El objetivo del presente trabajo fue establecer la frecuencia de genotipos de resistencia y susceptibilidad a padecer la enfermedad de scrapie en ovejas Junín. Se analizaron 56 muestras de sangre de ovinos de una unidad de producción privada de la región central del Perú. Se extrajo el ADN y los productos de PCR fueron analizados mediante digestión enzimática con endonucleasas de restricción que reconocen los polimorfismos del gen PrP. Se pudo identificar 4 de los 5 haplotipos reportados para el gen PrP: ARR (48.2%), ARQ (35.7%), AHQ (10.7%) y VRQ (5.4%). Además, se encontró 7 de los 15 genotipos reportados para el gen Prión: ARR/ARR (17.9%), ARR/AHQ (10.7%), ARR/ ARQ (42.9%), ARQ/AHQ (10.7%), ARQ/ARQ (7.1%), ARR/VRQ (7.1%) y ARQ/VRQ (3.6%). El 82.1% de las ovejas presentaron genotipos de resistencia a la enfermedad y el 17.9% presentó genotipos de susceptibilidad.
A reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly in culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent media, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 x 50 mm, 3.5 μm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 x 12.5 mm, 5μm), using 0.1% TFA and 2% n-propanol as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile as mobile phase B. The column temperature was 80ºC, the flow rate 1 ml/min and the absorbance monitored at 280 nm. The procedures and capabilities of the method were evaluated against the present criteria for linearity, limit of detection (LOD), accuracy, precision, and robustness of the International Conference on Harmonization (ICH) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The methods showed good linearity (R2 = 0.9996), a lower LOD of 2.4 μg/ml, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.
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