182호-02.fm

Transcription

1 J. Fd Hyg. Safety 18(2), (2003) Vibri parahaemlyticus w Immunglbulin ylk (IgY) p *Á½x *Á **Á z***á ****Á y* * w tœw ** w w *** û y **** w t w t w Prductin and Specificity f Imunglbulin ylk (IgY) n Vibri parahaemlyticus Wn-B Shim*, Hye-Jung Kim*, Sen Ja Park**, Dng Hn Kang***, Jin-Sn Kang****, and Duck-Hwa Chung* *%FQBSUNFOUPG'PPE4DJFODFBOE5FDIOPMPHZ(ZFPOHTBOH/BUJPOBM6OJWFSTJUZ(ZFPOHOBN,PSFB ***OTUJUVUFPGBHSJDVMUVSFMJGFTDJFODFT(ZFPOHTBOH/BUJPOBM6OJWFSTJUZ+JOKV,PSFB ***(ZFPOHTBOHOBNEP1SPWJODJBM(PWFSONFOU1VCMJD)FBMUI&OWJSPONFOUBM3FTFBSDI*OTUJUVUF4BSJNEPOH$IBOHXPO,PSFB ****%FQBSUNFOUPG'PPEBOE/VUSJUJPO+JOKV*OUFSOBUJPOBM6OJWFSTJUZ+JOKV(ZFPOHOBN,PSFB ABSTRACT This study was cnducted t prduce the egg ylk immunglbulin (IgY) n Vibri parahaemlyticus frm immunized hen with lipplysaccharide (LPS). Vibri parahaemlyticus is cnsidered as a ptentially pathgenic bacteria, the causative agents f the gastrenteritis. Accrding as the LPS antigens were injected int laying hens in rder t prduce antibdies against Vibri parahaemlyticus in egg ylk. After chickens were immunized fur times in 2 weeks interval and three times bster in 2 weeks interval, the prfile f antibdy prductin was examined by ELISA. The prductin f antibdy in egg ylk was started in 1 week after the first immunizatin, reached peak in 7 weeks and maintained until 13 weeks later. The antibdy titre in serum shwed similar tendency as IgY. N significant difference in antibdy titre when the titre cmpared t water diluted IgY and cmmercial IgY kit. Purified IgY reacted with nly Vibri parahaemlyticus, but ther Vibri species and fd-brne pathgenic bacteria. In cnclusin, we shwed that it is pssible t btain a high antibdy titre in chicken with quite lw amunts f LPS antigen. These results suggested that egg ylk antibdies culd be a gd surce fr prductin f specific antibdies t pathgenic bacteria inducing epidemic gastrenteritis. Key wrds: Vibri parahaemlyticus, LPS (lipplysaccharide), IgY, ELISA Vibri parahaemlyticus 1950, j ³. ³ w w ³ 2~5%. w ƒ ù w, w z w w, p (crab), (yster), (shrimp) ƒ (lbster) t w. w ú, w (epidemic gastrenteritis) ³. 1) ³ ù w q w š š, w ³ w šƒ w Authr t whm crrespndence shuld be addressed. t ƒw š. 2), w w ùyw w mw š. 3) ùy w z w w ƒ ùy w,, x x w, q,, s IgG wù yw ƒ immunglbulin ylk(igy) š. 4) w k l z w IgY 1500 mg p w 2~10%, 200 mg IgG p w 5% w ù z» w q. 5) IgG x ù w, ùy 61

