CN1246336C - P 28 GANK antibody used for liver cancer identification and diagnosis and its preparation method - Google Patents

Abstract

The present invention relates to the technical field of a medical detection reagent, which is a new rabbit anti-human examination record object p28<GANK> antibody for confirming hepatocellular carcinoma in the pathological differential diagnosis of hepatic carcinoma clinically. The p28<GANK> antibody of the present invention can recognize the hepatocellular carcinoma or hepatic duct cell hybridism hepatic carcinoma, and differentiate the hepatocellular carcinoma and the hepatic duct cell hybridism hepatic carcinoma from cholangiccarcinoma of the liver or metastatic hepatic carcinoma, and therefore, the present invention can be used for preparing a reagent for the differential diagnosis of the hepatic carcinoma, and can also be combined and applied with other hepatic carcinoma biomarkers to further improve the accuracy of hepatic carcinoma diagnosis.

Description

Liver cancer differential diagnosis p28 GANKAntibody and preparation method thereof

Technical field

The present invention relates to medical detection reagent technical field, is the anti-people p28 of a kind of new marker rabbit that in the liver cancer pathological differential diagnosis clinically hepatocellular carcinoma is confirmed ^GANKAntibody makes a distinction hepatocellular carcinoma and bile duct cell or secondary liver cancer, so that symptomatic treatment.

Background technology

Liver cancer roughly can be divided into three types on the tissue origin, i.e. hepatocellular carcinoma, bile duct cell liver cancer and secondary liver cancer.Because the pathology characteristics of liver cancer are feature to lose normal liver lobule structure, therefore on weave construction, be difficult to judgement is made in the source of disrupted tumour cell, especially in low differentiated liver cancer.And the biopsy specimen of surgery brings difficulty also for the pathological diagnosis of liver cancer because be subjected to organizing the restriction in the visual field.At present in hepatocellular carcinoma commonly used HepParl, AFP and CD34 as diagnosis antibody, and in bile duct cell liver cancer often with CK18 and CK19 as diagnosis antibody.Report to the hepatocarcinoma mark thing recall rate of present application in the real work is had nothing in common with each other.Though classical AFP mark high specificity, susceptibility is not high, and the actual positive rate in liver cancer tissue only is 25%～50%.

Summary of the invention

The present invention uses the p28 that filters out according to the principle of antigen and antibody specific combination from the human liver cell liver cancer tissue ^GANKThe p28 of gene preparation ^GANKThe proteantigen immunizing rabbit, the anti-people p28 of preparation rabbit ^GANKAntibody is because p28 ^GANKAntibody can the specific recognition hepatocellular carcinoma pernicious liver cell, therefore can be with the anti-people p28 of rabbit ^GANKAntibody is used to detect the human liver cell liver cancer in liver cell source, and hepatocellular carcinoma and bile duct cell liver cancer, secondary liver cancer are made a distinction.

People's liver cance high-expression p28 ^GANKGene is that people such as Fujita in 2000 use the new gene (GenBank:D83197) that the subtractive hybridization method filters out from the human liver cell liver cancer tissue, the protein of the 25kDa that 226 amino acid of its open reading frame coding are formed, wherein contain 6 repeat ankyrin sequences and 1 Rb protein binding domain (Nature Medicine 2000,6:96-99.).Its structure is seen aminoacid sequence table 1.

One, the anti-people p28 of rabbit of the present invention ^GANKThe preparation method of antibody is as follows:

1.RT-PCR method clone p28 ^GANKGene

1.1 synthetic primer:

Forward primer H1: 5 '-gcg GatccIts 5 ' end of agtagttgctgggacagc-3 ' is introduced BamHI restriction enzyme site ggatcc

Reverse primer H2: 5 '-cg CtcgagIts 5 ' end of cttgagtattaaacccagg-3 ' is introduced XhoI restriction enzyme site ctcgag

1.2 the synthesizing single-stranded DNA of reverse transcription reaction (adopting Gibco/BRL Corporation's Super script reverse transcription test kit):

Reaction is undertaken by following condition: the total RNA of people's liver cancer tissue (presses the operation of Life Technologies company's T rizol RNA purification kit, preparation) (1 μ g/ μ l) 2 μ l, Hexanucleotide primer (10 μ mol/L) 7 μ l at random, deoxyribonucleotide dNTPs (10mmol/L) 1 μ l, burnt ethylene carbonate (DEPC) treating water 2 μ l, 5 * the first chains synthesize damping fluid 4 μ l, dithiothreitol (DTT) DTT (0.1mol/L) 2 μ l, RNA enzyme inhibitors RNasin 1 μ l, RNA relies on DNA synthetic enzyme Superscript II 1 μ l, total reaction volume is 20 μ l, 42 ℃ of water-baths 50 minutes, 70 ℃ of deactivations are after 15 minutes, and it is standby that the reverse transcription product of gained is put-20 ℃ of preservations.

