Expression and diagnostic application of p12 protein of African swine fever virus by recombinant baculovirus

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(1)³¤¥Èó. * .* a. (2005) 45 1 Korean J Vet Res(2005) 45(1) : 63~70. Ò`CBDVMPWJSVTö~j*ÒæR.¢:ÊQ Wî~B*êÏ. R;+ ÁR;ëÁBÏ. ]ã>~¦ö ²Òß: 2005j 2ú 21¢). (. Expression and diagnostic application of p12 protein of African swine fever virus by recombinant baculovirus Kang-Seuk Choi*, Cheong-up Choi, Yong-Joo Kim National Veterinary Research and Quarantine Service, Ministry of Agriculture and Quarantine Service, Anyang 430-826, Korea (Accepted= February 21, 2005) Abstract : African swine fever (ASF) is an infectious disease of domestic and wild pigs for which there is no vaccine in the world. A proper surveillance of viral activity and a timely response to ASF outbreaks depend upon the rapid diagnosis of ASF viral infection. Internationally prescribed enzyme-linked immunosorbent assay (ELISA) is a fast, sensitive test routinely used in the diagnosis of the ASF. However, inactivated whole ASF virus antigen used in this test is a tedious to prepare and has a risk of outside exposure of infectious virus by laboratory accident during the preparation. An ASF virus noninfectious recombinant antigen is a safe and easily produced alternative antigen for use in diagnostic assay. We have cloned the ORF O61R gene of the ASF virus to generate a recombinant baculovirus producing the p12 protein in insect cells under control of the polyhedrin promoter as non-fusion protein. When used in an indirect ELISA, the p12 antigen showed reactivity with all known ASF positive pig sera but not with negative pig sera. Our results indicated that the p12 protein would be one of alternative antigens for diagnosis of the ASF. Key words : African swine fever, p12, recombinant baculovirus, ELISA, diagnosis. * V. "æöB 100%~ ~ÒNj ¾æâ > ® . î ÷f · O»ö ~~ * & Ú î> ®æò &¦ª "æ* 7/ $º êV(Ornithodoros spp)~. Bö ~~ Úê [20]. *Ò î÷ö 6"B "æöB OÚË NB 7zÚ& W>æ pbæ , ¢ .O W BB>Ú ®æ jî ;. [12, 14, 15, 16]. æ, î÷ Bö ~ ãB' b¢ ²z~º F~ O»f 6"æ~ 6>W &»~ ÿB z®Ú 6"»~ ³ ¦Âö ~ ê Ú¾ªj ~º © . ASFº "ö j*Ò Ò~¢Òï Îæ" îÒj, Ê¾ æ7 næöB &¦ª B~& [20, 23]. ê' G. j*Ò"æR.¢(African swine fever: ASF)º "æ öB ¸f ~N ~ÒNj º :ÊW *" ÷ . *"÷~ ÷öÚº Asfarviridae~ Asfivirus ö ³~º j*Ò"æR.¢:Ê(African swine fever virus, ASFV) [13]. :Êº £ 170 kb~ DNA genome, .V mRNA Öö jº Î², Ò 50B~ \Wî \W>Ú ® [10, 24]. ASF 6" "æöB~ ªç Ãçf /W;öB òW ;ræ ·~ "æR.¢(classical swine fever: CSF) f FÒ~ [16, 20, 23]. ß®, /W; ASF~ ãÖ 6". *Corresponding author: Kang-Seuk Choi National Veterinary Research and Quarantine Service, Ministry of Agriculture and Quarantine Service, Anyang 430-826, Korea [Tel: +82-31-467-1860, Fax: +82-31-449-5882, E-mail: [email protected]]. 63.

(2) R;+ÁR;ëÁBÏ". 64. öB ASFV ßö ¦Âf ªçÃç B*>º 6" »j ïÂ~º ®Ú FÏ > ASFV ßÚ ¦ Â .Ó¦Òº BæöB~ :ÊFÿ> [9, 17, 23]~ ïÂ¾ jB æöB~ î÷ .Vö B j>' . *Ò î÷~ êö &Ë ÎN' Ú¦Â O»b B &¦ª enzyme-linked immunosorbent assay(ELISA) & ÒÏ> ®b, ASF~ ãÖ ®z:Êöj Ï ELISA& B'b ÒÏ> ® [7, 19]. " ®z:Ê ö ö p73 [19], p72 [22], p54 [4, 18] 5 p30 [8, 18] Wî ê öbB BB> Ú ÒÏ> ® . ¾, jçræ p12 Wî ASFV Ú¦Âj * ELISAö 'ÏB Òf& B :& ì . ASFV Wî p12º 61B~ jÖb W >Ú ®b ORF O61Rö z^z>Ú ®b, º ? "^

