Construction and expression of a Rev-dependent TNF-R1 expressing HIV-infected-cell injurious vectors

Construction and expression of a Rev-dependent TNF-R1 expressing HIV-infected-cell injurious

vectors

SHI Wei-min;Dean Baylis;Damian Purcell;Paul U Cameron

【期刊名称】《中华医学杂志（英文版）》 【年(卷),期】2005(118)024

【摘要】Background Rev is necessary for exporting unspliced and incompletely spliced intron containing HIV mRNAs and for HIV replication. The aim of this study is to develop a kind of selective suicide construct that can specifically and directly induce HIV infected cells into apoptosis based on the high affinity of Rev and Rev response element (RRE). Methods Molecular-cloning technique was used to synthesis Rev dependent TNF-R1 expression construct pDM128-TNF-R1 (pT128) that contains RRE and TNFR1 gene. Restriction digestion, Polymerase Chain Reaction (PCR) and DNA sequencing were processed and the exactness and correctness of the inserted TNF-R1 gene in pT128 were confirmed repeatedly. The expression of pT128 co-transfected with different combination of other plasmids by calcium phosphate-DNA co-precipitation in Helas and by gene gun transfection in keratinocytes was further tested by flow-cytometry and cell counted under microscope. Results The new plasmid specifically expressed TNF-R1 in Helas when co-transfected with pRev but did not when without pRev. Indirect