pCAGGS-SIVct/HN, pCAGGS-SIVct+HN BioVector NTCC质粒载体菌种细胞基因保藏中心

pCAGGS-SIVct/HN, pCAGGS-SIVct+HN

293T cells were plated in a 6-well plastic culture plate at a cell density of 5×105 cells/well, and then incubated under 10% CO2 at 37° C. for 48 hours. The culture medium was changed with 800 μl/well of DMEM containing 1% bovine serum albumin. The cells were then used in transfection. In the combinations as indicated in Table 6 below, 1200 ng of gene transfer vector (pGL3C/CMVL.U3G2/RREc/s/CMVF EGFP/3LTRΔU3), 360 ng of packaging vector (pCAGGS/SIVagm gag-tat/rev), Sendai virus HN, F protein expression plasmid pCAGGS-SIVct/HN, pCAGGS-SIVct+HN, pCAGGS-Fct4, pCAGGS-Fct14, pCAGGS-Fct27, pCAGGS-Fct4/SIVct11, pCAGGS-Fct14/SIVct11, and pCAGGS-Fct27/SIVct11 were dissolved in 100 μl of Opti MEM (Gibco BRL) in each well. Then, 6 μl of PLUS Reagent (Gibco BRL) was added to the well. The mixture was stirred, and then allowed to stand still at room temperature for 15 minutes. A solution obtained by diluting 4 μl of LIPOFECTAMINE Reagent (Gibco BRL) with 100 μl of Opti MEM was added to the mixture. The mixed solution was stirred, and then allowed to stand still at room temperature for another 15 minutes. The resulting mixture was added dropwise to the 293 T cells prepared above while being stirred gently. The cells were then incubated under 10% CO2 at 37° C. for 3 hours. 1 ml of DMEM containing 1% bovine serum albumin and 15 μg/ml trypsin (Gibco BRL) was added to each well. After culture under 10% CO2 at 37° C. for 16 to 24 hours, the culture medium in each well was changed with 2 ml of DMEM containing 1% bovine serum albumin and 7.5 μg/ml trypsin (Gibco BRL). After subsequently culturing for 24 hours, culture supernatant was collected. This supernatant was then filtered with a filter having a 0.45 μm diameter and the resulting solution was used as a vector solution.

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