peak; Lane3: balance peak; Lane4: elution peak.

Fig. 2 is the western-blot qualification result of the anti-CK-19 polyclonal antibody among the present invention.Last sample is HepG2 cell (Bel7402) lysate.

Embodiment

Design of the present invention and synthetic CK-19 polypeptide antigen, with blue carrier proteins (KLH) coupling of maleinamide activatory keyhole blood, immune new zealand rabbit behind desalting and purifying, by repeatedly immunity and ELISA tire detect after, gather rabbit anteserum and through proteinG gel-purified column purification.This polyclonal antibody is through evaluations such as ELISA, western blot, but the CK-19 albumen that the specific recognition human liver cancer cell is expressed for diagnosing cancer of liver is offered help, and can instruct its clinical prognosis to judge and the selection of treatment plan.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are used to illustrate the present invention, limit the scope of the invention and be not used in.Under the prerequisite that does not deviate from technical solution of the present invention, any change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.

The design of embodiment 1:CK-19 polypeptide and synthetic

1.CK-19 aminoacid sequence

Obtain people's CK-19 aminoacid sequence (P08727) according to GenBank: shown in SEQNO.2.

The result: people CK-19 albumen contains 400 amino acid.

2.http the www.uniprot.org/uniprot of: //on-line analysis CK-19 structural domain (table 1):

Table 1

The result: people CK-19 albumen contains 9 structural domains.

3. with DNAstar software analysis CK-19 protein characteristic (table 2):

Table 2

The result: people CK-19 molecular weight of albumen is 44091.06 dalton, and iso-electric point is 4.97, is acidic protein.

4. with DNAstar software analysis CK-19 immunogenicity, hydrophilic and hydrophobic and surperficial accessibility:

The result: people CK-19 protein N terminal has 14 amino acid antigenicities, wetting ability and surperficial accessibility stronger.

5.CK-19 synthetic peptide sequence:

Through above-mentioned analysis, the peptide sequence of selecting for use is MTSYSYRQSSATSS(1aa-14aa).Embodiment 2: synthetic and and the carrier protein couplet of polypeptide

Embodiment 2: the synthetic and coupling of polypeptide

1. polypeptide is synthetic

For ease of with carrier protein couplet, synthetic polypeptide adds halfcystine, that is: a MTSYSYRQSSATSSC at the C end.Polypeptide is synthetic by Shanghai gill biochemical corp.

2. polypeptide and carrier protein couplet

The polypeptide antigen of chemosynthesis is a small molecules, itself is difficult to have good antigenicity, can only produce very weak immune response by induced animal, thus crosslinked with carrier proteins be very important.Carrier proteins contains a lot of epitopes, can stimulate t helper cell, and then induces the B cell response.Be used for having multiple with the crosslinked carrier proteins of polypeptide, wherein the carrier that the most generally uses is keyhole limpet hemocyanin (keyhole limpet hemacyanin, KLH), bovine serum albumin (bovine serumalbumin, BSA), ovalbumin (ovalbumin, OVA) and bovine thyroglobulin (bovine thyroglobulin, THY).KLH has higher antigenicity, is the crosslinked carrier of polypeptide the most commonly used.BSA also is commonly used to as peptide carrier, but is made the antibody of this method production exist certain limitation on using owing to BSA often is used as the blocker that detects test.

Polypeptide and carrier protein couplet:

A. with 200ul ultrapure water dilution one pipe maleimide activatory mcKLH, make into the solution of 10mg/ml.(annotate: it is translucent that above-mentioned solution is pearl opal, must not vibrate or heat, otherwise can cause the mcKLH precipitation.)

B. the haptens that contains sulfydryl with the coupling Buffer dissolving that is equivalent to solution 1.0-2.5 times volume in the step 1. for example: the 2mg haptens with 200-500ul coupling Buffer dissolving, is joined mcKLH.If polypeptide is Yi Rong, can join in the mcKLH suspension with solid.

(annotate: molten if haptens is difficult for, can add DMSO increases dissolving.DMSO concentration in the coupling lysate is lower than 30%, otherwise the carrier proteins volatility.）

C. mixing polypeptide, mcKLH immediately are then at room temperature reaction 2h.

Coupled product purifying: come the purifying conjugate by the gel chromatography desalting column

If conjugate a week with interior injection, use the PBS purifying.If conjugate is frozen, centrifugal with purifying buffering salt purifying if when coupling, form precipitation, collect supernatant, keep precipitation.Purifying supernatant only.The conjugate of purifying is combined with precipitation.

