Dataset Information

Expression of recombinant Rhipicephalus (Boophilus) microplus, R. annulatus and R. decoloratus Bm86 orthologs as secreted proteins in Pichia pastoris.

ABSTRACT: Rhipicephalus (Boophilus) spp. ticks economically impact on cattle production in Africa and other tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The R. microplus Bm86 protective antigen has been produced by recombinant DNA technology and shown to protect cattle against tick infestations.In this study, the genes for Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) were cloned and characterized from African or Asian tick strains and the recombinant proteins were secreted and purified from P. pastoris. The secretion of recombinant Bm86 ortholog proteins in P. pastoris allowed for a simple purification process rendering a final product with high recovery (35-42%) and purity (80-85%) and likely to result in a more reproducible conformation closely resembling the native protein. Rabbit immunization experiments with recombinant proteins showed immune cross-reactivity between Bm86 ortholog proteins.These experiments support the development and testing of vaccines containing recombinant Bm86, Ba86 and Bd86 secreted in P. pastoris for the control of tick infestations in Africa.

SUBMITTER: Canales M

PROVIDER: S-EPMC2262073 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Expression of recombinant Rhipicephalus (Boophilus) microplus, R. annulatus and R. decoloratus Bm86 orthologs as secreted proteins in Pichia pastoris.

Canales Mario M de la Lastra José M Pérez JM Naranjo Victoria V Nijhof Ard M AM Hope Michelle M Jongejan Frans F de la Fuente José J

BMC biotechnology 20080214

<h4>Background</h4>Rhipicephalus (Boophilus) spp. ticks economically impact on cattle production in Africa and other tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The R. microplus Bm86 protective antigen has been produced by recombinant DNA technology and shown to protect cattle against tick infestations.<h4>Results</h4>In this study, the genes for Bm86 (R. microplus), Ba86 (R. annulatus) and Bd8 ...[more]

PMID: 18275601

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Project description:For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually beta-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus (Boophilus) microplus ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against R. microplus were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against R. appendiculatus. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility.The transcription levels of nine potential reference genes: beta-actin (ACTB), beta-tubulin (BTUB), elongation factor 1alpha (ELF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutathione S-transferase (GST), H3 histone family 3A (H3F3A), cyclophilin (PPIA), ribosomal protein L4 (RPL4) and TATA box binding protein (TBP) were measured in all life stages of R. microplus and R. appendiculatus. ELF1A was found to be the most stable expressed gene in both species following analysis by both geNorm and Normfinder software applications, GST showed the least stability. The expression profile of Bm86 in R. appendiculatus and R. microplus revealed a more continuous Bm86 antigen abundance in R. microplus throughout its one-host life cycle compared to the three-host tick R. appendiculatus where large variations were observed between different life stages.Based on these results, ELF1A can be proposed as an initial reference gene for normalization of quantitative RT-PCR data in whole R. microplus and R. appendiculatus ticks. The observed differences in Bm86 expression profile between the two species alone can not adequately explain the lack of a Bm86 vaccination effect in R. appendiculatus.

Dataset Information

Expression of recombinant Rhipicephalus (Boophilus) microplus, R. annulatus and R. decoloratus Bm86 orthologs as secreted proteins in Pichia pastoris.

Publications

Expression of recombinant Rhipicephalus (Boophilus) microplus, R. annulatus and R. decoloratus Bm86 orthologs as secreted proteins in Pichia pastoris.

Similar Datasets

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