LI Jian-bin, MI Zhi-qiang, AN Xiao-ping, TAN Li, CHEN Bin, WANG Xiao-na, FAN Hua-hao, ZHANG Wen-hui, ZHANG Bo, FANG Xiang, TONG Yi-gang
China Biotechnology. 2012, 32(09): 48-54.
To screen HIV-1 LTR related host factors by constructing random RNAi library based on the lentiviral vector and HIV-1 LTR reporter cell line in which TK suicide gene can express stably. Methods: Oligo deoxynucleotides were chemically synthesized which formed hairpin structure that contain 19 random bases and were then fused with a linker sequence. The joined product was amplified by PCR and the PCR product was cloned downstream of U6 promoter of a lentiviral vector pLenti-U6 to prepare random shRNA Library. HIV-1 LTR and HSV-TK gene were amplified by PCR and then joined by overlapping PCR. The joined product was cloned into pcDNA3.1 expression vector. The recombinant plasmid obtained was transfected into HEK293 cells, and stable cell lines were selected with G418. Lentiviral particles were pachaged in 293FT cells by transfection with random shRNA library plasmid along with pLP1, pLP2 and VSV-G, using Lipofectamine 2000. HEK293/TK cells were plated and infected with enriched random lentivirus shRNA library and then treated by GCV. Results: The surviving cells were collected after about two months, and genomic DNAs were extracted from the surviving cells to amplify integrated shRNAs by nested PCR. After verifying the validity of the interference sequence by Western blotting, a clone was isolated that expressed shRNA which inhibited the expression of TK gene. The shRNA sequence was determined by sequencing, and this sequence is likely to inhibit a host factor required for HIV-1 LTR function. Conclusion: By this strategy, it is possible to identify host factors assistant HIV-1 LTR, which could be potential host cellular targets for preparing novel anti-HIV drug.