2 62 8PO#P4IJNFUBM w IgG w IgY w y ù w, w IgY w š. 6) ƒ š wx y ƒ ƒ w IgY, w p j p w (research), (diagnstics), w (antibitics-alternative therapy) s š. IgY γ-livetin š 64 KDa heavy chain 28 KDa light chain š IgG ƒ¾ x w IgG» w š š. ùy α-livetin, β- livetin. p w LPS (uter membrane prtein) w Vibri y w w w š» 7) ƒ xk w w ³ g w e w ƒ šwš, w p j p w e ƒ j ñš. w z w» w V. parahaemlyticus LPSd w w w k k z w ùy wš w ƒ y p w šwš w. LPS w w w ³ œw l Vibri parahaemlyticus KCTC 2729, 3% NaCl ƒ tryptic sy brth(tsb), 37 C 24 k w. V. parahaemlyticus LPSd Westphal Jann 8) phenlht water extractin methd w k 2,500 rpm (30 min, 4 C)z w 0.85% 10 ml. 2,500 rpm w 75 C ht water 10 ml 75 C 5 e g. 75 C ht phenl 10 ml 75 C 15 e g. w z water d z w 3 3 n k plyethylene glycl(peg) w z g w w w. w LPS w (10 mg/ml) Larssn et al.(1998) 9) xw Freund's cmplete adjuvant(fca) 1:1 y(emulsified) k z 1 ml 19 k(hylain Variet) x w w w š, ù x w, n ƒƒ 0.5 ml 1 w. z w 2, 3 Freund's incmplete adjuvant (FIA) w 1:1 y(emulsified) g w w w š, 4 w w. ƒ (bster) ³ z 2 6 w. w 1 z d w. w» (12 ) w š, Akita Nakai(1992) water-dilutin w 10) anti-lps IgY w., ùy ù l w wš 3mM HCl 10 w z, 10% acetic acid w ph 5 w. Cld chamber 6 e z 10,000 g 15 w ammnium sulfate 60% ƒw 15 w 10,000 g 15 w. d š e 60% ammnium sulfate 50 ml 10,000 g 15 w. d wš phsphate-buffered saline(pbs) 10 ml e xkw w. w x ww IgY sƒ w EGGstract TM IgY purificatin system kit(prmega, USA) IgY w, water-dilutin w IgY xw. ELISA w x w ùyw ƒ d V. parahaemlyticus w wx IgY ƒ w» w Larssn et al.(1998) k 9) k ú wx w, y(1998) 11) xw indirect cmpetitive ELISA w ƒ d w., V. parahaemlyticus frmalin killed cell(fkc) carbnate buffer 10 8 cell/100 µl w catingw 4C w e gš, PBST 3z z PBS 100 ml 0.5% bvine serum albumin(bsa) 200 µl 4C w blcking w. Phsphate Buffer Saline Tween

3 1SPEVDUJPOBOE4QFDJGJDJUZPG*NVOPHMPCVMJOZPML *H:PO7JCSJPQBSBIBFNPMZUJDVT 63 20(PBST) 4z wš ƒ well ƒƒ ƒ 1,000 wx IgY 100 µl 37æ 1 k z PBST 5z w. Phsphate Buffer Saline(PBS) 10,000 perxidasecnjugated affinity pure rabbit anti-chicken IgY 100 µl 37 C 1 k z» [ABTS; 2,2- azin-bis(3-ethylbenz-thiazlin)sulfnic acid] 100 µl ƒ w 30 g. k z ELISA Reader 405 nm Ÿ d w. d IgY V. parahaemlyticus Vibri V. parahaemlyticus ³ (Listeria, Salmnella, Staphylcccus, E-cli)» w ELISA x w. FKC carbnate buffer 10 8 cell/100 µl catingw micrtitre plate 4 C, vernight g. k micrtitre plate PBST 3z z Vibri ³ 200 w 37 C 1 k z w. 1:10,000 perxidase-cnjugated affinity pure rabbit antichicken IgY 37 C 1 k z» ABTS ƒw z 30 w ƒ w ELISA Reader 405 nm Ÿ d w. Electrphresis Fryer et al.(1986) 12) w, SDS-PAGE Laemmli 13) 5% stacking gel 12% separating gel(sdium ddecyl sulfateplyacrylamide gel) w, Cmassie brilliant blue R-250 w band y w. š ELISA w x w ƒ d V. parahaemlyticus LPS d w w k 1 z 1, 7 (1 ), 21 (3 ), 35 (5 ), 49 (7 ), 63 (9 ), 77 (11 ), 91 (13 ) x, x w 10,000 k wx w ƒ indirect cmpetitive ELISA d w. Fig. 1 1 l V. parahaemlyticus w w ƒ» w 3 z l ƒ ƒ w ƒ, 5 w ƒ š wš z w ù w. 2 Fig. 1. Antisera titratin f LPS immunized hen (Antisera was diluted as 10,000 ). z d w w s³ Ÿ 2.023» w j w 1.721(S.D ) x w š. (1999) 14) Streptcccus mutans-specific IgY 2 5 z w specific IgY z ƒ 30 { šw., w stress, ƒ w q. x 19, w 2 4z ³ 56 (8 ), 70 (10 ), 84 (12 ) 3z ƒ ƒƒ w ƒ 3 ƒ û ùkû. p ƒƒ ùkù 1» 2 w x w LPS ƒ w w w y w. w k ùy p w swwš w ù w w w ww w wš ù z w p. z w š š Akita and Nakai(1993) 15) w anti-lps IgY w SDS-PAGE y w. Fig KDa heavy chain 28 KDa light chain IgY y w š IgY γ-livertin Shimazi(1988) 16) ew. IgY w e w z 10% ƒ j z. w χ-carrageenan ƒ w ù z. 17)

