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Expression of a Haemonchus contortus cysteine protease in the baculovirus system Estefan Miranda-Miranda* María Hortensia Murillo-Sánchez Raquel Cossío-Bayúgar *Corresponding author Financial support: This research was supported by SAGARPA-CONACYT, Grant No. 2004-C01-6/B-1. Keywords: haemonchosis, molecular cloning, recombinant protease, synthetic substrates, western blot.
A Haemonchus contortus recombinant Cysteine Protease (CP) was expressed in the baculovirus system. The CP gene was isolated by PCR from H. contortus cDNA, the PCR amplicon was cloned downstream to the polihedrin promoter within a bacterial expression vector, Sf9 insect cells were used for simultaneous co-transfection with the CP-vector and baculovirus naked DNA, which originated recombinant viruses by homologous recombination capable to express recombinant CP in an insect cell culture. A recombinant protease was identified as a fusion protein with a Ni lithium affinity 6XHis group. Recombinant CP was purified by affinity chromatography to obtain active recombinant protease identified by H. contortus experimentally infested ovine sera on a western blot as a 37 kDa protein, as well as by enzyme activity on PAGE-gelatin. Cysteine protease activity was assayed against synthetic substrates including the dipeptides: Phe-Arg, cathepsin B substrate: Arg-Arg, the caspase tetrapeptide substrate: Tyr-Val-Ala-Asp. Maximum CP activity was detected at pH 6.0 for all synthetic substrates and total inhibition was achieved by E-64 but not by EDTA, pepstatin or PMSF. Recombinant H. contortus CP can be obtained in large amounts from transfected insect cell culture and may be applied to control experiments of ruminant Haemonchosis. |
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