(3) ö ¦O~º attachment proteinb rJ^ ®. [1, 2, 6, 10, 11]. $ p12 Wîf ¶ 6"B "æö B ; öWj Fê~º ©b >î [3]. öBº Ò baculovirus¢ Ï~ ASFV p12 Wîj B*~, B*B Ò p12 Wî Ú¦ÂÏ êöbB~ FÏW ¦¢ ELISA ï &~& .. Òò 5 O». B*Ç8

(4) ~ ·9 ASFV ORF O61R F*¶& ã«B B*Ç8. pBacPAK/ASFV.O61Rj ·W~V * ÒÏB viral DNA º ®zB ASFV Brasil-78"( National Veterinary Service Laboratory~ Dr. A. HouseB)¦8 ºÂ~&. . 7Î²ê>w»(polymerase chain reaction, PCR) ö ~ ORF O61R DNA~ Ã j *~ PCR primers O61R-F(5'-CGGGATCCAATACGCAAAATGGCAC-3') 5 O61R-R(5'-GCTCTAGAGCGGACCATGTACTCTGAC3')& ÒÏ>î . primerº ORF O61R ®º EcoRI-XbaI Ö.Þ Ú ORF O61R F*¶ *Ú¢

(5) ~ Ã >ê :¶~ W>î (Fig. 1). $ ORF O61R DNA~ ÎN' j *~ forward 5 reverse primer 5' öö BÎ² BamH1" Xba1 . ¦* "VBj ã«~& . PCRö ~ ORF O61R DNA Ã Ö>f BamH1 5 Xba1 .~ pUC19 plasmid(Gibco, USA)ö ã«~ cloning~& (pUC19/ ASFV.O61R). -² cloningB ORF O61R DNAº ÿ ¢ BÎ² .~ pUC19/ASFV.O61R¦8 º Â~ pBacPAK8(Clontech, USA)ö ã«~ F*¶Ò B*Ç8(pBacPAK/ASFV.O61R)¢ ·W~& . B *Ç8Ú ORF O61R DNA~ R: ã«¦º PCR », BÎ².» 5 "VBªC~ O»ö ~~ {~& . Ò`

(6) ~ ·9 ASFV ORF O61Rj

(7) ~º Ò baculovirusº B*Ç8 pBacPAK/ASFV.O61Rf BacPAK6 linear DNA (Bsu36I digested)(Clontech, USA)¢ Spodoptera frugipera (Sf9) Ï^

(8) (Invitrogen, USA)ö ClontechÒöB. Fig. 1. EcoRI restriction map of ASFV BA71 genome. DNA fragment containing ORF O61R was amplified by PCR to generate recombinant baculovirus (BacPAK/ORF61R)..

(9) Ò baculovirusö ~ j*Ò æR.¢:Ê p12 9î~ ** ê' 'Ï. ²Ë~º O»ö V¢ co-transfection Î ê Ï^

(10) ¢ 27 CöB &NV·~ ·W~& . Ò baculovirusº co-transfection 72* ê Ï^

(11) V·ç [b¦8 jë~ 2¢»(plaque assay)b ~& . ASFV p12Wîj B*~º Ò baculovirus(BacPAK/ORF61R)º ;7Ú»b ASFV ê "æ.Ó"~ >w¦¢ {bB FB ~& . Ò` ö~ *` Ò ASFV p12 Wî(rASFVp12) öf BacPAK/ ORF61R¢ 6"Î Sf9 Ï^

(12) ¦8 ºÂ~ B ~& . ¯, Ò V· Sf9 ^

(13) ö 5 M.O.I (multiplicity of infectivity)~ Ò baculovirus(BacPAK/ ORF61R)¢ 7« ê 27 C &NV·VöB V·~ & . 6" 5¢ V· ^

