1、Agricultural Sciences in China2008,7(12):1516-1523 2008,CAAS.All rights reserved.Published by Elsevier Ltd.December 2008Establishment of the Method of Immunohistochemistry Assay for the Detectionof Scrapie in Chinese Short-Tailed Han Sheep by Monoclonal AntibodyZHANG Yong-qiang1,WANG Zhi-liang1,WU X
2、iao-dong1,LIU Yu-tian1,ZHANG Hai-tao1,2 and BAO En-dong21 National Diagnostic Center for Exotic Animal Disease/China Animal Health and Epidemiology Center,Qingdao 266032,P.R.China2 College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,P.R.ChinaAbstractThe method of immunohist
3、ochemistry assay for the detection of scrapie in Chinese Short-tailed Han sheep was establishedusing monoclonal antibody.Genomic DNA was isolated from Chinese Short-tailed Han sheep blood.Using the polymerasechain reaction technique,PrP27-30 gene sequence was amplified from Chinese Short-tailed Han
4、sheep genomic DNA.Byrecombinant DNA technology,the recombinant protein of Chinese Short-tailed Han sheep PrP27-30 was obtained.Then,using standard methodology of myeloma cell fusion,a panel of monoclonal antibodies was generated.With mAbs,scrapie in Chinese Short-tailed Han sheep was detected by imm
5、unohistochemistry assay.The recombinant protein ofChinese Short-tailed Han sheep PrP27-30 was obtained and a panel of six hybridoma cell lines secreting specific antibodiesto Chinese Short-tailed Han sheep PrP27-30 related to scrapie was obtained with one fusion between myeloma Sp2/0 andspleen cells
6、 from mice immunized with the purified recombinant protein.Four hybridoma cell lines can be used inimmunohistochemistry assay for the detection of scrapie in Chinese Short-tailed Han sheep.So that the special monoclonalantibody developed in author s institute can be used to detect PrPSc of scrapie i
7、n Chinese Short-tailed Han sheep byimmunohistochemistry in China.Key words:Chinese Short-tailed Han sheep,PrP27-30,scrapie,monoclonal antibody,immunohistochemistry assayINTRODUCTIONTransmissible spongiform encephalopathies(TSE)areneurodegenerative diseases in human and animals,in-cluding Creutzfeldt
8、-Jakob disease(CJD),fatal familialinsomnia(FFI)in human,scrapie in sheep and goat,bovine spongiform encephalopathy(BSE)in cow,andother diseases in different species,it is now widely ac-cepted that many structurally diverse proteins canmisfold and cause so-called“conformational diseases”(Prusiner 199
9、8;Klug et al.2008;Avuso et al.2007;van Keulen et al.2008;Siqurdson 2008;WeissmannThis paper is translated from its Chinese version in Scientia Agricultura Sinica.ZHANG Yong-qiang,Ph D,Tel:+86-532-87839922,E-mail:zhangyq_;Correspondence WANG Zhi-liang,Tel:+86-532-87839922,E-mail:Z2004;Martinez-Lage e
10、t al.2005;Aguzzi 2006;Liberske2008).In these diseases,the key protein,prion,under-goes a major conformational change from its cellularnormal structure(PrPC)to an alternative state,whichforms fibrillar aggregates,generally denoted as scrapieisoform(PrPSc)(Kazlauskaitea et al.2005).Now strongevidence
11、indicates that the conversion of a normal soluble?-helical monomeric protein into a?-sheet-rich oligo-meric structure and further fibrillar aggregation are thekey events in the disease pathogenesis(Noinville et al.2008).In normal healthy conditions,prion is a harm-less plasma membrane protein predom
12、inantly expressedin the brain and implicated in copper binding and cellEstablishment of the Method of Immunohistochemistry Assay for the Detection of Scrapie in Chinese Short-Tailed1517 2008,CAAS.All rights reserved.Published by Elsevier Ltd.signaling(Muller et al.2005;Redecke et al.2005).Al-though
13、PrPC and PrPSc share the same amino acidsequence,distinctive physicochemical characteristicsdifferentiate the two isoforms.As opposed to PrPC,PrPSchas a high?-sheet content(Kazlauskaitea et al.2005)and is partially resistant to protease treatment,which isthe principle to detect TSE,and the segment t