4 64 8PO#P4IJNFUBM Fig. 3. Anti-LPS IgY titratin f LPS immunized hen (IgY was diluted as 1,000 ). Fig. 2. SDS-PAGE patterns f purified IgY frm egg ylk. Lane 1: Standard prtein (mlecular masses are in kildaltns), Lane 2: Nrmal IgY befre immunizatin, Lane 3: IgY after LPS immunizatin. H: heavy chain, L: light chain). w Stalberg et al.(2001) 18) chicken antibdy ùy ü w ƒ ƒ j» tritn X-100 phsphate w detergentƒ t w d š IgY phsphateƒ t w d IgY z 97% w 25% wƒ š š w. ù, water-dilutin IgY š z š 15) w IgY w zw š q. ELISA w IgY ƒ d x w y z k l ùy l IgY 1,000 k anti-lps IgY w ƒ ELISA d w. Fig. 3 ùy l IgY k x ƒ w ƒ š, ƒ x û ùk û. ù IgY w x w ƒ z 1 l» w ù p k w w 7 IgY l ƒ d w. Fig. 3 7 anti-lps IgY ELISA ùkù Ÿ ƒ d, z 13 ¾ IgY w 7 IgY û ùkû. ù z w k w, w p w 2 l 6 ¾ IgY w ƒ wx w w w w k k p f w IgY q. 14) 7 l anti-lps IgY ƒ» x w ƒ IgYƒ 7 ELISA w 1.863(S.D ) j ùküš. LPS w j ELISA ƒ ùkù IgY w sww ƒ IgY matrix effect ù kû» ƒ. Lee (2000) 19) Yersinia ruckeri w (rainbw trut) IgY z w 1 z 30~40 š w, whle cell w w w ƒ w, purified LPS k whle cell w w w ùkü š w. w (2000) 20) ùy w 1 z 2 l ùkù» w 6~8 š e wš ƒ 12 ¾ š šw. ù purified LPS w w 1 z 1 l w ƒ» w, 7 še w š, w ƒ 13 (91 ) IgY V. parahaemlyticus w w. Akita Nakai(1992) 10) IgY w x ph IgY w w ph IgY, ph û IgY ƒ k w j z ƒ, salt precipitatin 60% w p x š šw. IgY w ph % salt precipitatin zw š q, SDS-PAGE w p

5 1SPEVDUJPOBOE4QFDJGJDJUZPG*NVOPHMPCVMJOZPML *H:PO7JCSJPQBSBIBFNPMZUJDVT 65 Fig. 4. Cmparisn f EGGstract TM IgY purificatin system kit and water dilutin methd fr the extractin f IgY. y w (Fig. 2). wr, x IgY w w» w 9 w ƒ ƒ w ùy w q EGGstract TM IgY purificatin system kit(prmega, USA) w w IgY x water-dilutin w IgY ƒ d w Fig. 4., Fig. 4 ùkù ƒ û EGGstract TM IgY purificatin system kit w ƒƒ, 10 5 water-dilutin w IgY ƒƒ, w IgY ƒ j ƒ y water-dilutin w ƒ š. LPSd w w w V. parahaemlyticus Vibri V. parahaemlyticus ³ Listeria, Salmnella, Staphylcccus w p w. Fig. 5 anti-lps IgY ƒ ¼ w Fig. 5. Crss-reactivity f anti LPS-IgY n V. parahaemlyticus and ther Vibris (A), V. parahaemlyticus and nn- Vibris (B). V. parahaemlyticus 10,000 cell/ml l, Vibri ùkü. w Vibri Listeria, Staphylcccus, E-cli Salmnella xw anti-lps IgY ƒ V. parahaemlyticus k w w ƒƒ ùkûš, p 10 4 cell/ml l w. anti-lps IgY V. parahaemlyticus w z y y w ù V. parahaemlyticus g Vibri w w ƒ ƒw., Vibri parahaemlyticus w Y(IgY) w» w w. Vibri parahaemlyticus w ³, j ³., ùy Vibri parahaemlyticus w w w» w w LPS(lipplysaccharide) k w. 2 4 ¾ w, ƒ 2 3 ¾ w z ELISA w ƒ d w. ùy w 1 z 1 l ùkù» w, 7 š ƒ w š, w ƒ 13 z¾ ùkû. wr x w ƒ ùy w w. k IgY water-dilutin IgY kit w j ù

