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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 11, No. 2, 2008
Bioline Code: ej08021
Full paper language: English
Document type: Research Article
Document available free of charge

Electronic Journal of Biotechnology, Vol. 11, No. 2, 2008

 en Expression of a Haemonchus contortus check for this species in other resources cysteine protease in the baculovirus system
Miranda-Miranda, E.; Murillo-Sánchez, M.H. & Cossío-Bayúgar, R.

Abstract

A Haemonchus contortus check for this species in other resources 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.

Keywords
haemonchosis, molecular cloning, recombinant protease, synthetic substrates, western blot

 
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