14、hat canresist to protease treatment is well known as PrP27-30.The effective methods to detect TSE are immunohis-tochemistry(IHC)and Western blotting.Immunohis-tochemistry is a method that can be carried out on for-malin-fixed tissue to detect PrPSc in situ(O Rourke et al.2000).This sensitive tool re
15、lies not only on detectingthe presence of PrPSc but also on its distribution in thebrain.Debeer et al.(2001)reported that IHC remains asensitive tool applicable to BSE diagnosis even in brainsamples with very advanced autolysis.Wang et al.(2005)and his colleague expressed recombinant Chi-nese yellow
16、 cattle prion protein,developed anti-PrP mono-clonal antibody named as 4C11,and established themethod of IHC assay for the detection of BSE in China.In the present study,we expressed PrP27-30 fusionprotein of Chinese Short-tailed Han sheep,generatedanti-PrP27-30 monoclonal antibodies(mAbs)by im-muni
17、zing Prnp-null-mice,and finally,established themethod of IHC assay to detect scrapie in Chinese Short-tailed Han sheep in China.MATERIALS AND METHODSRecombination of Chinese Short-tailed Hansheep PrP27-30 in E.coliMaterials E.coli DH5?,E.coli BL21(DE3),andplasmid pET-32a were stored in the center la
18、boratoryof China Animal Health and Epidemiology Center.TaqDNA polymerase,T4 DNA ligase,restriction endonu-clease EcoR I and Hind III,and DNA marker DL2000were purchased from TaKaRa company.SpinPrep GelDNA Kit,Ni-NTA HiBind Purification Kit,and T7 pro-moter were from Novagen.Antiprion mAb F89 wereobt
19、ained from TSE Laboratory of United States De-partment of Agriculture.Antimouse IgG+IgM(HRP)antibody was obtained from Sigma.Primers One set of primers of Chinese Short-tailedHan sheep PrP27-30 gene with two terminal codens(TAATAA)were designed,and the EcoR I and Hind IIIsites were inserted to 5 and
20、 3 ends,respectively.Thesequence of primers is as follows:Up-stream primer:5-TCGGAATTCGGTCAAGGTGGTAGC-3 EcoR IDown-stream primer:5-GGCAAGCTTTGTTTATTAGCCTGGGAT-3 Hind IIIPCR amplification of Chinese Short-tailed Hansheep PrP27-30 gene Whole gene was extracted from20 mL fresh blood of Chinese Short-ta
21、iled Han sheep,and PrP27-30 gene subunit was amplified using PCR(94C,7 min;94C,1 min;55C,0.5 min;72C,0.5min;30 cycles;72C,10 min).Construction of expression vector carrying PrP27-30 gene of Chinese Short-tailed Han sheep Thebacterial expression vector pET32a and PCR productswere digested using restr
22、ictive enzymes EcoR I andHind III,respectively,to generate cohesive ends.ThePrP27-30 gene of Chinese Short-tailed Han sheep wasinserted into the vector pET32a by T4 DNA ligase afterboth of them were purified.The recombinant vectorcontaining PrP27-30 gene was developed providing anN-terminal polyhist
23、idine tag and was termed as PrP-pET-32a.The sequence of inserted fragment was con-firmed by DNA sequencing.Fusion gene expression of PrP27-30 of ChineseShort-tailed Han sheep Plasmid PrP-pET-32a wastransformed into E.coli BL21(DE3).Recombinantstrain was selected using agar plate containing ampicil-l
24、in(AMP)and confirmed by restriction enzyme mapping.Bacterial culture TB(with 100?g mL-1 AMP)wasgrown until they reached an optical density of 1.5 atOD600.The recombinant protein expression was in-duced for 1,3,4,and 5 h,and overnight at 25C afterthe addition of 1 mM isopropyl?-D-thiogalactopyranosid
25、e(IPTG)to the culture.Bacteria was lysed by ultra-sonic wave and removed by centrifugation.The solublefraction was loaded onto a Ni2+-column preequilibratedto purify the fusion protein.Analysis and identification of fusion protein Thewhole bacteria and the purified fusion protein were ana-lyzed by S