1.3 with above-mentioned reverse transcription product is template, pcr amplification people p28 ^GANKGene cDNA:

Reaction system is in following ratio: reverse transcription product 2 μ l, 10 * PCR damping fluid, 5 μ l, MgCl ₂(25mmol/L) 3 μ l, deoxyribonucleotide dNTPs (10mmol/L) 1 μ l, primer H1 (10 μ mol/L) 1 μ l, primer H2 (10 μ mol/L) 1 μ l, polysaccharase Taq polymerase 0.5 μ l, deionized water 36.5 μ l, total reaction volume is 50 μ l.The PCR program is: 94 ℃ of sex change 4 minutes; 94 ℃ 40 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, totally 35 circulations; 72 ℃ were extended 8 minutes.Reclaim purified pcr product by agarose gel electrophoresis routinely then.

1.4 make up p28 ^GANKGene induced recombinant expression pProEX-HTb-p28 ^GANK.

With BamHI and XhoI difference double digestion 6 * Histidine fusion expression vector pProEX-HTb (Gibco/BRL, Life Technologies, NY) and above-mentioned PCR purified product, separate, reclaim DNA test kit recovery enzyme with QIAGEN company glue and cut dna fragmentation through 1.5% sepharose.The pProEX-HTb carrier of then enzyme being cut behind the purifying is connected through the T4 ligase enzyme with PCR product after enzyme is cut purifying, obtains connecting product.

1.5 make up engineering bacteria HT-p28 and identify recombinant plasmid:

Routinely, with above-mentioned connection product transformed competence colibacillus bacillus coli DH 5 alpha, be coated with penbritin LB flat board, 37 ℃ of incubators spend the night, and extract plasmid DNA after the picking list bacterium colony amplification cultivation, carry out enzyme with BamHI/XhoI and cut with dna sequencing and identify.Qualification result shows that its recombinant expression plasmid is: pProEX-HTb-p28 ^GANK, its corresponding engineering bacterium is HT-p28.

2.p28 ^GANKProteic expression and purification

Routinely with engineering bacteria HT-p28 with 0.6mmol/L isopropylthio-(IPTG) inducing culture after 4 hours, the process specifications purifying p28 that provides according to German QIAGEN company histidine tagged protein purification kit ^GANKAlbumen obtains p28 ^GANKAlbumen.

The present invention H1, the p28 that the H2 primer increases from the total RNA reverse transcription of people's liver cancer tissue product ^GANKSequence is the cDNA between (GenBank:Dg3197) 78bp-761bp, its total length 684 bp, expressed p28 ^GANKAlbumen contains 221 amino acid.Structure is seen aminoacid sequence table 2.The p28 that forms by 226 amino acid shown in itself and the aminoacid sequence table 1 ^GANKAlbumen is compared 5 amino acid that lacked the C end, but the effect of antigen immune is constant.

3. the anti-people p28 of rabbit ^GANKThe preparation of antibody, purifying and evaluation

Polyclonal Antibody Preparation by " molecular cloning experiment guide " (second edition, Sa nurse Brooker J. etc., Science Press, Beijing, 1997, down with) method introduced carries out.

3.1 immunization

Get above-mentioned p28 ^GANKAlbumen 2mg is dissolved in 1.5ml physiological saline and isopyknic complete Freund's adjuvant, and (Gibco/BRL, Life Technologies is NY) after the emulsification, at the back of new zealand white rabbit intracutaneous multi-point injection.In 3 weeks after the first immunisation, new zealand white rabbit is carried out booster immunization respectively three times, each 2 weeks at interval.Each dosage is: p28 ^GANKAlbumen 1mg is dissolved in 1.5ml physiological saline, and (Gibco/BRL, LifeTechnologies NY) inject after the emulsification with isopyknic non-complete Freund's adjuvant.

