การวิเคราะห์การสังเคราะห์โปรตีนทั้งหมดภายในเซลล์เจ้าบ้านที่ติดเชื้อระยะเริ่มแรกของไวรัสก่อโรคเริมชนิดที่ 2

Abstract

Herpes simplex virus-type 2 (HSV-2) is the main cause of genital herpes worldwide including Thailand and it affects the infected individual and public health. HSVs have long been studied not only for their pathology and associated diseases but also as a model system for molecular processes and as tools for investigating important cellular regulatory proteins and pathways. Many studies have provided important information insights into our understanding. In this study, the total protein expression of African monkey kidney cell or Vero cell during early stage of HSV-2 infection was investigated using proteomic approaches. Firstly, the different protein extraction buffers were compared in term of their reproducibility and representation in the global proteome. Three types of extraction buffers were used which consisted of CHAPS-Urea-Thiourea buffer, Nonidet P-40 (NP-40) buffer and NP-40-Urea-Thiourea buffer. Total proteins from each of the extraction buffer were separated by 12.5% SDS-PAGE to determine the range of protein molecular weight (MW) and protein patterns, and followed by 2-dimensional gel electrophoresis (2-DE) to investigate the reproducibility and reliability of the protein extraction buffers. It was found that CHAPS-Urea-Thiourea buffer was the suitable extraction buffer giving the observed proteins with molecular weight between 30 and 97 kDa with total of 531 spots. Secondly, the different growth media with supplemented serum were compared in term of cell growth and cell proliferation. Three types of serum were used which consisted of iron supplemented calf serum, bovine calf serum and fetal bovine serum. It was found that fetal bovine serum was the suitable supplement serum for Vero cell growth during HSV-2 infection. The levels of total protein expression in Vero cell during early stage of HSV-2 infection were subsequently studied using 2-DE. The 2-DE gels were analyzed by ImageMasterTM 2D Platinum software version 5.0. It was revealed that thirty-nine protein spots were changed in expression levels at least 1.2 folds during HSV-2 infection. Fourteen protein spots were up-regulated and twenty-five protein spots were down-regulated. These proteins had pI between 4.44 and 7.24 with MW ranging from 15.3 to 98.5 kDa. The 26 protein spots were further analyzed by LC-MS/MS. The peptide mass fingerprints (PMF) were used for protein identification using NCBI database with MASCOT program. It was revealed that only eight protein spots were identified. There were vimentin, reticulocalbin-1, annexin A4, malate dehydrogenase, 14-3-3 protein beta/alpha protein, ubiquitin thioesterase L1 and 6-phosphogluconolactonase.