Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal beta 1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) crossreacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.

Phylogenetics and biogeography of a spectacular Old World radiation of butterflies: the subtribe Mycalesina (Lepidoptera: Nymphalidae: Satyrini)

Background: Butterflies of the subtribe Mycalesina (Nymphalidae: Satyrinae) are important model organisms in ecology and evolution. This group has radiated spectacularly in the Old World tropics and presents an exciting opportunity to better understand processes of invertebrate rapid radiations. However, the generic-level taxonomy of the subtribe has been in a constant state of flux, and relationships among genera are unknown. There are six currently recognized genera in the group. Mycalesis, Lohora and Nirvanopsis are found in the Oriental region, the first of which is the most speciose genus among mycalesines, and extends into the Australasian region. Hallelesis and Bicyclus are found in mainland Africa, while Heteropsis is primarily Madagascan, with a few species in Africa. We infer the phylogeny of the group with data from three genes (total of 3139 bp) and use these data to reconstruct events in the biogeographic history of the group.,Results: The results indicate that the group Mycalesina radiated rapidly around the Oligocene-Miocene boundary. Basal relationships are unresolved, but we recover six well-supported clades. Some species of Mycalesis are nested within a primarily Madagascan clade of Heteropsis,while Nirvanopsis is nested within Lohora. The phylogeny suggests that the group had its origin either in Asia or Africa, and diversified through dispersals between the two regions, during the late Oligocene and early Miocene. The current dataset tentatively suggests that the Madagascan fauna comprises two independent radiations. The Australasian radiation shares a common ancestor derived from Asia. We discuss factors that are likely to have played a key role in the diversification of the group. Conclusions: We propose a significantly revised classification scheme for Mycalesina. We conclude that the group originated and radiated from an ancestor that was found either in Asia or Africa, with dispersals between the two regions and to Australasia. Our phylogeny paves the way for further comparative studies on this group that will help us understand the processes underlying diversification in rapid radiations of invertebrates.