CMV envelope protein packaging lentiviral vector and application thereof
A technology of lentiviral vector and envelope protein, applied in the direction of virus/phage, virus, vector, etc., can solve the problem of ineffective transfection of NK cells, and achieve stable transfection function, high virus titer, and high-efficiency transfection Effect
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Embodiment 1
[0040] Embodiment 1: Plasmid vector construction
[0041] Snapgene software was used to analyze the restriction sites of all cloned DNA, and then CMV-G and other sequences were inserted into a complete new expression plasmid designed and constructed using conventional molecular cloning techniques in the field. Each gene sequence was amplified by high-fidelity PCR (Phanta Super-Fidelity DNA Polymeras, Nanjing Vazyme Company) or obtained from plasmid digestion. Among them, the amplification primers were synthesized by Suzhou Synbio (see Table 1 below), and the PCR reaction system is shown in Table 2 below.
[0042] Table 1. Primers used to construct vectors
[0043]
[0044]
[0045] Table 2. PCR reaction system
[0046] Reagent Added volume (μL) 5×Buffer 10 5xdNTP 4 10nM F primer 2 10 nM R primer 2 Phanta Polymeras (Vazyme) 0.5 total capacity 50
[0047] 1. Construction of MSCV-IRES-BleoR vector
[0048] 1. Using pTr...
Embodiment 2
[0075] Example 2: Viral packaging
[0076] The lentiviral vector is pLL3.7 (Addgene), which contains eGFP and can be used as a marker for transfection success. The traditional fake virus packaging process is attached image 3 , the pseudovirus packaging process of the present invention is shown in the attached Figure 4 . The traditional lentivirus system (i.e. VSV-G membrane protein packaging) pLL3.7 and packaging plasmids (pLP1, pLP2, pLP-VSVG) transfected 293T cells; and the lentivirus system of the present invention (CMV-G packaging) pLL3.7 and 293T packaging cells expressing gL / gO were transfected with packaging plasmids (pLP1, pLP2, pgH).
[0077] The specific steps were according to the instructions of Invitrogen, when the virus was packaged: 9 μg of packaging plasmid, 3 μg of vector plasmid, and the CPT high-efficiency transfection reagent was purchased from Wuhan Vinosai Biotechnology Company. The day before transfection, inoculate 5×10 6 293T cells / 100mm Petri d...
experiment example 1
[0078] Experimental Example 1: Determination of Virus Titer
[0079] After digestion and counting of 293T cells in good growth state, spread 24-well plate, 1×10 per well 5 cells. On the second day, the virus was diluted in a 10-fold gradient, and five consecutive dilutions were made and then put into the cells prepared above. On the third day, additional culture solution was added, and 500 μL of complete culture solution was added to each well. On the fifth day, the cells were collected, and the flow cytometer was loaded to detect the proportion of GFP-positive cells. Virus titer (TU / mL) = (10 5 Cell number×GFP+%)×1000×dilution factor.
[0080] Calculate virus titer, wherein, the titer of lentivirus CMV-G pseudovirus of the present invention is 6.8 * 106 TU / mL, the titer of traditional lentivirus VSV-G pseudovirus is 8×10 6 TU / mL shows that the transfection efficiency of the CMV-G pseudovirus of the present invention is significantly better than that of the VSV-G pseudovi...
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