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2007, Number 3

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Bioquimia 2007; 32 (3)

Use of the Baculovirus heterologous gene system expression on the generation of an hybrid recombinant antigen useful in the immunodiagnostics of ruminant hemonchosis

Miranda-Miranda E, Murillo-Sánchez MH, Cossío-Bayúgar R

Language: Spanish
References: 30
Page: 83-90
PDF size: 193.03 Kb.


ABSTRACT

We report a molecular cloning and expression in the baculovirus system of an Haemonchus contortus recombinant cystein protease (CP) gene, with the objective of obtaining a recombinant CP with biochemical and immunochemical properties similar to the natural one obtained from the parasite, with applications to the diagnosis prevention and control of sheep hemonchosis. The CP gene was isolated by PCR from H. contortus cDNA, the gene was cloned downstream to the polihedrin promoter within a bacterial expression vector, Sf9 insect cells were used co-transfected simultaneously with the CP-vector and baculovirus naked DNA which originated recombinant viruses by homologous recombination capable to express recombinant CP in vitro in an insect cell culture. A recombinant protease was identified as an inactive fusion protein with an affinity 6XHis moiety, it was purified by affinity chromatography in order to obtain enzymatically active recombinant protease which was identified by H. contortus experimentally infested ovine sera assayed by western blot and ELISA as well as by enzyme activity on PAGE-gelatin, the baculovirus expression system yielded 400 mg of recombinant diagnostic antigen per liter of culture media (400 mg L-1). We concluded that the obtained recombinant CP is similar to the natural one obtained from the parasite, can be obtained in large amounts from the transfected insect cell culture an may be applied in the immunodiagnostics and control of ruminant haemonchosis.


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