(14) j 500 × g 20ª* ö ªÒ~ cell pellet òj >{~& . ê cell pelletö 1/10 volume~ lysis buffer(0.01 M phosphate buffered saline containing 0.6% Triton X-100, 0.05% Tween 20 and protease inhibitor cocktail)¢ Î&~ âr>öB * ® .r ¾Ò(sonication) r 10ª* O~bB 6"^

(15) ¦8 rASFVp12 Wî ºÂ>² ~& . ê 500 × g 20ª* öªÒ~ cell debris¢ B r, rASFVp12 Wî FB ç[f ²ª~ þö ÒÏ~V *ræ -70 Cö &~& . ;ç Sf9 Ï^

(16) ¢ çVf ÿ¢ O»b ºÂ~ ö rW & ÒÏ~& . .Ó ASFV ê "æ.Óf National Veterinary Service Laboratory(NVSL)~ Dr. A. House¦8 BA ~ . .Óf öB ·W &.Ób ÒÏ> î . ASFV6" ¢"æF¾ .Ó 206f *·Ê Agence Française de Sécurité Sanitaire des Aliments (AFFSA) ²~ Dr. M. Remond¦8 BA~ . ASFV p12ö & ·W.Óf öB ÖB rASFVp12 Wîj Vîcö 7«bB ¶~. þ öB B~& . ¯, rASFVp12 Wî(v £ 1 mg)j Vîc 2vö 3" *Ï 2² b~ 7«~& . ê « 2"ö j.~ .Ój ªÒ r pool ~& . .Óö Ï ^

(17) lysates(1×10 /.Ó ml)¢ Î &~ NöB 1* ¾ÒbB .ÓÚ Ò~º jß ^

(18) Wî Ú¢ B~& . .Ó ö, j*Ò"æR.¢ jB æ ~ ê»Ë öB j "æ .Ó 2106j ASFV rW "æ ¢ .Ób ÒÏ~& . o. o. o. 8. 65.

(19)

(20). öB B*B rASFVp12Wî~ B*{j *~ ASFV ê ·W.Ó" Vîc p12 . Ój ÒÏ~ Western blotj ~& . b&, NuPAGE Novex Bis-Tris GelsöB rASFVp12 W îj Xcell SureLockTM Mini-cell(Invitrogen, USA)¢ Ò Ï~ BÒöB ²Ë~º O»ö ~~ *V'ÿ j ~& . ê, Xcell IITM Blot Module(Invitrogen, USA)j ÒÏ~ Wî ª³ j gel¦8 nitrocellulose ïb *8 . ê Wîª³ *B nitrocellulose ïj blocking buffer(5% skimmed milk& FB 0.01 M phosphate buffered saline(PBS)b 30ª* :ßÎ ê 1:100 C .Ó(ASFV p12 5 ASFV ö & ·W.Ó)" NöB 90ª* >w8 . ê Wî ª³" Ö Úº blocking buffer 1:1,000 C anti-species IgG(H+L) conjugated with alkaline phosphatase(Kirkegaard-Perry, USA) NöB 60ª >w* ¦Â r BCIP/NBT VîÏ (Kirkegaard-Perry, USA)¢ ÒÏ~ ö-Ú>wÖ> j Bï8 . *% ;%Ú» Ò baculovirus 6"Î Sf9 Ï ^

(21) öB~ p12Wî~ B* ¦¢ {~V *~ *7 ;7 Ú»j ~& . ÖF, Ò baculovirus¢ 6" Î ê 3¢ö 6"^

(22) ¢ >{~& . 6"^

(23) º 0.01 M PBS, pH7.4 *® ^¿ ê slide glassö êö~ 8 . ê êö ^

(24) º cold acetoneb -20 CöB 10ª* ;~& . rW&¢ *~ ; ç Sf9 Ï^

(25) ¢ ÿ¢ O»b ;8 . ASFV 6" Vero^

(26) slide glass~ ãÖ NVSL~ Dr. J. House¦8 BA~ . ;B Ò¢ö ASFV ê "æ.Ó(1:5,000V C)j Î&~ 37 CöB 1* ÿn >wÎ ê j>w Ö> j B~V * ~ 0.01 M PBS, pH 7.4 *® ^¿~& . . r FITC conjugated ant-swine IgG(H+L) Ï(Kirkegaard -Perry, USA; 1:500V C)j Î&~ 37 CöB 1* ÿn >wÚbB ö-Ú Ö Ö>j ¦Â~ê ~& . ¦Ò ^

(27) öB~ ;7"ï ¦º z. öB ;7*ã(Olympus, Japan)j Ï~ &V~ & . *% öB B* rASFVp12 Wî "æ .Ó ò¦8 ASF Ú¢ ¦Â > ®ºæ¢ Ò~V * ~ *7 ELISA¢ ~& . ÖF Maxisorp ELISA o. o. o.

