Globin gene dual-expression lentivirus vector and application thereof
A technology for expressing vectors and viral vectors, applied in the direction of viruses/bacteriophages, viruses, vectors, etc., to achieve the effects of avoiding homologous recombination, high-efficiency expression, and reducing homology
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Embodiment 1
[0078] The design of embodiment 1HBB and HBG2 fusion gene
[0079] According to the amino acid sequence of the wild-type HBB protein and the translation usage frequency of codons in the human proteome, redesign the coding sequence of the HBB protein, optimize the secondary structure of the 5'UTR, and remove minor cis-elements such as 3-HS, HBB - the enhancer of intron No. 2, etc., to obtain the first HBB expression cassette as shown in SEQ ID NO: 2, comprising a total of 50bp 5'-UTR and 441bp ORF;
[0080] Taking the first HBB expression cassette as the comparison object, mutating the wild-type HBB gene base by base, reducing the homology with the first HBB expression cassette, and obtaining the second HBB expression cassette as shown in SEQ ID NO:3;
[0081] The HBG2 expression cassette was initially optimized according to the codon usage frequency to obtain the first HBG2 expression cassette as shown in SEQ ID NO:4;
[0082] Optimize the 147 amino acids encoded by the ORF o...
Embodiment 2
[0086] The preparation of embodiment 2 lentiviral vectors
[0087] Artificially synthesized 1stHBB-P2A-T2A-2ndHBB, 1stHBB-P2A-T2A-1stHBG2, 1stHBB-P2A-T2A-2ndHBG2 fusion genes, and cloned HS2, HS3 and HS4 as shown in SEQ ID NO:6-8 from the genome , the HBB promoter βpro shown in SEQ ID NO: 9, the HBB polyA terminator shown in SEQ ID NO: 10, assembled by restriction endonuclease ligation and homologous recombination to obtain a complete globin gene double expression module, wherein, The full length of 1stHBB-P2A-T2A-2ndHBG2 is about 3.5kb, which saves about 2kb load compared with BB305. It is reverse loaded into the lentiviral vector and constructed as follows: figure 2 The indicated HBB and HBG2 double expression lentiviral vectors.
Embodiment 3
[0088] Example 3 Preparation of recombinant lentivirus
[0089] In this example, 293T cells were used to prepare recombinant lentivirus, and when the 293T cells spread to 80-90% of the bottom of a 100mm culture dish, the lentivirus was packaged:
[0090] 2 hours before virus packaging, replace the medium with DMEM containing 1% fetal bovine serum, and add 6mL / 100mm culture dish;
[0091] Prepare the plasmid mixture shown in Table 1:
[0092] Table 1
[0093] Reagent dose globin expression plasmid 7μg psPAX2 7μg pVSVG-Rev 7μg
[0094] Expression plasmids include: lentiviral vectors expressing 1stHBB-P2A-T2A-2ndHBB, lentiviral vectors expressing 1stHBB-P2A-T2A-1stHBG2, or lentiviral vectors expressing 1stHBB-P2A-T2A-2ndHBG2. Add to 500μL opti-MEM medium, mix well;
[0095] Add 36 μg PEI to another 500 μL opti-MEM medium, mix well, and let stand at room temperature for 5 minutes;
[0096] Mix the plasmid with PEI, mix well by pipetting, a...
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