3.2 preparation p28 ^GANKAntibody

For the third time a week behind the booster immunization, get rabbit ear edge venous blood 1ml, determining to tire reaches more than 1: 32.Behind booster immunization for the third time, carried out the carotid artery intubate on the 10th day again and get blood, exhaust, with blood put 4 ℃ 24 hours, draw the rabbit anteserum of separating out earlier, again with surplus blood in 4 ℃, 4000 rev/mins centrifugal 10 minutes, draw upper serum.With after the serum packing in-80 ℃ of preservations.Routinely, with the anti-people p28 of antigen affinity purification rabbit that is fixed on the fine film of nitre ^GANKAntibody.

3.3 identify p28 ^GANKAntibody

Identify the specificity of above-mentioned antibody purification with Western blotting, the concrete method of introducing by " molecular cloning experiment guide " is carried out, and with the antibody purified dilution, extent of dilution is 1: 50.It can specific recognition p28 ^GANKAntigen also comprises the p28 that expresses in the energy specific recognition eukaryotic cell lysate ^GANKAntigen.Qualification result shows that it is p28 ^GANKAntibody.

Two, p28 ^GANKThe immunohistochemical staining experiment of antibody differential diagnosis hepatocellular carcinoma

1. the immunohistochemical staining of liver cancer section:

Adopt Cell immunohistochemical staining method to detect the cell source of liver cancer tissue routinely.The test kit DAKO EnVision System that immunohistochemical methods employing U.S. DAKO company produces (DAKO Corporation, carpinteria, USA).

P28 ^GANKAntibody has liver cell specific stain preferably on tissue specificity, its staining reaction in human liver cell liver cancer is positive; And in people's bile duct cell liver cancer and secondary liver cancer, all negative to bile duct cell, vascular endothelial cell, lymphocyte, Kuffer Schwann Cells and the dyeing of liver mesenchymal cell.

With hepatocellular carcinoma (HCC), bile duct cell liver cancer (ICC), hepatic duct cytomixis liver cancer (C-HCC-CC) and secondary liver cancer (MHC) are used p28 respectively ^GANKAntibody, HepParl antibody, CK18 antibody and AFP antibody grouping carrying out immunohistochemical staining, coloration result is analyzed with the Crohn-Mantel-Haentzsel statistical technique, and comparative result sees Table 3.

Table 3 p28 ^GANK, Hep-Par1, CK18 and the dyeing of AFP antibody mediated immunity group are relatively

Types of organization	The example number	p28 GANK			HepPar1			CK18			AFP
														+	-	+ ％	+	-	+％	+	-	+ ％	+	-	+ ％
		Hepatocellular carcinoma (HCC) hepatic duct mixing with cells liver cancer (C-HCC-CC) bile duct cell liver cancer (ICC) metastatic hepatic carcinoma (MHC)	24 5 5 9	18 3 0 0	6 2 5 9	75 60 0 0	17 5 0 2	7 0 5 7	71 100 0 22	20 4 3 1	4 1 2 8	83 80 60 11	2 2 0 0													22 3 5 9	8 33 0 0
x 2，p														x 2＝16.44， p＝0.001			x 2＝20.05， p＝0.001			x 2＝13.54， p＝0.004			x 2＝5.29， p＝0.152

By table 3 as seen, to the HCC or the C-HCC-CC in liver cell source, the lower and p28 of AFP antibody staining rate ^GANkAntibody is the same with CK18 antibody with HepPar1 all staining reaction preferably, but to ICC and MHC, p28 ^GANKAntibody does not all dye, and HepPar1 antibody then still has part dyeing to MHC and CK18 antibody to ICC and MHC, and p28 is described ^GANKAntibody has actual application value in the differential diagnosis of the liver neoplasm of distinguishing the liver cell source.

