Avaliação de estratégias de produção em cultivo descontínuo da proteína quimérica multi-antigênicaTgAGS/BsT de toxoplasma gondii em escherichia coli BL21 star
Toxoplasmosis, despite advances in science and technology, is a disease that requires attention since there is no vaccine capable of immunizing humans and animals against all isolated types of Toxoplasma gondii. Thus, the use of chimeric proteins, which can contain multiple antigens, is a very promi...
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| Άλλοι συγγραφείς: | |
| Μορφή: | Dissertação |
| Γλώσσα: | pt_BR |
| Έκδοση: |
Universidade Federal do Rio Grande do Norte
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| Θέματα: | |
| Διαθέσιμο Online: | https://repositorio.ufrn.br/handle/123456789/44795 |
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| Περίληψη: | Toxoplasmosis, despite advances in science and technology, is a disease that
requires attention since there is no vaccine capable of immunizing humans and animals
against all isolated types of Toxoplasma gondii. Thus, the use of chimeric proteins, which
can contain multiple antigens, is a very promising alternative for the process of obtaining a
vaccine and diagnostic test for toxoplasmosis due to the great diversity of antigens presented
by T. gondii. In this context, the main focus of the present study is to evaluate batch culture
strategies in the production of the multi-antigenic chimeric protein TgAGS/BsT from
Toxoplasma gondii. To achieve the proposed objective, several exploratory cultures were
initially carried out to observe the kinetic behavior of E. coli BL21 Star in five different
medium compositions (2xTY, TB supplemented with glucose, TB supplemented with
glycerol, LB and M9) without the addition of IPTG (inductor). Then, cultures of E. coli B21
Star were carried out with 1.0 mM IPTG at different times of initiation of induction (0.5, 1
and 6 h) to evaluate the effects on cell growth, production of the protein of interest, pH and
acetic acid formation. The results showed that among the culture media evaluated, 2xTY
and TB supplemented with glycerol had the best cell concentration values of 3.42 ± 0.05
g/L and 5.48 ± 0.05 g/L, respectively. In the assays induced by IPTG, a higher expression
of TgAGS/BsT protein was observed, with induction beginning within 6 h of culture, with
a maximum concentration of protein of interest of 1.82 ± 0.02 g/L for the 2xTY and 2
medium. 2.49 ± 0.03 g/L for the TB medium. In addition, later induction by IPTG provided
greater stability of plasmid pET-TgAGS, remaining with values above 90% at the end of
culture. The expression of the TgAGS/BsT protein was confirmed by electrophoresis, with
a molecular mass of approximately 18.6 kDa. Therefore, the present study identified the best
conditions of culture medium composition and induction time for the production of
TgAGS/BsT protein in rotary incubator cultures, with results that encourage the expansion
to cultures in larger capacity bioreactors. |
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