Proteins of sheep abomasum that interact with Hc-SPI-I8

ABSTRACT: Freshly collected abomasum tissue of the naturally infected sheep was ground, filtered, and centrifuged at 3000 g at 4 °C for cell collection. The collected cells were lysed in NP-40 lysis buffer (Thermo Fisher Scientific) for 0.5 h with shaking, then centrifuged at 12000 g at 4 °C for supernatant collection. Coding sequence of Hc-spi-i8 (HCON_00067680) was amplified and inserted into pcDNA3.1(+) vector to produce FLAG-tagged Hc-SPI-I8 in HEK 293T cells. The recombinant Hc-SPI-I8-FLAG protein was isolated from lysed cells using anti-FLAG-conjugated magnetic beads (Bimake, Shanghai, China), and used to isolate and concentrate interactive proteins from the ovine tissue lysis. The isolated proteins were boiled, separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), from which proteins within a range of molecular weights from 30 to 45 kDa were subjected to digestion with trypsin (Promega), desalination with a sep-Pak C18 (Waters) and LC-MS/MS analysis with a Q-Exactive Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Peptides were identified and annotated using Proteome Discoverer v.2.1.

ORGANISM(S): Ovis Aries

SUBMITTER: Guangxu Ma  

PROVIDER: PXD055614 | iProX | Fri Sep 06 00:00:00 BST 2024

REPOSITORIES: iProX