Xia Yun, Shi Ying, Gao Yan, Guo Ji-Qiang, Song Xiang-Feng, Wang Hui
Research Center for Immunology, Xinxiang Medical College, Xinxiang 453003, Henan Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2008 Dec;16(6):1425-9.
This study was aimed to establish the cell lines of human T-cell leukemia virus type-1 (HTLV-1) tax gene expression and their biological activity. The eukaryotic vector pCMV-tax and pCMV-neo-Bam were transfected into Jurkat E6-1 by using liposome, following screening with G418, the tax gene expression cell line-TaxP and the negative cell lines-TaxN were selected and enriched. Then, the mRNA expressions of LAT, SLP70, ZAP70 and NF-kappaB (p65) were measured by using RT-PCR; the NF-kappaB bioactivity was tested after transfecting the pNF-kappaB-Luc plasmids into TaxP and TaxN cells; meanwhile CD25, CD69 expressions on surface of the two cell lines were tested by flow cytometry. The results showed that TaxP and TaxN cell lines were established and the tax gene expression was detected; as compared with TaxN, the mRNA expressions of LAT, SLP70 and ZAP70 in TaxP were enhanced (p<0.05), while the NF-kappaB bioactivity in TaxP cells was stronger than that in TaxN cells (p<0.01). The CD25, CD69 on TaxP cells surface was highly expressed. It is concluded that TaxP and TaxN cell lines are established, TAX protein could promote the activation of T cells and activate the NF-kappaB pathway.
本研究旨在建立人T细胞白血病病毒1型(HTLV-1)tax基因表达细胞系及其生物学活性。采用脂质体将真核载体pCMV-tax和pCMV-neo-Bam转染至Jurkat E6-1细胞,经G418筛选,挑选并富集tax基因表达细胞系TaxP和阴性细胞系TaxN。然后,采用RT-PCR检测LAT、SLP70、ZAP70和NF-κB(p65)的mRNA表达;将pNF-κB-Luc质粒转染至TaxP和TaxN细胞后检测NF-κB生物活性;同时,通过流式细胞术检测两种细胞系表面CD25、CD69的表达。结果显示,成功建立TaxP和TaxN细胞系并检测到tax基因表达;与TaxN相比,TaxP中LAT、SLP70和ZAP70的mRNA表达增强(p<0.05),而TaxP细胞中的NF-κB生物活性强于TaxN细胞(p<0.01)。TaxP细胞表面的CD25、CD69高表达。结论:成功建立TaxP和TaxN细胞系,TAX蛋白可促进T细胞活化并激活NF-κB通路。