Expresión recombinante en E. Coli de antígenos de Actinobacillus Pleuropneumoniae para vacunación y diagnóstico
Actinobacillus pleuropneumoniae is a gramnegative bacteria responsible for the porcine pleuropneumonia. In this work we have proceeded to the production and purification, using molecular biology techniques, of proteinaceous antigens from this bacteria and to its use in the formulation of a subunit vaccine and in an ELISA for the serological diagnostic. The four chosen antigens were two outer membrane proteins (Tbp1 y Tbp2) and two exotoxins (ApxI y ApxIII).
The first requisite was the localization and sequenciation of the tbp1gene in the A. pleuropneumoniae genome. The tbp2 gene was cloned to be used as a probe for the tbp1 detection, this gene is located upstream the tbp1. After its location, the tbp1 gene was cloned in the pUC119 vector and his complete sequence was obtained, this sequence was submitted to the Genbank con el accession code Z49708, the European patent number is EP0733708 and the American is 08/624655. These results were published (Daban et al., 1996; Medrano et al., 1997) (Anexos VII.1 y VII.2).
On the other side, for the ApxIa y ApxIIIa genes cloning a different strategy was employed. As their sequences were already published, PCR oligonucleotides were prepared with mutations inserted in order to generate restriction sites, allowing the amplification using the genome as template followed by cloning in expression vectors.
Once the four proteins coding genes cloned we proceeded to their heterologue production in E. coli. Diverse vectors were used to achieve this: pMAL, which yields a fusion protein with the MBP (Maltose Binding Protein) and pET range vectors, most of the work has been done with the late ones. The four recombinant antigens have been cloned and produced in the pET vectors, the whole antigens and also as peptides. The recombinant proteins were purified from the expression cultures by means of affinity chromatography making use of histidine fusion tags.
After the expression and purification of the four antigens (ApxI, ApxIII, Tbp1 y Tbp2) on a laboratory scale, an experimental vaccine was prepared. This vaccine was tested in three experimental protocols using pigs free form antibodies against A. pleuropneumoniae. The harmlessness and immunogenicity of this vaccine were evaluated.
Western-blot and ELISA were used to make a serological titration on samples obtained from the vaccination assays. Using these samples and other field sera collections obtained from swine with known diverse clinical reports, an indirect ELISA assay kit was developed for the detection of specific antibodies against A. pleuropneumoniae. The antigens used in the preparation of the assay are the recombinant proteins Tbp2 and ApxI. This assay is currently being commercialised under the brand CIVTESTSUISAPP.
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Advisor:Querol Murillo, Enrique; Daban Marín, Montserrat
School:Universitat Autónoma de Barcelona
Source Type:Master's Thesis
Keywords:406 departament de bioquimica i biologia molecular
Date of Publication:04/04/2003