Transcript PRESENTED BY Prof. c.o.n. ikeobi

MOLECULAR SCREENING OF MHC RESISTANCE
GENES IN WEST AFRICAN DWARF GOATS
An IFSERAR- sponsored project
Research Project Number: UNAAB/IFSERAR/IGR47
Investigation Team: Prof. C.O.N. Ikeobi, Prof. M.O. Ozoje, Dr. A.O. Talabi, Dr. M.
Okpeku and Dr. I.G. Imumorin
PRESENTED
BY
Prof. C.O.N IKEOBI
Department of Animal Breeding and Genetics,
College of Animal Science and Livestock Production
INTRODUCTION
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Nigeria’s population currently estimated at 140 million is the most
populous in Africa, commanding a ratio of one in five Africans and
growing at 2-3% annually.
It is estimated that livestock farming and herding accounts for
about 10% of Nigeria’s Gross Domestic Product.
Goats make substantial contributions to the subsistence sector of
the economy in very many ways, most notable of which includes
being easily adaptable to small-holder and subsistence management
on account of small size, low-input management based on browsing
rather than grazing in addition to being trypanotolerant in the
Nigeria’s sub-humid and humid tropical climates characterized by
high prevalence of tse-tse flies.
Despite these advantages, little attention had been paid to the
genetic characterization and possible improvement of goats in
Nigeria.
 Adu et al. (1979) studied reproductive performance and
Buvanendran et al. (1981) and Moruppa (1985) reported on goat
haemoglobin and transferrin alleles.
 These were followed more recently with other reports by
Ebozoje and Ngere (1995), Odubote and Akinokun (1992),
Odubote (1994) and Imumorin et al. (1999).
 The major histocompatibility complex (MHC) plays an important
role in the adaptive immune response of vertebrates (Trowsdale,
1993).
 The MHC is an organized cluster of tightly linked genes with
immunological and non-immunological functions, and is present in
all vertebrates, except in jawless fish (Tizard, 2004).
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The primary function of the MHC is to code for specialized
antigen-presenting receptor glycoproteins.
These molecules bind processed peptide antigens and present
them to T lymphocytes, thereby triggering immune responses.
The genes encoding these molecules are polymorphic, and in
sheep, goats and cattle, high levels of polymorphism are observed
in both the DRB and DQA genes (Ellis and Ballingall, 1999).
The major aim of this study is to carry out a molecular screening
of MHC resistance genes in West African Dwarf Goats.
OBJECTIVES
BROAD OBJECTIVE
 Molecular Screening of MHC Resistance gene in
West African Dwarf Goats.
SPECIFIC OBJECTIVES
To clone, characterize and sequence MHC genes in West Africa
Dwarf goats
 To identify genetic variants in MHC genes in West African Dwarf
goats
 To determine the association of MHC genotypes and quantitative
traits in West African Dwarf goats as potential tools for markerassisted selection.
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MATERIALS AND METHODS
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Study Area.
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Study Population: 200 WAD goats were sampled.
Data Collection: Data on both qualitative and quantitative traits as
well as Heat stress data were collected on each animal.
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Blood Collection
10ml= 5ml(EDTA)+ 5ml(Heparinised)
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(DNA)
( Serum Analysis)
DNA extraction was carried out using Zymobead genomic DNA
extraction kits.
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Primers: Primer3 was used to design the primer. This was then
ordered from the Bioneer Primers.
The Primer’s sequence for DRB 1 is
Genes
Primers
A-T
Product
Ref.
DRB 1
F: TATCCCGTCTCTGCAGCACATTTC
R: TCGCCGCTGCACACTGAAACTCTC
60oC
570 bp
Amills et al. (1995)
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A-T = Annealing temperature; bp = base pairs
PCR: PCR master mix from Bioneer was also used for the
reaction.
 These primers were used in a PCR consisting of 30 cycles of: 94
C for 1 min, 60 0C for 1min and 72 0C for 1 min. Reactions were
performed in 20 ml total volume.
 Agarose gel electrophoresis was performed using 1.5% agarose.
 The bands were snapped with the aid of a gel documentation
system.
 The remaining PCR products were send to Cornell University
core laboratory for sequencing.
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The results of the sequence was used to design the restriction
enzyme that will make a cut in the sequence using NEBCUTTER
(an online tool).
The resulting bands from the digested product was then
genotyped manually to know whether the animal was
homozygous or heterozygous for the DRB gene.
HpyAV 5'... C C T T C (N)6 ... 3' 3'... G G A A G (N)5 ... 5'
Helicobacter pylori 26695
RESULTS
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In the sequence, it was discovered that the sequence is in exon 2
of the DRB gene in Capra hircus.
Contig or consensus sequence
GGGTGCGGTTCCTGGACAGATACTTCTATAATGGACAAGAGACGTGCG
CTACGACAGCGACTGGGGCGAGTTCCGGGCGGTGGCCGAGCTGGGG
CGGCCGGACGCCAAGTACTGGAACAGCCAGAAGGAGATCCTGGAGG
ACAGGCGGRCCGAGGTGGACACGTTCTGCAGACACAACTACGGGGTC
ATTGAGAGTTTCAGTGTGCAGCGGCGAA.
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Protein translation
Amino acid sequence
VRFLDRYFYNGQEYVRYDSDWGEFRAVAELGRPDAKYWNSQKEILEDRRXEV
DTFCRHNYGVIESFSVQRR
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Amino acid sequence with nucleic acids
GTGCGGTTCCTGGACAGATACTTCTATAATGGACAAGAGTACGTGCGCTACGACAGCGAC
V R F L D RY FY N G Q EY V RY D S D
TGGGGCGAGTTCCGGGCGGTGGCCGAGCTGGGGCGGCCGGACGCCAAGTACTGGAACA
GC
W G E F R AV A E L G R P D A KY W N S
CAGAAGGAGATCCTGGAGGACAGGCGGRCCGAGGTGGACACGTTCTGCAGACACAACTAC
Q K E I L E D R R X EV D T F C R H NY
GGGGTCATTGAGAGTTTCAGTGTGCAGCGGCGA
GV I E S F SV Q R R
Protein BLAST (Basic Local Alignment
Search Tool)
 >dbj|BAA23121.1| - accession number in gene bank
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MHC class II DRB [Capra hircus]
 Length=89, Score = 124 bits (312), Expect = 1e-34, Method:
Compositional matrix adjust.
 Identities = 57/71 (81%), Positives = 64/71 (91%), Gaps = 0/71
(0%)
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GEL PICTURE AND SAMPLES WITH RESTRICTION
ENZYMES
RS
WAD
SH
WAD
References
Buvanendran,V, Sooriyamoorthy, T, Ogunsusi, RA and Adu IF. 1981.
Haemoglobin polymorphism and resistance to helminths in
red sokoto goats. Trop. Anim. Hlth. Prod. 13: 217 – 21.
 Ebozoje, MO and Ngere, LO 1995. Genetic analysis of preweaning
growth in West African Dwarf goats and their half-breds. Inter
J. Anim Sci. 10: 247 – 251.
 Ellis, S.A, and K.T.Ballingall. 1999. Cattle MHC: Evolution in
action? Immunol. Rev. 167:159-168.
 Tizard, I.R. 2004. Acquired Immunity: antigen-presenting
receptors. In: Veterinary Immunology: an introduction,
Elsevier, Philadelphia, PA, USA, 67-77.
 Trowsdole J. 1993. Genomic structure and function in the MHC.
Trends genet 9:117- 122.
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THANKS FOR
LISTENING