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BACKGROUND
Ticks and tick-borne diseases (TBDs) cause economic
losses to small farmers due to the high mortality in
susceptible breeds, reduced in milk production and
resilience in animal traction (plow-power). The need to
improve the local breeds of cattle in Uganda through cross
breeding with the exotic breeds has always been a challenge
because of the high mortalities by TBDs. Ticks control are
of economic importance mainly because the parasites they
transmit which cause huge losses as a result of high
mortalities. Tick control is of increasingly concern due to
the escalating costs of acaricide and increased selection of
acaricide resistance in ticks. Strategies of adopting anti-tick
vaccines therefore represents a promising alternative
including advantages, of environmental safety and low cost
of production. The aim of this abstract is to show the results
of production and characterization of recombinant protein
Ra92A from Rhipicephalus appendiculatus, a gut variant
Bm86 gene.
METHODS
The Ra92A gene was synthesized with codon usage
for Pichia pastoris, cloned in the plasmid pPICZalphaA,
and propagate in Escherichia coli TOP10F'. P. pastoris
GS115 was transformed with the plasmid (pPICZα-Ra92A)
by electroporation. The selection of recombinants occurred
in YPD plates plus Zeocin™. The confirmation of the
recombinants was made by PCR and sequencing with
5'AOX1 and 3'AOX1 primers, as well as by enzyme
restrictions. The confirmed clones were inoculated into 5 ml
of BMGY (1% yeast extract, 2% peptone, 100 mM
potassium phosphate, pH 6.0, 1.34 % YNB, 4 x 10-5%
biotin, 1% glycerol) and incubated at 28 °C overnight, 200
rpm. The cultures were centrifuged, ressuspended in
BMMY (1% yeast extract, 2% peptone, 100 mM potassium
phosphate, pH 6.0, 1.34 % YNB, 4 x 10-5% biotin, 1%
methanol) to OD600nm = 1.0 and incubated under the same
conditions for 3 days, supplemented with 1% of methanol
(v/v) every 24 hours. Finally, the cultures were centrifuged
at 3,000 x g for 5 min and supernatants stored at -20 °C.
The supernatants were subjected to dot blotting against
monoclonal anti-His (C-term) (Invitrogen) to check the
expression of rRa92A.
Characterization and Production of recombinant protein Ra92A from
Rhipicephalus appendiculatus aiming tick vaccine antigen in Uganda.
Renato Andreotti1, Margaret Saimo-Kahwa2, Rodrigo Casquero Cunha3, Jacqueline Cavalcante Barros1,
Fabio Pereira Leivas Leite3
1 – Embrapa Beef Cattle, Brazil; 2 – Markerere University, Uganda; 3 – Universidade Federal de
Pelotas, Brazil
Financial support:
Figura 1. pPICZalpha-Ra92A map.
Figura 2. Dot blotting of the transforming colonies: GS-Ra92A1-8;
Bm86-CG positive control (+C) and GS115 negative control (-C).
pPICZa lpha Ra 92A
5512 bp
6xHis
c-myc e pitope
alpha-fac tor signal peptide
Zeo(R)
Ra92A synthetic
3' AOX1 prime r
5' AOX1 prime r
alpha-fa ctor primer
TEF1 prom oter
AOX1 promoter
EM7 promoter
pUC origin
AOX1 trans cription terminator
CYC1 transcription ter minator
Eco
RI (1209)
Sac
I (209)
Xba
I (3191)
Bm86-CG (+C)
GS-Ra02A 1
GS-Ra02A 2
GS-Ra02A 3
GS-Ra02A 4
GS-Ra02A 5
GS-Ra02A 6
GS-Ra02A 7
GS-Ra02A 8
GS115 (-C)
RESULTS
Eight recombinants were confirmed. All with the
synthetic sequence of Ra92A integrated into the yeast genome.
In dot blotting was possible to detect protein by histidines tail
in the supernatant. The rRa92A protein is available to associate
an adjuvant to be evaluated for efficacy in immunization trials
aiming enhancing cattle productivity through control of ticks by
anti-tick vaccine in Uganda