The Eukaryotic Expression of HIV-1 Rev, Its Effect on the Lytic Cycle Replication of KSHV and the Relationship between KSHV and Prostate Cancer Cells
|School||Nanjing Medical University|
|Keywords||Human immunodeficiency virus type 1 Virus particle protein regulatory factors Human herpes virus type 8 Copy Infection|
Human immunodeficiency virus type 1 ( human as immunodeficiency or virus type 1 , of HIV-1 ) infection significantly with the increase of Kaposi's sarcoma -related herpes virus ( Kaposi's 's sarcoma cells in - associated herpesvirus , KSHV ) infection by occurrence of Kaposi economic sarcoma ( Kaposi's 's sarcoma cells in , KS ) of risk. KSHV infection is a necessary condition of KS occurred , but enough to trigger there is no when other cofactors role is KS, promote KSHV triggered KS factors , however , are not completely understood . Before our research has confirmed HIV-1 - trans- activating protein (transactivative transcription protein, Tat) is activated to KSHV lytic cycle replication factors . This paper plasmid containing the HIV-1 virus particle protein expression regulatory factor (regulator of virion protein expression, Rev) gene encoding the prev - Flag recombinant plasmids and restriction enzyme digestion and sequence determination ; prev- Flag recombinant plasmids were transiently transfected primary exudative lymphoma [primary effusion lymphoma, PEL, also known as body cavity lymphomas (body cavity-based lymphoma, BCBL)] sources BCBL-1 cells and mouse embryonic fibroblasts NIH/3T3 by RT - PCR , Western blot were detected from the mRNA and protein levels of the Rev gene expression ; BCBL-1 cell total RNA extract transiently transfected with the recombinant plasmid of pRev-Flag , Real-time PCR detection of KSHV minor capsid protein coding genes ORF26 KSHV cycles the copy switch gene ORF50 mRNA and RT-PCR detection the ORF26 mRNA transcription level ; BCBL - 1 cells and PC-3 culture change in cell morphology observed cultured PC-3 cells , RT-PCR was used to detect the expression of KSHV ORF26 . The nucleic acid sequence analysis showed that the cloned the Rev gene sequences in GenBank registered Rev sequence 100% homologous RT-PCR and Western blot analysis detected a specific band of in the Rev expected position . Real-time PCR and RT-PCR analysis showed that the Rev gene encodes a protein can reduce KSHV transcriptional level . The cells were cultured PC-3 cell shape change , RT-PCR detected ORF26 expression . This study successfully constructed a recombinant plasmid containing the sequence of Rev gene and obtain the correct expression in eukaryotic cells BCBL-1 and NIH/3T3 cells ; preliminary exploration showed that the the Rev gene encoding protein can inhibit KSHV lytic cycle replication ; KSHV can infect prostate cancer .