Journal: Infection and Immunity
Article Title: Cloning and Sequence Analysis of a Highly Polymorphic Cryptosporidium parvum Gene Encoding a 60-Kilodalton Glycoprotein and Characterization of Its 15- and 45-Kilodalton Zoite Surface Antigen Products
Figure Lengend Snippet: (A) Time course of gp15 mRNA expression in C. parvum -infected MDCK cell monolayers. Total RNA was isolated from infected MDCK cells at various times postinfection, DNase treated, and used as a template for RT-PCR (lanes 2 to 12) or control PCR (lanes 13 to 23), both primed with the gp15ATG and gp15STOP primers. The arrowhead indicates the position of the 1-kb gp15 amplicon. RNA template was isolated from infected MDCK cells at 0.5 h (lanes 3 and 14), 2 h (lanes 4 and 15), 4 h (lanes 5 and 16), 6 h (lanes 6 and 17), 9 h (lanes 7 and 18), 11 h (lanes 8 and 19), 24 h (lanes 9 and 20), and 48 h (lanes 10 and 21) postinfection; from uninfected MDCK cells (lanes 2 and 13); and directly from purified sporozoites (2 μg; lanes 11 and 22). Additional controls for the RT-PCR and PCRs lacked template nucleic acid (lanes 12 and 23 and 24, respectively). (B) Intracellular expression of gp15 protein occurs late in merogony. C. parvum ), or with anti-gp15 MAb 11A5 (F and H), CrA1 (J and L) or CrA2 (N and P) followed by biotinylated secondary antibodies and Cy3-conjugated streptavidin. Parasite nuclei in the same microscopic fields were stained with DAPI (lanes A, E, I, M, C, G, K, and O).
Article Snippet: CrA1/2 and 11A5 immunoprecipitations were performed by incubating a preformed ternary complex composed of primary MAb, biotin-conjugated secondary antibody (goat anti-mouse IgA or goat anti-mouse IgG), and streptavidin-agarose beads (EY Laboratories) with the extracts on a rotator for 1 h at room temperature.
Techniques: Expressing, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Purification, Staining