26、DS-PAGE and identified by Western blotting.The recombinant protein was separated by 12%SDS-PAGE and transferred on NC membrane.Free bindingsites were blocked by 5%nonfat dried milk in phos-1518ZHANG Yong-qiang et al.2008,CAAS.All rights reserved.Published by Elsevier Ltd.phate buffered saline(PBS)fo
27、r overnight at 4C priorto addition of antibodies.Mouse monoclonal antibodyF89 was diluted using PBS and incubated with NC mem-brane for 1.5 h at room temperature with constantshaking.After washing with PBST for three times,membrane was incubated with horseradish peroxidase(HRP)-conjugated antimouse
28、IgG+IgM(diluted withPBS)for 1 h.After washing it again for three times,membrane was displayed in 3,3-diaminobenzidine(DAB)solution to visualize the positive band.Generation of anti-Chinese Short-tailed Han sheepPrP27-30 mAbsAnimals Prnp-null-mice were given by the OIE refer-ence laboratory,Institute
29、 of Animal Neurology in BernUniversity,Switzerland.Balb/C mice were obtainedfrom the Experimental Animal Center of Shandong Uni-versity of Traditional Chinese Medicine,China.Reagent Complete Freund s adjuvant,incompleteFreund s adjuvant,DMEM,and PEG(MW4 000)werepurchased from Sigma Inosine,thymine,a
30、nd nucleo-side were from Gibico.Preparation of mouse myeloma cell line Sp2/0 Sp2/0cell was maintained at 37C in humidified 5%CO2 inHT medium supplemented with 10%FCS.Generation of monoclonal antibodies Eight-week oldPrnp-null-mice were subcutaneously immunized with30?g of recombinant Chinese Short-t
31、ailed Han sheepPrP27-30 protein emulsified in complete Freund s ad-juvant and boosted with an equal amount of antigen pro-tein emulsified in incomplete Freund s adjuvant with aninterval of two weeks.After two booster doses,eachimmunized mice was injected intravenously with 15?gprotein in 100?L PBS t
32、hree days before cell fusion.Mice were put to death,and single spleen cell suspen-sions were fused with Sp2/0 myeloma cells using poly-ethylene glycol(PEG)4 000.The fused cells were cul-tivated in DMEM supplemented with 10%FCS and HATand plated into 96-well cell culture plates.Test of positive well
33、by indirect enzyme-linkedimmunosorbent assay Hybridoma cell supernatant wasscreened by indirect ELISA.Polyvinyl plates werecoated for 2 h at 37C with 50?L of recombinant PrP27-30 solution(8?g mL-1 diluted in carbonate bufferedsaline,pH 9.6)and blocked with 2%bovine serum al-bumin(Sigma)for 1.5 h at
34、37C,washed in PBST,andincubated with 50?L of undiluted supernatant at 37Cfor 1 h.After washing three times,a horseradish peroxi-dase(HRP)-conjungated goat antimouse antibody(1:8 000)was added and was kept for 45 min at 37C.The reaction was visualized using 3,3,5,5-tetramethylbenzidine ELISA-substrat
35、e and blocked with1 M sulfidric acid.ELISA plates were read at 450 nm.The positive wells at first screening were amplified at24-well plates,and the positive wells at following screen-ing were cultivated at 96-well plates with one cell inone well approximately.After two limited dilution,thepositive w
36、ells were chosen for further studies.Identification of mAbs by Western blotting Recom-binant fusion protein and ovine brain homogenate wereused as source of PrP.Recombinant fusion proteinwas separated by 12%SDS-PAGE and transferred onNC membrane similar to the steps mentioned in“Analy-sis and identi
37、fication of fusion protein”.Ten percentovine brain homogenate was separated by 15%SDS-PAGE and blotted on PVDF membrane(Millipore).Freemembrane binding sites were quenched using 2%BSAin PBS with 0.1%(v/v)Tween 20(PBST).Six mono-clonal antibodies,2H3,4C6,5F11,7F1,7F8 and 7F11and secondary HRP-conjuga
38、ted antimouse immuno-globulins were diluted in PBST(0.1%Tween20,v/v)and incubated for 1 h with constant shaking at roomtemperature.Membranes were thoroughly washedbetween steps using PBST.Establishment of the method of IHC assay forthe detection of scrapieReagent Instrument for IHC and mounting medi
