Expression of a Haemonchus contortus cysteine protease in the baculovirus system

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.

Identification of gut antigens from female boophilus microplus ticks

Un extracto de intestino de hembras semi-repletas de garrapata Boophilus microplus, fue separado mediante electroforesis SDS-PAGE, identificándose al menos 18 bandas en un rango de peso molecular de 17 a 258 kDa; al practicar el ensayo inmunoenzimático de Dig-glicanos, las proteínas por arriba de 58 kDa, mostraron estar asociadas con carbohidratos. El análisis de Western blot permitió identificar cinco antígenos de intestino con un peso molecular en un rango de 89 a 208 kDa, utilizando un suero de conejo anti-intestino de garrapata. Estos antígenos fueron localizados en la superficie del intestino de la garrapata mediante inmunofluorescencia indirecta en cortes histológicos del mismo intestino. Los tres antígenos (99, 141, 189 KDa) identificados, son diferentes de los previamente reportados en la literatura y podrían ser utilizados en pruebas para inducir inmunidad en contra de las garrapatas en ganado bovino