(28) R;+ÁR;ëÁBÏ". 66. plate(NUNC, Denmark)ö 0.01 M PBS '; ³ê C rASFVp12 ö(C Wîï £ 10 µg/ml ö )j well 50 µl O Î&~ 37 CöB 1*ÿn O8 . ê, ^¿Ï buffer(0.002 M PBS containing 0.05% Tween 20) ELISA plate¢ 3² ^¿~ O >æ pf Ö> j B8 . ê blocking buffer (0.01 M PBS, 5% skimmed milk and 0.05% Tween 20) ';~² C ¦Ò.Ój rASFVp12ö O B plateö '' 2>b well 50 µl O Î&~ 37 C öB 1*ÿn >w8 . çVf ÿ¢ O»b ELISA plate¢ ^¿ ê peroxidase-conjugated anti-swine IgG(H+L) Ï(Kirkegaard-Perry, USA; 1:1,500V C) j well 50 µl O Î&~ 37 CöB 1*ÿn >w ÚbB ö-Ú ÖÖ>j ¦Â~² ~& . ê O-phenylenediamine(OPD) VîÏ(Sigma, USA)j well 50 µl O Î&~ NöB 10ª* >wÎ. r 1.25 M ÖÏb Bï>wj 7æ8 . ê ELISA plateº 492 nmöB 7ê¢ G;~& . ¦Ò Ö"º ' .Ó~ 7ê¢ T/PjN(¦Ò.Ó 7ê/· W&.Ó 7ê) *~~ êÖ~& . ò£ rASFVp12 ö wellöB ¦Ò.Ó~ T/P jN 0.3(r W.Ó~ ï T/PjN~ 2V)ç¢ ãÖ ASFV 6" Vero ^

(29) slide¢ Ï *7 ;7Ú»b ELISA jß>w ¦¢ Ò~& . o. o. o. Ö . Ò` 9î ~ ** ASFV p12 Wîj z^z~º genome F*¶ ¦* . º ASFV~ genome 7 EcoRI-XbaI Ö.Þ ¦* Úö Ò~, .Þ(913 bps) 7 ORF O61R DNA(183 bps) ¢

(30) ~º C 358 bp V~ DNA¢ Ã ~ê ~&. (Fig. 1). Ò baculovirus(BacPAK/ORF61R)º [ ;W B Sf9 Ï^

(31) ÚöB pUC19/ASFV.O61R Ç8f BacPAK6 linear DNA*~ F*¶ Òö ~~ W 'b ·W>î . BacPAK/ORF61Rö 6"B Ï^

(32) º 6" 3¢ Â] ^

(33) æWÎ"& &V>V · ~&b(Fig. 2A right), :Ê >¢ 6" 5¢ öº Ï^

(34) ~ 90% ç ^

(35) æWÎ"¢ ¾æÚî. . ¾ ;ç^

(36) öBº ^

(37) æWÎ"& &V>æ p ~ (Fig. 2A left). 6" ^

(38) ¢ ASFV ê "æ. Ó" >wÎ ê ;7"ï~&j r, 6"^

(39) º ; ;7>wj ¾æÚî (Fig. 2B right). > ;ç Ï ^

(40) öBº ;7>wj ¾æÚæ p~ (Fig. 2B left).. Ò` 9î ~ ö9 Ò baculovirus BacPAK/ORF61R 6" Ï^

(41) ¦8 Ò B*Wî(rASFVp12)¢ ºÂ r öWj Western blot»" *7 ELISA ªC~& . B*Wîj æW* *V'ÿ»b ª³Î ê Western blot»b p12 Vîc .Ó" >w8 j r, £ 12 kDa" 17 kDa~ Wî ª³ &V> î . &¦ª~ B* Wîf £ 12 kDa~ V&. (Fig. 3A lane 2). ?f O»b ASFV ê "æ. Ó" >w8j r ASFV Úº £ 12 kDa~ Wî ª³"ò £ >wj ¾æÚî (Fig. 3A lane 3). ;ç . . Fig. 2. Expression of ORF O61R gene of ASFV by recombinant baculovirus BacPAK/ORF61R. (A) BacPAk/ASFV.ORF61R infected cells (right) showing cytopathic effects. (B) BacPAk/ASFV.ORF61R infected cells (right) showing strong reactivity with hyperimmune ASFV pig serum in indirect immunofluorescence assay..

