Lentiviral vector, construction method and application thereof
A technology of lentiviral vectors and viruses, applied in lentiviral vectors, construction methods and their application fields, can solve the problems of low titer of lentiviruses and achieve broad application prospects
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Embodiment 1
[0048] The present invention uses LTR1.27-GEW as the backbone to construct a series of vectors, such as figure 2 shown. Among them, it is speculated that the reverse transcription process of Obio-01 is divided into image 3 as shown, image 3 Middle: RNA genome refers to "RNA genome", Minus strand strongstop synthesis refers to "negative strand strong stop synthesis", Minus strand transfer refers to "negative strand jump", Minusstrand extension refers to "negative strand extension", Plus strand strong stop synthesis refers to " Plus strand strong termination synthesis", Plus strand transfer refers to "positive strand jump", dsDNA synthesis refers to "DNA synthesis". The plasmid map of LTR1.27-GEW is as follows Figure 4 shown. The plasmid maps of Obio-01, Obio-03, and Obio-04 are as follows Figure 5-7 shown. The nucleotide sequences of Obio-01, Obio-03 and Obio-04 are respectively shown in SEQ ID NO: 2-4. The nucleotide sequence based on LTR1.27-GEW is shown in SEQ ID...
Embodiment 2
[0054] This embodiment discloses a method for virus purification, specifically including:
[0055] After 48 hours of transfection, collect the virus for the first time, collect the medium into a 50ml centrifuge tube, and replace the cells with fresh complete medium. After 72 hours of transfection, collect the virus for the second time, collect the medium into a 50ml centrifuge tube, and discard the cells.
[0056] Centrifuge the collected culture supernatant with a centrifuge at 3500rpm for 10min at room temperature, and pour the supernatant into a new 50ml centrifuge tube; centrifuge at 30,000rpm at 4°C for 2h with an ultracentrifuge, carefully discard the supernatant, and centrifuge the tube Put it upside down on sterilized absorbent paper, add DPBS to resuspend the precipitate, collect it into a 1.5ml EP tube, and store it in a -80°C refrigerator.
Embodiment 3
[0058] This embodiment discloses a titer detection method, which specifically includes:
[0059] The real time quantitative PCR method was used to determine the lentivirus titer, and the specific steps were as follows:
[0060] Preparation of samples: Press 1×10 5 Cells were seeded with 293T cells in each well of a 24-well plate; the virus was added the next day, and the fresh medium was replaced 12-20 hours later; 72 hours after infection, photos were taken to record the fluorescence, and then the cells were harvested to extract genomic DNA and perform quantitative PCR experiments to determine the titer.
[0061] Real-time PCR was done on ABI7500 instrument. Reagents SYBR Master Mixture from TAKARA were used.
[0062] 1. Configure the reaction system according to the following ratio:
[0063] SYBR premix ex taq: 10μl;
[0064] ROX: 0.4 μl;
[0065] Upstream primer (25 μM): 0.5 μl;
[0066] Downstream primer (25 μM): 0.5 μl;
[0067] Genomic DNA: 2.0μl;
[0068] 6.6 μl...
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