2. liver cancer differential diagnosis experiment:

In order further to estimate p28 ^GANKAntibody is used for the possibility of clinical liver cancer pathological differential diagnosis, with p28 ^GANKAntibody compares with HepPar1 and the CK18 antibody used always clinically, adopts Golden Standard diagnostic evaluationtest (evaluation test of golden index diagnosis marker) to analyze.Evaluation index has: susceptibility (sensitivity, write a Chinese character in simplified form Se), specific degree (specificity, write a Chinese character in simplified form Sp), misdiagnosis rate (mistake diagnostic rate writes a Chinese character in simplified form α), rate of missed diagnosis (omissiondiagnostic rate, write a Chinese character in simplified form β), (positive predictive value writes a Chinese character in simplified form PV to positive predictive value ⁺), (negative predictive value writes a Chinese character in simplified form PV to negative predictive value ^-), accuracy rate (validity rate writes a Chinese character in simplified form π), Younden index (YoundenIndex writes a Chinese character in simplified form YI).

Concrete operations are: with pathological diagnosis is that the liver neoplasm sample that liver cell is originated is classified as the positive group of diagnosis D ⁺, D ⁺=HCC+C-HCC-CC; Pathological diagnosis is that the liver neoplasm sample in non-liver cell source is classified as the negative group of diagnosis D ^-, D ^-=ICC+MHC.Detect the positive T that is classified as with antibody to be checked ⁺, T ⁺Comprise in the group that pathological diagnosis is the sample in liver cell source, i.e. the routine number a (making a definite diagnosis) that makes a definite diagnosis with antibody to be checked, a=HCC ⁺+ C-HCC-CC ⁺, comprise that also pathological diagnosis is the sample in non-liver cell source, promptly uses the routine number b (mistaken diagnosis) of antibody mistaken diagnosis to be checked, b=ICC ⁺+ MHC ⁺Detect the negative T that is classified as with antibody to be checked ^-, T ^-Comprise in the group that pathological diagnosis is the sample in liver cell source, i.e. the routine number c (failing to pinpoint a disease in diagnosis) that fails to pinpoint a disease in diagnosis with antibody to be checked, c=HCC ^-+ C-HCC-CC ^-, comprise that also pathological diagnosis is the sample in non-liver cell source, the routine number d (differential diagnosis) that promptly makes differential diagnosis, d=ICC with antibody to be checked ^-+ MHC ^-Evaluation result is as shown in table 4.HepPar1, CK18 and p28 are estimated in table 4 gold standard test (Golden Standard Test) ^GANKAntibody is 40

Performance in the differential diagnosis of example liver cancer

Test	Pathological diagnosis		Amount to
				D +	D -
	HepPar1 T + T -Amount to CK18 T + T -Amount to p28 GANK T + T -Amount to	a＝22 c＝7 a+c＝29 a＝23 c＝5 a+c＝28 a＝18 c＝7 a+c＝25				b＝1 d＝10 b+d＝11 b＝5 d＝7 b+d＝12 b＝0 d＝15 b+d＝15	a+b＝23 c+d＝17 a+b＝28 c+d＝12 a+b＝18 c+d＝22
HepPar1：Se＝0.76 sp＝0.91，α＝0.09 β＝0.24，π＝0.8000 YI＝0.67，PV +＝0.96 PV -＝0.59 CK18：Se＝0.85 Sp＝0.64，α＝0.36 β＝0.15.π＝0.7890 YI＝0.49，PV +＝0.85 PV -＝0.64 p28 GANK：Se＝0.70 Sp＝1.00，α＝0.00 β＝0.30，π＝0.8235 YI＝0.70，Pv +＝1.00 PV -＝0.70

Find out p28 by table 4 ^GANKAntibody is at specific degree (Sp), misdiagnosis rate (α), accuracy rate (π), Younden index (YI), positive predictive value (PV ⁺) and negative predictive value (PV ^-) etc. all be better than HepPar1 and CK18 antibody on the index.P28 is described ^GANKAntibody is applied to the superiority of clinical liver cancer pathological differential diagnosis.

Description of drawings

The recombinant plasmid pProEX-HTb-p28 that Fig. 1 makes up for the present invention ^GANKStructural representation.

Fig. 2 is p28 of the present invention ^GANKPolyacrylamide gel electrophoresis collection of illustrative plates behind the antigen purification.

Fig. 3 is p28 of the present invention ^GANKThe Western blotting of antibody identifies figure.

Fig. 4 is p28 of the present invention ^GANKAntibody is at the in-house immunohistochemical methods figure of hepatocellular carcinoma (200 *).