A. dissolve one bottle of purifying buffering salt, add the ultrapure water of the 60ml degassing.Preserve at 4 degree.

B. take away the lid of T﹠B of desalting column is discharged storage liquid.A but desalting column purifying 0.5ml sample.

C. the purifying damping fluid with 3-5 times of column volume (15-25ml) washes pillar.

D. the peptide carrier mixture with 0.5ml directly adds the post center.The purifying damping fluid that adds 0.5ml is collected each peak in separator tube.

E. under 280nm, measure absorbancy and contain conjugate to determine any part.Detected at first absorption peak will be the hapten conjugation thing.Mix the part that all contain conjugate

F. after the part that contains conjugate occurs, continue to add damping fluid in post, collecting does not have the link coupled haptens.

G. with the conjugate filtration sterilization, aseptic being kept at-80 spent.

The result: the Coomassie brilliant blue method records protein concentration and content after the coupling, every pipe 250ug packing, and-80 degree are preserved.

Embodiment 3: anti-CK-19 polypeptide rabbit polyclonal antibody preparation and purifying:

1. animal immune

Two new zealand male rabbits of every kind of coupled product immunity, body weight 2-2.5Kg.Not formula Freund's complete adjuvant and Fu Shi Freund are available from Sigma company.

Immune programme for children (table 3):

A. with PBS dilution 100ug antigen, to 1.25ml, with equal-volume adjuvant mixing, vortex vibration 100 minutes detects the emulsification result, gets an antigen and drips in waterborne, and one minute insoluble separates diffusion.

B. ear vein is got blood before the immunity, gets serum after 4 degree leave standstill, and-80 degree are preserved, and make negative control sera.

Table 3

Antibody titer ELISA detection method:

(1) experiment reagent

A. phosphate buffered saline buffer (PBS) (10 * concentrate)

Sodium-chlor (NaCl) 50g

Repone K (KCl) 1.25g

Potassium primary phosphate (KH ₂PO ₄) 1.25g

Sodium phosphate dibasic (Na ₂HPO ₄12H ₂O) 18.1g

Distilled water adds to 1000ml

Transfer pH to 7.2 with 1MHCL

B. confining liquid

Normal bovine serum 10ml

1 * PBS(does not contain tween) 90ml

C. lavation buffer solution

Tween 20 (Tween20) 0.2ml

1×PBS1000ml

D. substrate colour developing A liquid

Sodium-acetate (CH ₃COONa) 13.6g

Citric acid (C ₆H ₈O ₇H ₂O) 1.6g

Hydrogen peroxide (H ₂O ₂30%) 0.3ml

Distilled water adds to 500ml

E. substrate colour developing B liquid

Disodium ethylene diamine tetraacetate (EDTA-Na ₂) 0.2g

Citric acid (C ₆H ₈O ₇H ₂O) 0.95g

Glycerine (C ₃H ₈O ₃) 50ml

Tetramethyl benzidine (TMB) 0.2g

Distilled water adds to 500ml

F. stop buffer (2MH ₂SO ₄)

Get dense H ₂SO ₄27.62ml, slowly joining in the distilled water of 473ml, mixing gets final product.

G. antigen coated liquid (0.1M carbonate buffer solution) pH9.6

Yellow soda ash (Na ₂CO ₃) 1.59g

Sodium bicarbonate (NaHCO ₃) 2.93g

Sodium azide (NaN ₃) 0.2g

Distilled water adds to 1000ml

H. the goat anti-mouse igg of horseradish peroxidase-labeled (commercialization).

(2) experimental procedure

A. antigen coated: pure polypeptide antigen final concentration is generally 1-2 μ g/ml, add in each hole of polystyrene enzyme joint inspection drafting board with getting 100 μ l after the coating buffer dilution, 4 ℃ spend the night after, washing lotion washing 3 times.Advise that 4 ℃ of bags are spent the night

B. sealing: every hole adds 200 μ l or fills it up with confining liquid, and 4 ℃ are spent the night or 37 ℃ after two hours, wash 3 times, pat dry.Putting 4 ℃ of refrigerators preserves standby.

C.ELISA detects

(a) add testing sample: rabbit ear vein blood sampling back separation of serum, use the PBS doubling dilution, 50-100 μ l/ hole application of sample by in the good enzyme plate, simultaneously, is chosen the preceding negative contrast of rabbit anteserum of immunity respectively to bag, hatches 30min for 37 ℃, washs 3 times, pats dry.

(b) adding two resists: according to the selection extension rate of tiring of ELIAS secondary antibody, 30min is hatched for 37 ℃ in 100 μ l/ holes, washs 3 times, pats dry.

(c) colour developing: add A, each 80 μ l/ hole of B liquid, 37 ℃ of colour developing 15min.

