摘要
为建立稳定表达马TRIM5α(eqTRIM5α)的HEK293细胞系,并检测马TRIM5α(eqTRIM5α)的抗病毒功能,本研究将eqTRIM5α克隆于慢病毒表达载体pLPCX中,构建重组质粒pLPCX-eqTRIM5α-HA,同时构建恒河猴的pLPCX-Rh TRIM5α-HA作为阳性对照。利用VSV-G包装慢病毒,感染HEK293细胞,并利用嘌呤霉素筛选、纯化,建立了稳定表达eqTRIM5α和Rh TRIM5α的HEK293细胞系。经western blot和流式细胞术鉴定,该细胞系传代至20代后,仍然能够检测到eqTRIM5α和RhTRIM5α的稳定表达。将该细胞系应用于抗马传染性贫血病毒(EIAV)活性研究中,结果显示eqTRIM5α同RhTRIM5α一样均能够抑制病毒复制,表明eqTRIM5α是一种对EIAV具有较强限制作用的宿主蛋白。该细胞系的建立为进一步评价和研究eqTRIM5α的抗病毒作用和机制奠定了基础。
To establish HEK293 cell lines stably expressing equine TRIM5α（eq TRIM5α） and evaluate the antiviral activity of eq TRIM5α, the eq TRIM5α and rhesus monkey（Rh TRIM5α） genes were cloned to lentiviral expressing plasmid p LPCX,respectively. The recombinant plasmid, p VSV-G and pc GP were cotransfected into HEK293 T cells in order to package pseudovirion. The pseudovirion was used to infect HEK293 cells and cell lines stably expressing eq TRIM5α and Rh TRIM5α（HEK293-eq TRIM5α and HEK293-Rh TRIM5α） which were generated using colony-purification in present of puromycin. The cell lines expressing eq TRIM5α or Rh TRIM5α were confirmed by western blot and flow cytometry, indicating that eq TRIM5α and Rh TRIM5α were stably expressed in the HEK293-eq TRIM5α and HEK293-Rh TRIM5α cell lines. Furthermore, the cell lines were used to test retroviral restriction, it showed that the equine infectious anemia virus（EIAV） infection was inhibited by the eq TRIM5α. These data demonstrated that the established cell lines would facilitate future study on restriction mechanism of EIAV infection.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2016年第4期294-297,311,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(31222054)
中国博士后科学基金项目(2015M581223)