Fig. 5 is p28 of the present invention ^GANKThe specific stain figure (200 *) of antibody in liver cancer tissue.

Fig. 6 is p28 of the present invention ^GANKThe immunohistochemical methods figure (200 *) of antibody in the bile duct cell liver cancer tissue.

Fig. 7 is p28 of the present invention ^GANKAntibody is at the in-house immunohistochemical methods figure of secondary liver cancer (200 *).

Embodiment

Embodiment 1: the anti-people p28 of preparation rabbit ^GANKAntibody

1.RT-PCR method is cloned p28 from people's liver cancer ^GANKGene

1.1 synthetic primer:

Forward primer H1:5 '-gcg GatccIts 5 ' end of agtagttgctgggacagc-3 ' is introduced BamHI restriction enzyme site ggatcc

Reverse primer H2:5 '-cg CtcgagIts 5 ' end of cttgagtattaaacccagg-3 ' is introduced XhoI restriction enzyme site ctcgag

1.2 the synthesizing single-stranded DNA of reverse transcription reaction:

The total RNA of people's liver cancer tissue (presses the operation of Life Technologies company's T rizol RNA purification kit, 1 μ g/ μ l) 2 μ l, Hexanucleotide primer (10 μ mol/L) 7 μ l at random, deoxyribonucleotide dNTPs (10mmol/L) 1 μ l, burnt ethylene carbonate (DEPC) treating water 2 μ l, 5 * the first chains synthesize damping fluid 4 μ l, dithiothreitol (DTT) DTT (0.1mol/L) 2 μ l, RNA enzyme inhibitors RNasin 1 μ l, RNA relies on DNA synthetic enzyme Superscript II 1 μ l, and total reaction volume is 20 μ l, 42 ℃ of water-baths 50 minutes, 70 ℃ of deactivations are after 15 minutes, and it is standby that the reverse transcription product of gained is put-20 ℃ of preservations.

1.3 with above-mentioned reverse transcription product is template, pcr amplification people p28 ^GANKGene cDNA:

Reverse transcription product 2 μ l, 10 * PCR damping fluid, 5 μ l, MgCl ₂(25mmol/L) 3 μ l, deoxyribonucleotide dNTPs (10mmol/L) 1 μ l, primer H1 (10 μ mol/L) 1 μ l, primer H2 (10 μ mol/L) 1 μ l, polysaccharase Taqpolymerase 0.5 μ l, deionized water 36.5 μ l, total reaction volume is 50 μ l.The PCR program is: 94 ℃ of sex change 4 minutes; 94 ℃ 40 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, totally 35 circulations; 72 ℃ were extended 8 minutes.Reclaim the PCR product through the agarose gel electrophoresis separation and purification.

1.4 make up p28 ^GANKGene induced recombinant expression pProEX-HTb-p28 ^GANK:

With BamHI and XhoI difference double digestion 6 * Histidine fusion expression vector pProEX-HTb (Gibco/BRL, Life Technologies, NY) and the PCR product of above-mentioned purifying, separate through 1.5% sepharose, reclaim test kit recovery enzyme with QIAGEN company glue and cut dna fragmentation, then enzyme is cut the DNA that pProEX-HTb carrier behind the purifying and PCR enzyme are cut purifying, with the molecule number ratio is 1: 5, add T4 ligase enzyme damping fluid 2 μ l, add T4 ligase enzyme 2u (5u/ μ l), mend deionized water to total reaction volume 20 μ l, put 22 ℃ of water-baths and connect and spend the night, 65 ℃ of inactivators 10 minutes.

1.5 make up engineering bacteria HT-p28 and identify recombinant plasmid:

Routinely, with above-mentioned connection product transformed competence colibacillus bacillus coli DH 5 alpha, be coated with penbritin LB flat board, 37 ℃ of incubators spend the night, and extract plasmid DNA after the picking list bacterium colony amplification cultivation, carry out enzyme with BamHI/XhoI and cut, and order-checking is identified.The recombinant expression plasmid that qualification result obtained is: pProEX-HTb-p28 ^GANK, its corresponding engineering bacterium is HT-p28.

