Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (p > 0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62 μg/g (±2); biomass from cell suspension cultures 83 μg/g (±0.2); culture medium from suspensions 71.75 mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.Fil: Gomes, Marinna. Universidade Federal de Juiz de Fora; BrasilFil: Alvarez, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Maimónides; ArgentinaFil: Quellis, Leonardo Ramos. Universidade Federal de Juiz de Fora; BrasilFil: Laguía Becher, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Maimónides; ArgentinaFil: Castro, Juciane Maria de Andrade. Universidade Federal de Juiz de Fora; BrasilFil: Gameiro, Jacy. Universidade Federal de Juiz de Fora; BrasilFil: Caporrino, Maria Cristina. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Silva, Ana Maria Moura da. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Santos, Marcelo de Oliveira. Universidade Federal de Juiz de Fora; Brasi

Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs

<p>Abstract</p> <p>Background</p> <p>Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and are versatile toxins, targeting many important elements involved in hemostasis, such as basement membrane proteins, clotting proteins, platelets, endothelial and inflammatory cells. The functional diversity of SVMPs is in part due to the structural organization of different combinations of catalytic, disintegrin, disintegrin-like and cysteine-rich domains, which categorizes SVMPs in 3 classes of precursor molecules (PI, PII and PIII) further divided in 11 subclasses, 6 of them belonging to PII group. This heterogeneity is currently correlated to genetic accelerated evolution and post-translational modifications.</p> <p>Results</p> <p>Thirty-one SVMP cDNAs were full length cloned from a single specimen of <it>Bothrops neuwiedi </it>snake, sequenced and grouped in eleven distinct sequences and further analyzed by cladistic analysis. Class P-I and class P-III sequences presented the expected tree topology for fibrinolytic and hemorrhagic SVMPs, respectively. In opposition, three distinct segregations were observed for class P-II sequences. P-IIb showed the typical segregation of class P-II SVMPs. However, P-IIa grouped with class P-I cDNAs presenting a 100% identity in the 365 bp at their 5' ends, suggesting post-transcription events for interclass recombination. In addition, catalytic domain of P-IIx sequences segregated with non-hemorrhagic class P-III SVMPs while their disintegrin domain grouped with other class P-II disintegrin domains suggesting independent evolution of catalytic and disintegrin domains. Complementary regions within cDNA sequences were noted and may participate in recombination either at DNA or RNA levels. Proteins predicted by these cDNAs show the main features of the correspondent classes of SVMP, but P-IIb and P-IIx included two additional cysteines cysteines at the C-termini of the disintegrin domains in positions not yet described.</p> <p>Conclusions</p> <p>In <it>B. neuwiedi </it>venom gland, class P-II SVMPs were represented by three different types of transcripts that may have arisen by interclass recombination with P-I and P-III sequences after the divergence of the different classes of SVMPs. Our observations indicate that exon shuffling or post-transcriptional mechanisms may be driving these recombinations generating new functional possibilities for this complex group of snake toxins.</p