Composition for recovering measles virus, and kit, purpose and method thereof
A measles virus and composition technology, applied in antiviral agents, biochemical equipment and methods, virus/bacteriophage, etc., can solve problems such as difficulty in rescue, complicated rescue system and method, and reduce homology and excellent virus The effect of the rescue effect
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Embodiment 1
[0067] Embodiment 1: Construction of transcription vector and expression vector
[0068] 1. Nucleic acid sequence mutation modification
[0069] The original sequence of the vector plasmid pVAX1 (purchased from Invitrogen, with a CMV promoter) was modified, and the 34th base T mutation was designed to be G, and the 50th base A mutation was designed to be T, thereby eliminating the MluI enzyme Cutting site and SpeI restriction site, and its 725th-730th base sequence AGCTCG mutation is designed as ACGCGT, so that the multiple cloning site of the vector plasmid has a MluI restriction site. The designed vector plasmid sequence (produced by Shanghai Jierui Bioengineering Co., Ltd.) was synthesized and transformed, and it was named pVAXm. The carrier plasmid sequence was sequenced and identified (by Takara Company), and the obtained sequence is shown in SEQ ID No.: 1 in the sequence listing.
[0070] The hammerhead ribozyme sequence and hepatitis D virus ribozyme sequence were res...
Embodiment 2
[0084] Example 2: Screening for suitable host cells
[0085] 1. Cell Transfection
[0086] Vero cells (purchased from Shanghai Cell Resource Center) and 293T cells (purchased from Shanghai Cell Resource Center) were subcultured at 1:1, respectively, and 5×10 5 Cells were plated in the wells of a 6-well plate; BHK-21 cells (purchased from Shanghai Cell Resource Center) were subcultured at 1:2, and 5×10 5cells / well were plated in 6-well plates. When the cells in the six-well plate were fused to a density of 80-90%, the three cell lines were transfected with three expression vectors pVAXm-N, pVAXm-P and pVAXm-L, as follows:
[0087] Solution 1: Dilute 10 μl / well transfection agent (lipofectamine 2000, available from Invitrogen) with 240 μl / well serum-free medium, and mix gently at room temperature for 5 minutes; Solution 2: Dilute with 10 μl / well serum-free medium 4 μg / well expression vector; mix solution 1 with solution 2 and let stand at room temperature for 20 minutes. At ...
Embodiment 3
[0091] Example 3: Rescue of measles virus
[0092] 1. Co-transfection
[0093] Put the sterilized coverslips into the wells of the six-well culture plate, then add 2mLoptimem (available from GIBCO) medium, and add 8×10 5 293T cells were plated in the wells of a 6-well plate at 37°C, 5% CO 2 cultured overnight. Afterwards, co-transfect the cells with the transcription vector constructed above and the three expression vectors, as follows:
[0094] After mixing the transfection agent (lipofectamine 2000, which can be purchased from Invitrogen Company), 10 μl / well was pipetted, diluted with 100 μl optimem (GIBCO) medium, and allowed to stand at room temperature for 5 minutes. Afterwards, the four vectors were diluted with 100 μl of optimem (GIBCO) medium, and the proportions of the four vectors are shown in Table 1 (Groups 1-11). After mixing them with the diluted transfection agent, they were left at room temperature for 20 minutes. Add 500 μl / well of the above mixture dropwi...
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