2.p28 ^GANKProteic expression and purification

Routinely with above-mentioned engineering bacteria HT-p28 with 0.6mmol/L isopropylthio-(IPTG) inducing culture after 4 hours, the process specifications purifying p28 that provides by German QIAGEN company histidine tagged protein purification kit ^GANKAlbumen.Concise and to the point step is as follows:

The HT-p28 engineering bacteria that takes a morsel is inoculated in 20ml LB (the containing penbritin 100mg/ml) substratum, and 37 ℃ of following shaking culture are spent the night.Inoculum size by 1% with the bacterial classification of incubated overnight in 1 liter LB (containing penbritin 100mg/ml) substratum, 37 ℃ of following shaking culture are to absorbance value OD600nm about 0.5～0.7, add the IPTG that final concentration is 0.6mM then, through inducing culture after 4 hours, 4 ℃ down 6,000 rev/mins centrifugal 15 minutes.Bacterial precipitation lysate (50mM NaH ₂PO ₄PH8.0,300mM NaCl, 10mM imidazoles, 10mM beta-mercaptoethanol, 10% glycerine, 1mg/ml N,O-Diacetylmuramidase) 10ml is resuspended, on ice bath ultrasonic 10 minutes * 3 times, again 4 ℃ down 13,000 rev/mins centrifugal 15 minutes.Centrifugal back supernatant mixes with 1ml 50% (V/V) Ni-NTa suspension, and 4 ℃ were shaken 1 hour slowly, then with purification column on whole liquid, collects effluent liquid.With washing fluid (50mM NaH ₂PO ₄PH8.0,300mM NaCl, 20mM imidazoles, 10mM beta-mercaptoethanol, 10% glycerine) 10ml * 3 wash post.Use elutriant (50mMNaH at last ₂PO ₄PH8.0,300mM NaCl, 250mM imidazoles, 10mM beta-mercaptoethanol, 10% glycerine) 1ml * 5 wash-outs, six Histidine fusion roteins, collect albumen elution samples liquid.Get 10 μ l protein sample liquid and add 10 μ l sample-loading buffer (100mM Tris-Cl pH6.8, the 200mM dithiothreitol (DTT), 4% sodium lauryl sulphate, 0.2% bromjophenol blue, 20% glycerine), 100 ℃ of sex change 3 minutes, last sample 10% polyacrylamide gel (SDS-PAGE) electrophoresis, coomassie brilliant blue staining is identified.Electrophoretogram as shown in Figure 2, wherein label 1 is a bacterial lysate; 2 is the upper prop effluent liquid; 3 for washing post liquid; 4～7 is albumen elutriant successively.Find out the p28 of purifying by Fig. 2 ^GANKThe protein band correct position, purity is higher, and yield also can satisfy next step immunizing rabbit institute expense.

3.p28 ^GANKPolyclonal Antibody Preparation, purifying and evaluation

The method that Polyclonal Antibody Preparation is introduced by " molecular cloning experiment guide " is carried out.Get above-mentioned p28 ^GANKAlbumen 2mg is dissolved in 1.5ml physiological saline, and (Gibco/BRL, Life Technologies NY) after the emulsification, select 5 some branch injections at the back of new zealand white rabbit intracutaneous with isopyknic complete Freund's adjuvant.In 3 weeks after the first immunisation, new zealand white rabbit is carried out booster immunization respectively three times, each 2 weeks at interval.Each dosage is: p28 ^GANKAlbumen 1mg is dissolved in 1.5ml physiological saline, and (Gibco/BRL, Life Technologies NY) injects after the emulsification with isopyknic non-complete Freund's adjuvant.For the third time a week behind the booster immunization, get rabbit ear edge venous blood 1ml, determining to tire reaches more than 1: 32.Behind booster immunization for the third time, carried out the carotid artery intubate on the 10th day again and get blood, exhaust, with blood put 4 ℃ 24 hours, draw the rabbit anteserum of separating out earlier, again with surplus blood in 4 ℃, 4000 rev/mins centrifugal 10 minutes, draw upper serum.After the serum packing ,-80 ℃ of preservations.

