Objective: To develop an effective multi-genes pseudovirus model as positive control for detecting exogenous RNA virus (CV2117, HAV and HTLV-2) of biological products. Methods: Three-plasmid lentivirus packaging system, including enveloped plasmid (pMD2.G), packaging plasmid (psPAX2) and shuttle plasmid (pWPXL-EGFP), was used. Shuttle plasmid containing six gene fragments was synthesized and named as pCHH-EGFP, which was containing CV2117, HAV and HTLV-2 gene fragments. The mass ratio of enveloped plasmid-packaging plasmid-shuttle plasmid (1 ∶ 3 ∶ 4) was used to transfect into HEK293T/17 cells and fresh complete media was changed at 5 h after transfection. The nuclease was added into transfected cell culture at 24 h after transfection. Virus supernatant was collected at 48 h and 72 h after transfection. Virus supernatant was diluted 10 times with PBS for infecting HEK293T/17 cells. Fresh media was changed at 24 h after infection and virus infection titer was evaluated by fluorescence microscope, flow cytometry at 72 h post-infection. Reverse transcription quantitative real-time PCR (RT-qPCR) method was used to detect the gene copy number of pseudovirus as following: ①pretreatment at 42 ℃ for 2 min; ②reverse transcription at 25 ℃ for 5 min, 1 cycle; 42 ℃ for 30 min, 1 cycle;85 ℃ for 5 min, 1 cycle; ③PCR amplification at 37 ℃ for 2 min, 1 cycle; predenaturation at 95 ℃ for 10 min,1 cycle; denaturation at 95 ℃ for 15 s, annealea at 60 ℃ for 1 min, 40 cycles. The pseudoviruses were tested for sample suitability with human mesenchymal stem cell samples. Results: The three-plasmid lentivirus packaging system was used to transfect into HEK 293T/17 cells and the enhanced green fluorescent protein (EGFP) positive cells was more than 90%. The residual plasmid DNA in pCHH-EGFP pseudovirus was less than 0.005%. The EGFP positive cells of pCHH-EGFP lentivirus was 8.06% by flow cytometry, and the virus infection titer was 6.21×105 TU · mL-1. The copy number of the target gene was detected by RT-qPCR, the copy number of CV2117-1, CV2117-2, HAV-1, HAV-2, HTLV-2-1 and HTLV-2-2 were 2.99×104 copies · μL-1, 1.50×104 copies · μL-1, 2.40×104 copies · μL-1, 1.09×104 copies · μL-1, 2.03×104 copies · μL-1 and 1.92×104 copies · μL-1, respectively. The results of hMSCs sample suitability showed that lentivirus vector CV2117-HAV-HTLV2 (LV-CHH) pseudovirus could be used as the positive control, and the recovery rate was 70%-130%. Conclusion: The lentivirus pseudovirus, LV-CHH, containing multiple RNA viral gene fragments was constructed successfully. It can be used as a positive control for PCR detection of exogenous virus in stem cell products.
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