(42) Ò` baculovirusö ~ j*Ò æR.¢:Ê p12 9î~ ** ê Ï. 67. Fig. 3. Antigenicity of rASFVp12 produced by recombiant baculovirus BacPAK/ORF61R. (A) Western blot analysis of rASFVp12 protein using anti-p12 GP serum (lane 2) and hyperimmune ASFV pig serum (lane 3). Mock infected insect cells showed no reactivity with hyperimmune ASFV pig serum (lane 1). (B) ELISA reactivity of rASFVp12 protein using anti-p12 guinea pig serum and hyperimmune ASFV pig serum.. Ï^

(43) F¾ Wîj ÿ¢ O»b ºÂ~ >w 8j r ASFV Úf >w~º Wî ª³f &V >æ p~ . >, *7 ELISA O»b B* Wî (rASFVp12)~ >wWj Ò~&j r, ASFV ê "æ.Ó" p12 Vîc .Óf Îv 1:10,000V ç(P/N ratio 2.0 ç V&)~ ¸f &¢ ¾æÚî. (Fig. 3B). Ò` 9îj Ï ö ~ Ú¦Â öB B* Ò p12Wî ASFV Ú ¢ ¦Â~V * êöbB FÏ æ ¦¢ * 7 ELISA Ò~& . Checkerboard titration»j. Ö" Ò p12ö" ¦Ò .Ó~ '; \C V>º '' 1:150 5 1:100î (Fig. 4). 6 ';³ê ~ rASFVp12 ö 5 ¦Ò.Ój ÒÏ~ *7 ELISA¢ {ã~, rASFVp12 ö ASFV ßÚ¢ ¦Â > ®º æ ¦¢ Ò~&. (Fig. 5). FÎW ï&¢ *~ ASFV Ú·W "æ .Ó 206" ASFV ÚrW "æ.Ó 2106j ÒÏ~ & . ASFV Ú·W "æ.Ó 2067 156(75%)f 0.90 ç~ ¸f T/P jNj ¾æÚî . &Ë Ôf > wWj ·W.Ó~ T/P jNf 0.56î . > rW .Ó~ 88.5%(186/2106)º 0.2~~ Ôf T/P j .

(44) . . Fig. 4. Checkerboard titration using antigen (rASFVp12) and control positive and negative sera. T/P ratio was calculated by dividing ASFV positive serum into ASFV negative serum.. Nj ¾æÚîb, 76~ rW.Óf 0.3 ç~ ç& 'b ¸f T/P jN(& 0.46 T/PjNj ª)j ¾ æÚî . ¸f T/PjN(0.3ç)j rW "æ.Ó. ~ ãÖ ASFV6" Vero ^

(45) ¢ Ï *7 ;7 Ú»b vN¦Ò Ö" Îv rW>wj ¾æÚî. (data not shown)..

(46) R;+ÁR;ëÁBÏ". 68. Fig. 5. Distribution of T/P ratio of test serum samples in indirect ELISA. The arrow indicates T/P criteria between ASFV positive and ASF negative sera in the ELISA.. V. ASFV «¶º receptor B endocytosisö ~ 6>W ^