(d) stop: add stop buffer 80 μ l/ holes.

(e) reading: measure each hole OD value with the single wavelength of 450nm, exceed greater than 2.5 with ratio (P/N), as the stagnation point that is judged as the positive or determines to tire with negative control hole OD value

Rabbit anteserum is collected:

Adopt the carotid artery depletion method, non-anti-freezing rabbit whole blood is hatched 2h at 37 ℃ after collecting,

Antibody purification:

A, saturated ammonium sulphate method

(1) preparation saturated ammonium sulphate solution (SAS)

With 767g (NH ₄) ₂SO ₄Slowly be added to while stirring in 1 liter of distilled water.Be transferred to sulfuric acid pH7.0 with ammoniacal liquor or sulfuric acid.This is that saturation ratio is 100% ammoniumsulphate soln (4.1mol/L, 25 ℃).

(2) precipitation

A. the centrifugal 30min of sample (serum) 20000 ' g removes cell debris;

B. keep supernatant liquor and measurement volumes;

C. slowly add isopyknic SAS while stirring in supernatant liquor, final concentration is 1:1

D. solution is placed on and stirred 6 hours on the magnetic stirring apparatus or stir and spend the night (4 ℃), protein is fully precipitated.

(3) dialysis

A. the centrifugal 30min of protein soln 10000 ' g (4 ℃).Abandon supernatant and keep precipitation;

B. precipitation is dissolved on a small quantity in (10-20ml) PBS-0.2g/L sodium azide.Put into dialysis tubing behind the resolution of precipitate to 24-48 hour (4 ℃) of PBS-0.2g/L sodium azide dialysis, changed dialysis buffer liquid once every 3-6 hour, thoroughly to remove sulfate of ammoniac;

C. dialyzate is centrifugal, measures protein content in the supernatant liquor.

B, Protein G affinity purification

(1). required reagent and specific installation

1.0mol/LTris (pH8.0); 100mmol/LTris (pH8.0); 10mmol/LTris (pH8.0); 50mmol/L glycine (pH3.0); Simple column chromatographic instrument.

(2). operation steps.

A. the 1.0mol/LTris (pH8.0) that adds 1/10 volume transfers the pH value to 8.0 that contains antibody sample (serum, tissue culture supernatant liquor or ascites).

B. antibody-solutions is passed through albumin A or Protein G microballoon post.In these posts, every milliliter wet microballoon can be in conjunction with about 10-20ml antibody (1 albumin A or Protein G microballoon molecule in conjunction with 2 molecular antibodies).About volume of record dress post microballoon.Because the amount of cleaning and elution buffer is used in the decision of the volume of microballoon post.

C. use 100mmol/LTris (pH8.0) the washing microballoon of 10 times of column volumes.

D. use 10mmo1/LTris (pH8.0) the washing microballoon of 10 times of column volumes.

E. use 50mmol/L glycine (pH3.0) wash-out microballoon post, add the damping fluid of about 1/2 column volume at every turn, gradation adds.Test tube with the 1mol/LTris that contains 1/10 column volume (pH8.0) is collected elutriant, and each pipe is slowly shaken up, and makes its pH value return to neutrality.

The collection tube that f. will contain antibody mixes, and surveys total protein with SDS-PAGE and coomassie brilliant blue staining.

The result: antibody purification is after the SDS-PAGE protein electrophoresis is identified (Fig. 1), and it is 0.4mg/ml that ultraviolet spectrophotometer is measured antibody concentration.Antibody is stored in-20 ℃ behind the purifying, in the 0.01MPBS damping fluid, contains 40% glycerine and 0.5%BSA in the solution.

The evaluation of embodiment 4:CK-19 polyclonal antibody

1.CK-19 the ELISA of polyclonal antibody identifies

With synthetic CK-19 polypeptide serves as to detect antigen, coated elisa plate, with the not immune rabbit anteserum of 1:2000 dilution as negative control, with the antibody doubling dilution behind rabbit anteserum and the purifying, use the ELISA method and detect, be judged as the positive greater than 2.1, calculate monoclonal antibody and tire with ratio with negative serum.As a result, tiring of the anti-CK-19 antibody of purifying reaches 1:4000(table 4).

The anti-CK-19 antibody titer of table 4

2. the Western-Blot of anti-CK-19 polyclonal antibody identifies

Collect HepG2 cell 1 * 10 ₇Individual, after its cracking, lysate carries out the 10%SDS-PAGE electrophoresis, with reference to the western blotting method described in " molecular cloning ", the electrotransfer condition is 100V electrophoresis 1h, and confining liquid is the TBST that contains 5% skim-milk, and one anti-is the CK-19 polyclonal antibody (final concentration 0.5 μ g/ml) among the present invention, two anti-are the sheep anti-mouse igg antibody of horseradish peroxidase-labeled, carry out immunoblotting assay by the ECL Color Appearance System.

