Establishment of disease resistant strain using MHC class I gene

Transcription

1 Establishment of disease resistant strain using MHC class I gene Mitsuru Ototake Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency, JAPAN

2 Background of Research Fish diseases are becoming a serious problem. There is an increasing need for the establishment of disease-resistant fish, as it is the basic step for solving this problem. However, unlike in domestic animal farming, there are still very few indexes which have been identified as disease resistant. There are some positive aspects of fish farming. Fish spawn much more than cattle, and clone technology is already established in fish.

3 virus Function of MHC class I in mammals Partial digestion of Antigens Proteasome AA Antigen presentation MHC Ia TCR 9AA Killer T-Cell Response Cytotoxicity

4 Major Histocompatibility complex (MHC) gene in mammals Major Histocompatibility complex gene (MHC) is a very important molecule in mammals and birds, for its role is to present the information on antigens to the T-cell. There are extremely large varieties of types in MHC. MHC types have some relation with disease resistance in several diseases. Fish MHC

5 Variation of α1 domain among groups of classical MHC class I AA identity with I ⅠVTHSLKYFYTASS-EVPNFPEFVVVGMVDGVQMVHYDSN--SQRMVPRQDWMNKAAETLPQYWESQTGILKGTQQTYKASIDIVKQRFNQSG Ⅱ...R...TT-GI.D...D..V.N.KVISY...I--TK.K..K.S..EENLN--Q...NQG.DQ...AE..F..N.Q..QT...T ⅢA...R.V...T.-G..D...SL.I...M.IDY...--TK.V..K...A.TEG--SD...R..QNSI.DE..F..N..VA...T Ⅳ...G.T-GIEG..Q..A..I...M.IDYF..V--.EKN.LK.S..EGVRD-----EKMI.NTR..N...F..NVE.GM...TT-- 47 ⅤA...V.-GDID...TA..L..KG.FMYF..S--TKTA..KTE..KREGA---D..DR..QG.I.AH..F.VN.QTL.D...KST 46 ⅥA.NT.Q...T.-GID...TM.I.N.H.ID...I--TK.AIQKAE.ISG.VD--.D..KRN.QTYAAEEPVFLNN.K.A.S...T Ⅶ.I..W.A.L...T-GLSD...ALNL..DEL.GYF.TK--TN.FEGK.S.VEEKLG--Q..M.R.EN..R..S.SF.VNVG..ME...TK-- 40 Ⅷ...MQ.IV..V.-GLDEI..HTE...Q.F.Y...V--LKKII.KT..IE.NVD--AS..KRE.DRNIA.E..F.SNVA.AMT...TR-- 42 HLA-A2 GS..MR..F.SV.RPGRGE.R.IA..Y..DT.F.RF..DAA...E..AP.I-EQEG--.E..DGE.RKV.AHS..HRVDLGTLRGYY...EA- 36

6 Frequency of groups of MHC type in rainbow trout No-mark Albino Steelhead Donaldson's Nikko 0% 50% 100% A B C D E F G H I

7 Expression of classical MHC class I gene up-regulated by IHN-virus infection Onmy-UBA Agarose Northern blot

8 Rainbow trout MHC regions Onmy-IA and Onmy-IB. IB region (111,126 bp) IA region (103,035 bp) UDA UCA TAPBP pseudogene % nt identity in ORFs UBA UEA 90 UFA LMP7 MECL1 95 LMP2d LMP2d.1 MECL1.1LMP2.1 LMP7.1 TAP2B LMP2 TAP2B 93

9 Fluorescence in situ hybridization (FISH) mapping of Onmy-IA

10 Fluorescence in situ hybridization (FISH) mapping of Onmy-IB

11 Correlation between MHC type and phenotype (method) Preparation of the experimental Fish Select parents according to MHC type. Fertilize Example: BF X BF F1 siblings MHC BB : BF : FF = 1 : 2 : 1 Other genes are expected to be mixed evenly Observation of correlation between MHC type and phenotype.

12 Functions of trout MHC Classical MHC class I Grafts rejection (Sarder et al. 2003) Efficacy of Vaccine Behavior and growth (Azuma et al. in press) Non-classical MHC class I Disease resistance

13 Mortalities of siblings in the challenge test after the DNA vaccination with IHNV N-protein Classical MHC Class I Type Dead (fish) Live (fish) Mortalit y BB % * BF % FF % * *: significant difference (p < 0.01)

14 Mortalities of siblings after the challenge with IPN virus Non-classical MHC class I type Dead (fish) Live (fish) Mortalit y A1 A % * A1 A % * *: significant difference (p<0.001)

15 BB and FF display different Locomotor patterns 9 Feed Feed Activity (count / 10 min.) 6 3 FF FB BB Time of day

16 Summary of MHC class I in rainbow trout Rainbow trout has two MHC class I regions: Classical region on 18th chromosome Non-classical region on 14th chromosome In classical region, there is only one locus of classical class I, which is quite different from mammals in number. Other characteristics of classical MHC such as the sequence of key amino acid, gene structure, expression pattern, the rejection of transplanted tissue are almost the same as those in mammals. The classical MHC gene type correlates to the vaccine effectiveness, behavior and growth. The non-classical MHC type correlates to disease resistance against IPN.

17 Collaborations Fisheries Research Agency Dr. I. Kiryu Dr. J.M. Dijkstra Dr. Y. Yoshiura Dr. A. Fujiwara Dr. R.I. Sarder Dr. T. Somamoto Dr. T. Azuma Fujita Health University Dr. K. Hashimoto Nagano prefecture Dr. Motonishi Insel Riems (Germany) Dr. Uwe Fischer Mr. Bernd Koellner China agricultural university Dr. C. Xia Nippon University Dr. T. Nakanishi Tokyo university of Fisheries Dr. N. Okamoto Dr. T. Aoki Tokai University Dr. T. Shiina