39、umwere purchased from DAKO company.Protease Kand protease K inhibitor were obtained from Sigmacompany.Scrapie-affected ovine brain sections wereprovided by TSE laboratory of United States Depart-ment of Agriculture.Preparation of standard positive and negativesamples Scrapie brain was from TSE labor
40、atory ofUnited States Department of Agriculture,negative ovinebrain was collected from normal and healthy ovine inChina.Protocols for detection of scrapie by IHC The pro-tocols are according to the national standard GB/Establishment of the Method of Immunohistochemistry Assay for the Detection of Sc
41、rapie in Chinese Short-Tailed1519 2008,CAAS.All rights reserved.Published by Elsevier Ltd.T19180-2003.Standard of positive and negative results Positiveresult:Red immunostaining were observed in the dor-sal nucleus of vagus nerve,nucleus of solitary tract,and spinal nucleus of trigeminal nerve.Negat
42、ive resultis on the contrary of the positive result mentioned above,nonspecific background staining and redimmunostaining in the places mentioned above.RESULTSExpression of PrP27-30 gene of Chinese Short-tailed Han sheep in E.coliThe recombinant plasmid PrP-pET-32a was transformedinto E.coli BL21(DE
43、3)and the recombinant strain BL21(DE3)was obtained.SDS-PAGE and Western blottingof the bacterial protein showed that a specific proteinband with a molecular weight of 35 kD emerged in thebacterial protein(Figs.1 and 2).SDS-PAGE gel-scan-ning showed that the expressed PrP27-30 fusion pro-tein increas
44、ed with the time and accounted for 46.9%of the total bacterial protein in the overnight inducedgroup.All of fusion protein is in inclusion.Generation and identification of anti-ChineseShort-tailed Han sheep PrP27-30 monoclonalantibodiesProduction of Chinese Short-tailed Han sheepmonoclonal antibodie
45、s Six hybridoma lines secretingthe mAbs against Chinese Short-tailed Han sheep PrP27-30 were obtained,which were designated as 2H3,4C6,5F11,7F1,7F8,and 7F11,respectively.Identification of mAbs by Western blotting West-ern blotting analysis showed that all of six mAbs re-acted with recombinant PrP27-
46、30,and a positive bandappeared at molecular weight of 35 kD(Fig.3)and couldrecognize PrPC in brain homogenate of Chinese Short-tailed Han sheep(Fig.4).Establishment of immunohistochemical methodfor the detection of scrapiePositive immunostaining by mAbs 2H3,4C6,5F11,and7F11 immunohistochemical analy
47、sis were detected inthe scrapie-affected ovine brain on the obex symmetri-cally(Fig.5-A,B,C,D).There was no significant dif-ference between positive control(Fig.5-E)and the testgroup of monoclonal antibodies as shown in Fig.5.Incontrast,there was no immunostaining in the negativecontrol(Fig.5-F).The
48、se pictures showed that 2H3,4C6,5F11,and 7F11 could recognize PrPSc in scrapie-affected ovine tissue after the digestion of PK.DISCUSSIONMonoclonal antibodies are useful tools for diagnosisand study of prion diseases.Silke Harmeyer(GroschupFig.1 The cellular localization of Chinese Short-tailed Han
49、sheepPrP27-30 in E.coli BL21(DE3).1,pET-32a negative control;2,osmotic shock suspension;3,cell lysis suspension;4 and 5,wholecell;6 and 7,insulion;M,protein molecular weight marker;8 and9,purification of fusion proteins.Fig.2 Identification of fusion Chinese Short-tailed Han sheepPrP27-30 protein by
50、 Western blotting.1 and 2,insulion detectedby Western blotting;3 and 4,purification of fusion protein detectedby Western blotting;5 and 6,insulion;7 and 8,purification offusion protein;M,protein molecular weight marker.1234567M8948 kD33 kD25 kD48 kD33 kD12M453M76848 kD33 kD25 kD1520ZHANG Yong-qiang