(47) Ú «~ [2], ";öB p12Wîf ^

(48) receptor¢ ~º VËj ~º ©b rJ^ ®. [11]. ASFV p12 Wîf ASFV~ BV· ";öB ?"^

(49) attachmentf &NB j ~º © ö 6"B "æöB &Ë Ò ?"Ú Ú¢ Fê~º Wî {>î [3, 21]. $ p12Wîf : Ê" *~ æ& 'f Wî >î [5]. p12 Wî~ öW" F*' n;Wf ¢ êöbB 'Ï > ®º &ËWj B "º © . Ö" ASFV Ú·W .Ó" ASFV Úr W .Ó*ö Ò p12 Wî(rASFVp12)f~ Â] ELISA >w ;ê~ N& &V>î . ASFV6" " æ.Ó~ ãÖ &¦ª p12Wî" ; >w(0.9 ç ~ T/PjN)j ¾æÚî . ¢¦ 6" "æ.Óf ç& 'b Ôf T/PjN(~ 0.56)j &æò, T/P jNj "æ rW.Ó(T/PjN 0.46) º ; >wj ¾æÚî . ç&'b ¸f T/PjNj " æ rW .Ó f *7 ;7Ú»öB ASFVf~ ö Ú>w &V>æ pf ©b j jß ELISA >wö ~ ç&'b ¸f ELISA Bï>wj ¾æ Þ ©b ÒòB . Ö'b öB Ö Ò p12Wîf ASFV ÚêÏ ELISA~ ê öbB FÏ ©b 6B . Ö"º Ò rASFVp12 ö ELISA êöbB 'Ï &Ë. ~ º ©j ¾rb B~& º 6öB ê'b 7º ~~¢ æî ® .. ¶ ~ ö ~~ baculovirus¢ Ï~ B* ãÖ Ò p12Wî B*ï Ö>~, ASFV~ native p12f FÒ >' VËj ¾æÚº ©b >î [6, 10, 11]. ©f Ò baculovirus ö ~ B*B p12Wî êöbB 'j B~º © . öB Ò baculovirusö ~ ÖB Ò rASFVp12 Wîf &¦ª £ 12 kDaö ~º Wî ; B*>îb, ¢¦ 17 kDa~ Wî ;ê B*>î . jÖ 61B W>Ú ®º p12Wîf .ç ª¶ï 6.5 kDa [2]ª j J " r öB B*B Ò p12Wî f monomer ; º dimer(12 kDa)¾ trimer(17 kDa) ; B*Nj r > ®î . Ö"º Angulo [6] Ò p12Wî~ immunoprecipitaion Ö "f ¢~~&, &ËB* p12 WîöBê &V> î [1]. æ, p12 Wî~ multimerization f Angulo [6] æ' :f ? glycosylation~ ' Ë º disulfide Öö V ©b 6B . öB Ö Ò rASFVp12 Wîf ¦Ò O»ö V¢ öW~ N& &V>î . ¯, *7 ELISAöB~ >w Ö"f Ò, Western blotöBº ASFV ê "æ.Ó" Ö £ >wj ¾æÚî. . ²ê Western blotöB~ ö-Ú>wf £ 12 kDa~ Wî ª³öB &V>îæò 17 kDa~ Wî ª³öBº &V>æ p~ . Ö"f FÒ Ö"& Pastor [19] 5 Carrascosa [10]ö ~ Bê B : ® . Immunoprecipitation assay(RIPA) öB~ p12Wî~ ; öWj &æò Western blotb ~&j r öW~ &¦ªj ç ~& . ¯ ö Wî~ æW¾Ò";ì öWj ¦Ò ELISA¾ RIPAf Ò, Western blot~ ãÖ 2-ME(2mercaptoethanol)f ?f zB rASFVp12 öj ¾ Ò(æW)Î ê Wîj ª³~ öWj Ò~&. º 6j J " r, Wî æW ¾Òº p12 W îö Ò~º öW ; «Ú' ö~ ç j .¾~º ©b Îê . ²ê p12 B *Wî Vîc.Óf ASFV6""æ.Ó" Ò Western blotöBê ; ö Ú>wj ¾æÚî. . Ö"º linear epitope ASFV virionöB º 'b ¦ Â>Ú ®æ pj >wj. Ö £~² ¾æÚº >, Ò p12WîöB º linear epitopes ¦ ÂB ; Ò~ >wj Fê~V r^ ©b 6B . ¯, ASFV6" "æöB~ p12ö & >wf linear.

