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| Author(s): |
Débora Colombi
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2003-03-17 |
| Examining board members: |
Paulo Lee Ho;
Chuck Shaker Farah;
Luis Carlos de Souza Ferreira;
Sylvia Luisa Pincherle Cardoso Leão;
Mari Cleide Sogayar
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| Advisor: | Paulo Lee Ho |
| Abstract | |
Bordetelfa pertussis, the agent of whopping cough, establishes infection by attaching to the ciliated epithelial cells of the respiratory tract. The bacterial adherence is mediated by pertussis toxin and filamentous hemagglutinin (FHA). FHA is the major adhesin of B. pertussis and displays multipie adherence activities. FHA contains four distin\'ct domains that exhibit specific affinities for different ligands or receptor, the amino-terminal end, the RGD triplet (FHA1097-1099), the lectin domain (FHA1141-1279) and the heparin-binding domain (FHA442-863). In this study, two overlapping regions of the heparin-binding domain, Mal80 (FHA299-873) and Hep (FHA442-873), were amplified by peR and subcloned in pAE expression vectors for E. coli. The fusion proteins in pAE were transformed in E. coli BL21 SI, induced with NaCI 0,3 M and purified using a nickel-charged metal chelating resin. The purified protein has low heparin affinity and does not have hemagglutination activity. The purified protein HEP was used to produce polyclonal antibodies in mouse. The anti-HEP antibodies are able to recognize the HEP, MAL80 and FHA proteins in ELISA and western assays, but anti-FHA only recognized the FHA protein. The genetically detoxified S1 subunit of pertussis toxin and Hep domain were amplified by the TAP Express method. There are two PCR reactions involved in the TAP processo At the end of the process the fragment of interest will carry a CMV promoter and a SV40 terminator and is ready to be introduced into animals or cell by transfection. Groups were immunized with proteins and/or DNA, challenged i.c. with a lethal dose of live Bordetelfa pertussis and the survival was monitored. No groups were protected against the challenge. The recombinant protein HEP were also expressed in Lactobacilfus aiming the development of potential mucosal vaccines. The polyclonal antibodies produce in mouse immunized with DNA and protein Hep expressed in E. coli and Lactobacillus were able to inhibition the FHA hemagglutination activity. (AU) | |