Abstract: 【Objective】 BHK-21 cell line stably expressing T7 RNAP was constructed by establishing a lentivirus vector system overexpressing T7 RNA polymerase (T7 RNAP).【Method】 In this study,T7 RNAP gene was amplified using the primers designed according to Escherichia coli BL21 (DE3) genome sequence published in GenBank (accession No.:NZ_CP081489.1),and then cloned into lentiviral vector to obtain the recombinant plasmid pCDH-CMV-T7 RNAP.After screening positive clones and sequencing identification,the packaging system and auxiliary packaging plasmid containing recombinant plasmid pCDH-CMV-T7 RNAP were co-transfected into HEK-293T cells by liposome,and the lentivirus was packaged and purified to obtain a recombinant lentivirus with high virus titer.The collected virus solution was infected with BHK-21 cells with multiplicity of infection (MOI)=1,5,10 and 20 respectively.The fluorescence was observed after 72 h to screen the best MOI.The recombinant lentivirus was infected with BHK-21 cells under the optimal MOI conditions.The positive rate of infected cells was observed by the expression of green fluorescent protein copGFP.4 μg/mL puromycin was used as the resistance screening material for multiple rounds of screening,and finally BHK-21-T7 RNAP cell line was obtained.Further,three pairs of detection primers were designed for RT-PCR detection of the selected cell lines,and the ability of T7 RNAP to drive the downstream gene of T7 promoter was reflected by detecting the activity difference of sea kidney luciferase (hRluc) reporter gene between test and blank control groups.【Result】 T7 RNAP gene in the genome of Escherichia coli BL21 (DE3) cells was successfully amplified,the recombinant lentivirus vector was successfully constructed,and the titer of the recombinant lentivirus was 1.0×10⁸ TU/mL.BHK-21-T7 RNAP cell line was successfully screened.The specific DNA sequence of T7 RNAP could be detected by RT-PCR.After transfection of plasmid containing T7 promoter driven hRluc (pT7-hRluc) into this cell line,it was found that the activity of hRluc in BHK-21-T7 RNAP cell line was extremely significantly higher than that blank control BHK-21 cells (P<0.01).【Conclusion】 In this study,BHK-21 cell lines stably expressing T7 RNAP were obtained,and T7 RNAP in the selected BHK-21-T7 RNAP cell lines could drive the transcription of the downstream gene hRluc of pT7 promoter.The results laid a cellular foundation for the establishment of RNA virus reverse genetics platform.

Key words: T7 RNA polymerase; lentiviral vector; cell strain; stable expression