The result: the CK-19 polyclonal antibody among the present invention can detect the protein band about the 44KD size in the HepG2 cell pyrolysis liquid, conforms to people CK-19 molecular weight of albumen size (Fig. 2).

SEQUENCE LISTING

＜110〉Nanfang Medical Univ

＜120〉a kind of peptide sequence, polyclonal antibody and application that is used to prepare anti-liver neoplasm mark CK-19 antibody

<130>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 14

<212> PRT

＜213〉artificial sequence

<400> 1

Met Thr Ser Tyr Ser Tyr Arg Gln Ser Ser Ala Thr Ser Ser

1 5 10

<210> 2

<211> 400

<212> PRT

＜213〉people CK-19 albumen

<400> 2

Met Thr Ser Tyr Ser Tyr Arg Gln Ser Ser Ala Thr Ser Ser Phe Gly

1 5 10 15

Gly Leu Gly Gly Gly Ser Val Arg Phe Gly Pro Gly Val Ala Phe Arg

20 25 30

Ala Pro Ser Ile His Gly Gly Ser Gly Gly Arg Gly Val Ser Val Ser

35 40 45

Ser Ala Arg Phe Val Ser Ser Ser Ser Ser Gly Gly Tyr Gly Gly Gly

50 55 60

Tyr Gly Gly Val Leu Thr Ala Ser Asp Gly Leu Leu Ala Gly Asn Glu

65 70 75 80

Lys Leu Thr Met Gln Asn Leu Asn Asp Arg Leu Ala Ser Tyr Leu Asp

85 90 95

Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val Lys Ile

100 105 110

Arg Asp Trp Tyr Gln Lys Gln Gly Pro Gly Pro Ser Arg Asp Tyr Ser

115 120 125

His Tyr Tyr Thr Thr Ile Gln Asp Leu Arg Asp Lys Ile Leu Gly Ala

130 135 140

Thr Ile Glu Asn Ser Arg Ile Val Leu Gln Ile Asp Asn Ala Arg Leu

145 150 155 160

Ala Ala Asp Asp Phe Arg Thr Lys Phe Glu Thr Glu Gln Ala Leu Arg

165 170 175

Met Ser Val Glu Ala Asp Ile Asn Gly Leu Arg Arg Val Leu Asp Glu

180 185 190

Leu Thr Leu Ala Arg Thr Asp Leu Glu Met Gln Ile Glu Gly Leu Lys

195 200 205

Glu Glu Leu Ala Tyr Leu Lys Lys Asn His Glu Glu Glu Ile Ser Thr

210 215 220

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

225 230 235 240

Pro Gly Thr Asp Leu Ala Lys Ile Leu Ser Asp Met Arg Ser Gln Tyr

245 250 255

Glu Val Met Ala Glu Gln Asn Arg Lys Asp Ala Glu Ala Trp Phe Thr

260 265 270

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

275 280 285

Leu Gln Met Ser Arg Ser Glu Val Thr Asp Leu Arg Arg Thr Leu Gln

290 295 300

Gly Leu Glu Ile Glu Leu Gln Ser Gln Leu Ser Met Lys Ala Ala Leu

305 310 315 320

Glu Asp Thr Leu Ala Glu Thr Glu Ala Arg Phe Gly Ala Gln Leu Ala

325 330 335

His Ile Gln Ala Leu Ile Ser Gly Ile Glu Ala Gln Leu Gly Asp Val

340 345 350

Arg Ala Asp Ser Glu Arg Gln Asn Gln Glu Tyr Gln Arg Leu Met Asp

355 360 365

Ile Lys Ser Arg Leu Glu Gln Glu Ile Ala Thr Tyr Arg Ser Leu Leu

370 375 380

Glu Gly Gln Glu Asp His Tyr Asn Asn Leu Ser Ala Ser Lys Val Leu

385 390 395 400

Claims (2)

1. a polypeptide that is used to prepare anti-liver neoplasm mark CK-19 antibody is characterized in that its aminoacid sequence is shown in SEQ NO.1.

2. as the described polypeptide of claim 1, it is characterized in that described peptide sequence connects a halfcystine at the C end.

3. a polyclonal antibody is characterized in that it is obtained by the described polypeptide immune animal of claim 1.

4. the application of the described polyclonal antibody of claim 3 in preparation diagnosing cancer of liver medicine.

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