重组慢病毒载体介导KSHV分子开关蛋白Rta在BCBL1细胞中的表达及其功能鉴定

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Abstract Objective: To construct the recombinant lentivirus carrying Kaposi′s sarcoma-associated herpesvirus (KSHV) replication and transcriptional activator（Rta） gene. Methods: The fragment of Rta gene from expression vector pcDNA3.1-Rta was cloned into the lentivirus vector pHAGE-CMV-MCS-IzsGreen, then the recombinant vector pHAGE-Rta, packaging vector psPAX2 and envelope vector pMD2.G were cotransfected into the 293 T cells. Filtered culture media were harvested and the viral titer was checked by observing the expression of green fluorescent protein (GFP). BCBL-1 cells were infected with the LentivirusRta of which multiplicity of infection (MOI) was 1.0, 5.0 and 10.0, respectively, and Rta protein was detected by western blotting; meanwhile, the expression of KSHV lytic protein viral interleukin-6 (vIL-6) and the production of KSHV virion were examined by western blotting and virion release assay, respectively. Results: Recombinant lentivirus with high titer and efficient infection were obtained by the lentivirus vectors system. BCBL-1 cells could be efficiently infected, and the expression of Rta protein in BCBL-1 cells infected with Lentivirus-Rta was detectable. The expression of Rta could promote KSHV lytic replication,upregulate the expression of vIL-6 protein and the production of KSHV virion. Conclusion: The recombinant lentivirus vector carrying Rta gene was constructed successfully; and the expression of Rta in BCBL-1 cells infected with Lentivirus-Rta could reactivate KSHV from latency successfully.

Received: 19 October 2013

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