6 66 8PO#P4IJNFUBM kü, Vibri parahaemlyticus Vibri ³ p w. ùkù, LPS w ƒƒ w ƒ w, w ùy w w ³ p w w œ ƒ. š x 1. Chiu, A., Chen, and Chen, S.K.: Fdbrne illness in Taiwan, Fd Aust., 43, (1990). 2. Yang, H.C., Hng, S.S., Kim, K.H., Chi, S.H. and Chung, H.J.: Distributin f Vibri vulnificus in Chnnam castal area, Kr. J. Appl. Micrbil. Bitechnl., 27, 70-74(1999). 3. Weltzin R., Mnath T.P.: Intranasal antibdy prphylaxis fr prtectin against viral disease. Clin. Micrb. Rev., 12(3), (1999). 4. Hsu E., Flajnik M.F., Pasquier L.D.: A third immunglbulin class in amphibians. J. Immunl., 135(3), (1985). 5. Schade, R., Burger, W. Schneberg, T., et al.: Avian egg ylk antibdies. The egg -laying capacity f hens fllwing immunizatin with antigens f different kind and rigin and the efficiency f egg ylk antibdies in cmparisn t mammalian antibdies. Altex Alternative Zu Tierexp., 11, (1994). 6. Hansen, P., J.A. Scble, B. Hansn and N.J. Hgenraad: Islatin and purificatin f immunglbins frm chicken eggs using thiphilic interactin chrmatgraphy. J. Immunl. Methds, 215, 1-7 (1998). 7. Tetsur Kga, Tmi Kawata: Islatin and Characterizatin f Outer Membrane frm Vibri parahaemlyticus. J. General Micrbilgy, 129, (1983). 8. Westphal, O., Jann, K.: Bacterial lipplysaccharides. Methds Carbhydr. Chem., 5, (1965). 9. Larssn, A.: David Carlander and Martin Wilhelmssn: Antibdy respnse in laying hens with small amunts f antigen. Fd and Agricultural Immunlgy, 10, (1998). 10. Akita, E. M. and Nakai, S.: Immunglbulin frm egg ylk: islatin and purificatin. J. Fd Sci., 57, (1992). 11. y, w, ½, w ³,», ûx: ùy l w e w p. w t wz. 30, (1998). 12. Fryer, H.J., Davis, G.E., Manthrpe M., Varn, S.: Lwry prtein assay using an autmatic micrtiter plate spectrphtmeter. Anal. Bichem., 153(2), (1986). 13. Laemmli, U. K.: Cleavage f structural prteins during the assembly f head f bacteriphage T4. Nature, 227, (1970). 14. w, ½, w ³, ûx,», y:» ùy Streptcccus mutans p w w. Kr. J. Anim. Sci., 41(5), (1999). 15. Akita, E. M., Nakai, S.: Cmparisn f fur purificatin methds fr the prductin f immunglbins frm eggs laid by hens immunized with an entertxigenic E. cli strain. J. Immunl. Methds, 160, (1993). 16. Shimizu M., Fitzsimmns R. C. and Nakai S.: Anti-E. cli immunglbulin Y islated frm egg ylk f immunized chickens as a ptential fd ingredient. J. Fd Sci. 53, (1988). 17. ½ y, k,, x: (IgY). w œwz, 14(6), (1999). 18. Stalberg J. and Larssn A.: Extractin f IgY frm egg ylk using a nvel aqueus tw-phase system and cmparisn with ther extractin methds. Ups J. Med. Sci., 106(2), (2001). 19. Seung Bae Lee, Yshinri Mine: Effects f Hen Egg Ylk Immunglbulin in Passive Prtectin f Rainbw Trut against Yersinia ruckeri. J. Agric. Fd Chem. 48, (2000). 20. ù, ½, w : ùyw w y» w I. Brdetella brnchiseptica, Pasteurella multcida Actinbacillus pleurpneumniae IgY. w wz. 40(3), (2000). 21. Tsung C. Chang, Chi H. Chen, and Hui C. Chen: Develpment f Latex Agglutinatin Test fr the Rapid identificatin f Vibri parahaemlyticus. J. Fd Prtectin, 57(1), (1994). 22. T. Hnda, Y. Ni, M. Yh, and T. Miwatani: Prductin f mnclnal antibdies against thermstable direct hemlysin f Vibri parahaemlyticus and applicatin f the antibdies fr enzyme-linked immunsrbent assay. Med. Micrbil. Immunl., 178, (1989). 23. Shahjahan Kabir: Cmpsitin and Immunchemical prterties f the Cell Surface Prteins f Vibri Chlerae. J. General Micrbilgy, 132, (1986). 24. Antni Verdliva, Giancarl Basile, Girgi Fassina: Affinity purificatin f immunglbulins frm chicken egg ylk using a new synthetic ligand. Jurnal f Chrmatgraphy, 749, (2000).