The antigen affinity purification p28 on the fine film of nitre is fixed in utilization ^GANKSpecific antibody, concrete steps are as follows: p28 ^GANKSample 10%SDS-PAGE on the antigen samples 100 μ g, electrotransfer are to nitrocellulose NC film, and ponceau dyeing marks p28 ^GANKThe antigen zone position.Cut antigen zone, 1 * TBS flush away dyestuff is after 5% skim-milk (1 * TBS preparation) sealing 2 hours, with rabbit anti-serum (dilution in 1: 20) incubated at room 3 hours, change the middle rinsing of 1 * PBS (1%Tween-20) 10 minutes * 3 times over to, use 1 * PBS rinsing again 10 minutes * 2 times.Get a plastics plate,, the NC film is put thereon, on the NC film, drip elution buffer (0.2mol/L glycine pH2.8,1mmol/L EGTA) 200 μ l, place wet box, room temperature 20 minutes at bottom tiling parafilm film.Collect whole elutriants and put the 1.5ml centrifuge tube, add 1/10 volume 1mol/LTris alkali (pH10), 1/10 volume, 10 * PBS, 1/10 volume 2%BSA, 1/100 volume, 4% sodium azide.Renew bright antiserum(antisera) and continue aforesaid operations, wash-out antibody is collected in gradation.

With the specificity of Western blotting purification Identification antibody, carry out purifying p28 by the method that " molecular cloning experiment guide " introduced ^GANKAntibody dilution is 1: 50.The results are shown in Figure 3, wherein each antigen is respectively, 1:BSA antigen, 2～4:His-p28 ^GANKAntigen, 5:SK-Hep1 cell pyrolysis liquid, 6: the HEK293 clone lysate of stable transfection pcDNA3.1,7: stable transfection Myc-p28 ^GANKHEK293 clone lysate.Used antibody is respectively, and 1,2,5～7 use p28 ^GANKAntibody (1: 50); 3 usefulness in advance with His-p28 ^GANKP28 after antigen is hatched ^GANkAntibody; P28 after 4 usefulness are hatched with BSA antigen in advance ^GANKAntibody.P28 ^GANKAntibody capable specific recognition p28 ^GANKAntigen, this specific reaction can be by p28 ^GANKAntigenic in advance in conjunction with and end.P28 ^GANKThe p28 that antibody is expressed in also can specific recognition eukaryotic cell lysate ^GANKAntigen.

Embodiment 2:p28 ^GANKAntibody differential diagnosis hepatocellular carcinoma

The test kit DAKO EnVision System that immunohistochemical methods employing U.S. DAKO company produces (USA), operation is carried out routinely for DAKOCorporation, carpinteria, and concrete steps are as follows:

1. make the used section of liver cancer tissue immunohistochemical methods:

Get people's liver cancer tissue piece 1cm * 1cm * 1cm, paraffin embedding is cut into slices with 5 μ m thickness routinely.After 60 ℃ of baking boxs of paraffin section spend the night, by following flow process dewaxing entry, 5 minutes → distilled water of (2) 10 minutes → dimethylbenzene of (1) 10 minute → dimethylbenzene of dimethylbenzene (3) 10 minutes → 100% ethanol 5 minutes → 95% ethanol 5 minutes → 85% ethanol, 5 minutes → 75% ethanol 5 minutes.

2. immunohistochemical staining:

Section 3%H ₂O ₂Methanol solution is handled, and room temperature was placed after 20 minutes, washed 5 minutes * 3 times with distilled water.The row antigen retrieval, section is put into the 0.01M citrate buffer and was boiled 5 minutes, ceases fire 10 minutes, boils 5 minutes again, naturally cools to room temperature, and distilled water is washed 5 minutes * 3 times.37 ℃ were sealed 30 minutes with 1% bovine serum albumin (BSA) down, got rid of deblocking liquid, did not wash, and directly added p28 of the present invention ^GANKAntibody (one anti-, 1: 10), put in the wet box 37 ℃ 30 minutes, put 4 ℃ of refrigerators then 16 hours.After the taking-up, room temperature rewarming 15 minutes is washed 5 minutes * 3 times with 0.01mol/L PBS then.Drip DAKO goat-anti rabbit two anti-(DAKOCorporation, carpinteria, USA), 37 ℃ 45 minutes, wash 5 minutes * 4 times with 0.01mol/L PBS, the DAB colour developing was washed 1 time with 0.01mol/L PBS after 2～10 minutes, Hematorylin was redyed 10 seconds, put tap water and returned indigo plant, put distilled water immersion.The section dehydration is transparent, the neutral gum mounting, and cover glass covers.