(50) Ò baculovirusö ~ j*Ò æR.¢:Ê p12 9î~ ** ê' 'Ï epitopeö ~ >w º conformational epitopeö ~ &¦ª Úê º ©j ~ ~Æ . æ, conformational epitope~ ö¢ 2ZÒ > ®º Wî öæW ¾Ò";j ~æ pº êO »j J , Ò p12Wî~ ê' 'Ïf. Ö FÏ ©b 6B . 6, öB Ö Ò p12Wîf ASFV Ú¢ ¦Â~º êöbB r" ?f 6j B . Ñ, ®z:Ê ö" Ò Ò p12 Wî öf öÖ";öB *"W ®º ASFV ¢ /~æ pbæ ¢> þ öBê £² ê öj Ö > ® . ~, ®z :Êöj B ~V *~ BEI(binary ethyleneimmine)f ?f Bz W ®zB~ / j>'¾, Ò p12Wî ö~ B";öBº BzW zB¢ ÒÏ~æ pbæ /¶ö & Ú*ê& Ö Ô . q , baculovirus¢ Ï~ Ö~º p12 Wî öf ³ê £² Ö > ®V r^ö öÖj& & Z~ [10]. Ö'b, öB B*B Ò p12Wî öf êöbB öW Ö>~, n*~, £² Ö > ®º Ë6j &æ ® .. ò, öB 'Ï p12Wî öj Ï ELISA j ASF ê¾ .V Ï'b ÒÏ~V *Bº . · ¢.Ó ò j {~ º&' FÎ W ï&& jº~ .. 2.. 3.. 4.. 5.. 6.. 7.. Ö V. ~ ORF O61R F*¶¢ ã« Ò ¢ ·W~& . ·WB Ò baculovirusö ~ Ï^

(51) öB B*B Ò Wîf &¦ª 12 kDa~ dimer;&b, ¢¦º 17 kDa~ trimer ; & . (2) Ò baculovirusö ~ B*B Ò p12W îj *7 ELISA~ êöb ÒÏ Ö" ASFV Ú·W "æ.Óf T/PjN 0.5çj ¾æÚÚ, 0.5 ò~ T/P jNj ¾æÞ ASFV ÚrW "æ.Ó" ~ 6ê &Ë~& . æ, öB Ö Ò p12Wîf ASFV ÚêÏ ELISA~ ê öbB FÏ ©b 6B . (1) ASFV baculovirus. ^^ò 1. Alcami A, Angulo A, Lopez-Otin C, Munoz M, Freije JM, Carrascosa AL, Vinuela E. Amino acid sequence and structural properties of protein p12, an. 8.. 9.. 10.. 11.. 12. 13.. 69. African swine fever virus attachment protein. J Virol 1992, 66, 3860-3868. Alcami A, Carrascosa AL, Vinuela E. The entry of African swine fever virus into Vero cells. Virology 1989, 171, 68-75. Alcaraz C, De Diego M, Pastor MJ, Escribano JM. Comparisn of a radioimmunoprecipitation assay to immunoblotting and ELISA for detection of antibody to African swine fever virus. J Vet Diagn Invest 1990, 2, 191-196. Alcaraz C, Rodriguez F, Oviedo JM, Eiras A, De Diego M, Alonso C, Escribano JM. Highly specific confirmatory western blot test for African swine fever virus antibody detection using the recombinant virus protein p54. J Virol Methods 1995, 52, 111-119. Angulo A, Vinuela E, Alcami A. Comparison of the sequence of the gene encoding African swine fever virus attachment protein p12 from field virus isolates and viruses passaged in tissue culture. J Virol 1992, 66, 3869-3872. Angulo A, Vinuela E, Alcami A. Inhibition of African swine fever virus binding and infectivity by purified recombinant virus attachment protein p12. J Virol 1993, 67, 5463-5471. Arias M, Sanchez Vizcaino JM. Manual de diagnostico serologico de la peste porcina africana (Manual of diagnostic serology for African Swine fever.). pp.1-44, Ministry of Agriculture, CISA-INIA, Valdeolmos-28130, Madrid, 1992. Barderas MG, Wigdorovitz A, Merelo F, Beitia F, Alonso C, Borca MV, Escribano JM. Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae. J Virol Methods 2000, 89, 129-136. Bech-Nielsen S, Fernandez J, Martinez-Pereda F, Espinosa J, Perez Bonilla Q, Sanchez-Vizcaino JM. A case study of an outbreak of African swine fever in Spain. Br Vet J 1995, 151, 203-214. Carrascosa AL, del Val M, Santarén JF, Viñuela E. Purification and properties of African swine fever virus. J Virol 1985, 54, 337-344. Carrascosa AL, Sastre I, Viñuela E. African swine fever virus attachment protein. J Virol 1991, 65, 22832289. Coggins L. African swine fever virus. Pathogenesis. Prog Med Virol 1974, 18, 48-63. Dixon LK, Costa JV, Escribano JM, Rock DL,.

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