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目的:初步探索人类免疫缺陷病毒1型(HIV-1)反式激活蛋白(Tat)对卡波济肉瘤相关疱疹病毒(KSHV)感染人神经母细胞瘤细胞系SK-N-SH细胞(SK细胞)的影响。方法:将原发性渗出性淋巴瘤(PEL)细胞系BCBL-1细胞与SK细胞共培养,观察SK细胞的细胞病变效应(CPE)并采用Real-timePCR检测KSHV在SK细胞中的复制。重组质粒Tat+pcDNA3.1(+)和空载体pcDNA3.1(+)分别转染SK细胞,经G418筛选获得抗性克隆,以RT-PCR和Westernblot检测Tat基因的表达。通过BCBL-1细胞与SK-Tat/SK-vector细胞共培养,采用Real-timePCR检测SK-Tat/SK-vector细胞中KSHV裂解期基因ORF50和26的mRNA表达。结果:细胞共培养后,SK细胞出现CPE且SK细胞中检测到KSHV的基因。RT-PCR和Westernblot均在Tat预期位置检测到特异性条带。Real-timePCR检测显示Tat降低KSHVORF50和26mRNA转录水平。结论:初步探索表明,在细胞共培养过程中Tat蛋白抑制KSHV在SK细胞中的复制。
OBJECTIVE: To investigate the effect of HIV-1 transactivator (Tat) on the proliferation of human neuroblastoma SK-N-SH cells (SK cells) infected with Kaposi’s sarcoma-associated herpesvirus (KSHV) )Impact. Methods: BCL-1 cells of primary exudative lymphoma (PEL) cells were co-cultured with SK cells. The cytopathic effect (CPE) of SK cells was observed and the replication of KSHV in SK cells was detected by Real-time PCR. The recombinant plasmid Tat + pcDNA3.1 (+) and empty vector pcDNA3.1 (+) were transfected into SK cells respectively. The resistant clones were screened by G418. The expression of Tat gene was detected by RT-PCR and Western blot. The BCL-1 cells were co-cultured with SK-Tat / SK-vector cells and Real-time PCR was used to detect the mRNA expression of KSHV lytic stage genes ORFs 50 and 26 in SK-Tat / SK-vector cells. RESULTS: After co-culture of cells, CPE was found in SK cells and KSHV was detected in SK cells. Specific bands were detected in the expected sites of Tat by both RT-PCR and Western blot. Real-time PCR showed that Tat reduced KSHVORF50 and 26 mRNA transcription levels. CONCLUSIONS: Preliminary exploration indicates that Tat protein inhibits the replication of KSHV in SK cells during cell co-culture.