3. observations:

(1) immunohistochemical staining of hepatocellular carcinoma cancerous tissue and corresponding adjacent tissues the results are shown in Figure 4.Fig. 4 shows the stained positive regional centralized at the liver cell endochylema, and a matter lymphocyte is negative.Cancerous tissue (HCC) staining power more same case cancer beside organism (Para-HCC) is strong.

(2) immunohistochemical methods of hepatocellular carcinoma and Yihong-haematoxylin dyeing results of comparison is seen Fig. 5.Found out that by Fig. 5 left side figure is the liver cell stained positive, bile duct epithelial cell, vascular endothelial cell, Kuffer Schwann Cells and mesenchymal cell dyeing are negative.Right figure is Yihong-Hematorylin (H﹠amp of same sample continuity section; E) dyeing.

(3) immunohistochemical staining of bile duct cell liver cancer the results are shown in Figure 6.Be shown as negative staining.

(4) immunohistochemical staining of secondary liver cancer the results are shown in Figure 7.Show metastasis dyeing feminine gender among the figure, the other hepatic tissue stained positive of cancer from colorectal carcinoma.

The The above results explanation, p28 ^GANKAntibody is stained positive in human liver cell liver cancer, and the expression in people's bile duct cell liver cancer and secondary liver cancer is all feminine gender.Liver cell specific stain is preferably arranged, and bile duct cell, vascular endothelial cell, lymphocyte, Kuffer Schwann Cells and the dyeing of liver mesenchymal cell are all negative on tissue specificity.

P28 of the present invention ^GANKAntibody capable is discerned the liver cell property or the hepatic duct cytomixis liver cancer in liver cell source preferably, and they and bile duct cell or secondary liver cancer are made a distinction.In the pathological diagnosis of clinical liver cancer, actual application value is arranged, also can with other liver cancer marker combined utilization, improve the accuracy rate of diagnosing cancer of liver, so that formulate suitable treatment plan at corresponding liver cancer type.

Table 1 p28 ^GANKThe Argine Monohydrochloride sequence table

MEGCVSNLMVCNLAYSGKLEELKESILADKSLATRTDQDSRTALHWACSAGHTEIVEFL

LQLGVPVNDKDDAGWSPLHIAASAGRDEIVKALLGKGAQVNAVNQNGCTPLHYAASK

NRHEIAVMLLEGGANPDAKDHYEATAMHRAAAKGNLKMIHILLYYKASTNIQDTEGNT

PLHLACDEERVEEAKLLVSQGASIYIENKEEKTPLQVAKGGLGLILKRMVEG

Table 2 p28 ^GANKProteantigen aminoacid sequence table

MEGCVSNLMVCNLAYSGKLEELKESILADKSLATRTDQDSRTALHWACSAGHTEIVEFL

LQLGVPVNDKDDAGWSPLHIAASAGRDEIVKALLGKGAQVNAVNQNGCTPLHYAASK

NRHEIAVMLLEGGANPDAKDHYEATAMHRAAAKGNLKMIHILLYYKASTNIQDTEGNT

PLHLACDEERVEEAKLLVSQGASIYIENKEEKTPLQVAKGGLGLILK

Claims (1)

1. the anti-people p28 of rabbit ^GANKAntibody is used to prepare the purposes of hepatocellular carcinoma differential diagnosis reagent, the anti-people p28 of said rabbit ^GANKThe preparation method of antibody is as follows:

(1) synthetic primer

Forward primer: H ₁: 5 '-gcg GatccAgtagttgctgggacagc-3 '

Reverse primer: H ₂: 5 '-cg CtcgagCttgagtattaaacccagg-3 '

(2) the synthesizing single-stranded DNA of the personnel selection total RNA reverse transcription of liver cancer tissue;

(3) be template with above-mentioned reverse transcription product, use primer H ₁And H ₂Pcr amplification people p28 ^GANKGene cDNA;

(4) make up p28 ^GANKGene induced expression plasmid pProex-HTb-p28 ^GANK

(5) make up engineering bacteria HT-P28;

(6) express and purifying p28 with engineering bacteria HT-P28 ^GANKAlbumen;

(7) use p28 ^GANKThe protein immunization rabbit makes to produce the anti-people p28 of rabbit ^GANKAntibody;

(8) by the anti-people p28 of immunize rabbit serum pref rabbit ^GANKAntibody.

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