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Master's Dissertation
DOI
https://doi.org/10.11606/D.87.2016.tde-07032016-134221
Document
Author
Full name
Marina Katia Ferreira Mazzoni
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2015
Supervisor
Committee
Mendonça, Ronaldo Zucatelli (President)
Kfoury Junior, José Roberto
Wolff, José Luiz Caldas
Title in Portuguese
Clonagem e expressão de proteína antiapoptótica presente na hemolinfa de Lonomia obliqua WALKER 1855 (Lepdoptera: Saturniidae) em Escherichia coli.
Keywords in Portuguese
Escherichia coli
Lonomia obliqua
Antiapoptótica
Clonagem
Expressão gênica
Proteína recombinante
Abstract in Portuguese
A lagarta L. obliqua tem se destacado por apresentar em sua hemolinfa proteínas com atividade biológica demonstrada em cultivos celulares. A literatura disponível não apresenta trabalhos sobre a expressão da proteína antiapoptótica de L. obliqua, então este estudo objetiva a clonagem e expressão da proteína antiapoptótica presente na hemolinfa de L. obliqua, em sistema bacteriano Escherichia coli. O RNAm extraído do tegumento de L. obliqua deu origem a um cDNA obtido por RT-PCR, o qual foi clonado em vetor pCR II-TOPO para posterior transformação de bactérias E. coli JMQC. Na expressão heteróloga, o fragmento foi subclonado em vetor pET28a e transformadas bactérias E. coli BLQC. A indução de expressão foi realizada com IPTG 1 mM. A APLOrEC purificada por cromatografia foi identificada por Western Blot. A atividade biológica da APLOrE foi analisada em células VERO e L929 após indução de morte e verificou-se que esta protegeu os cultivos induzidos com 4mM de H2O2 portanto, eficaz na manutenção estrutural do citoesqueleto destas células.
Title in English
Cloning and expression of antiapoptotic protein present in the hemolymph of Lonomia obliqua Walker 1855 (Lepidoptera: Saturniidae) in Escherichia coli.
Keywords in English
Escherichia coli
Lonomia obliqua
Antiapoptotic
Cloning
Gene expression
Recombinant protein
Abstract in English
The caterpillar L. obliqua has become known for performing in their hemolymph proteins with biological activity demonstrated in cell cultures. The available literature does not provide studies on the expression of antiapoptotic protein L. oblique, so this study aims cloning and expression of anti-apoptotic protein present in the hemolymph of L. oblique, bacterial system in Escherichia coli. The extracted mRNA L. obliqua husk gave a cDNA obtained by RT-PCR, which was cloned into pCR II-TOPO vector for subsequent transformation of E. coli bacteria JMQC. The heterologous expression, the fragment was subcloned into pET28a vector and transformed E. coli bacteria BLQC. The induction of expression was performed with 1 mM IPTG. The APLOrEC purified by chromatography was identified by Western blot. The biological activity was examined in APLOrE VERO and L929 cells after death induction, and it was found that this protected crops H2O2 induced with 4mM therefore effective in the structural maintenance of the cytoskeleton of such cells.
 
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Publishing Date
2016-03-07
 
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