A kind of autologous hematopoietic stem cell modified by hbb fusion gene, preparation method and application thereof
A technology of hematopoietic stem cells and fusion genes, applied to genetically modified cells, cells modified by introducing foreign genetic material, botanical equipment and methods, etc., can solve the problems of lack of hypersensitive sites and low expression of hemoglobin
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Embodiment 1
[0032] Example 1 Construction of recombinant expression vector pCDH-HBB
[0033] Comments on HBB fusion gene module figure 1 (See Appendix SEQ ID NO. 1 for the complete nucleic acid sequence).
[0034] The sequence of each module of HBB fusion gene
[0035] (1) DNase I hypersensitive site HS2 (SEQ ID NO.2)
[0036] (2) DNase I hypersensitive site HS3 (SEQ ID NO.3)
[0037] (3) DNase I hypersensitive site HS4 (SEQ ID NO.4)
[0038] (4) Promoter (SEQ ID NO.5)
[0039] (5) Enhancer (SEQ ID NO.6)
[0040] (6) HSb T87Q (SEQ ID NO.7)
[0041] Above-mentioned DNase I hypersensitive sites HS2-HS4 are core sequences cut from full-length sequences;
[0042] The present application has carried out codon optimization on the nucleotide sequence of HSb T87Q;
[0043] According to the above SEQ ID NO.1 sequence, Shanghai Jierui Bioengineering Co., Ltd. was entrusted to synthesize its entire expression cassette, inserted into the pCDH vector (provided by the Chinese Center for Disease...
Embodiment 2
[0046] Example 2 Packaging and titer determination of lentivirus
[0047] 1) Recovery of packaging cell lines
[0048] The packaging cell line used in the present invention is 293T cells. Take out the cryopreserved 293T cells from the liquid nitrogen tank, quickly throw them into a 37°C water bath and shake quickly, and try to dissolve the cell solution completely within 2 min. Transfer 1 mL of cell solution to a 50 mL centrifuge tube, add 35 mL of normal saline to wash DMSO, mix well and centrifuge under the following conditions: 1500 rpm, 5 min. Remove the supernatant, add 5 mL of fresh high-glucose DMEM (containing 10 Vol% FBS) medium to resuspend the cells, and transfer them to T75 flasks. Continue adding high-glucose DMEM (containing 10 Vol% FBS) medium to each flask. volume to 10mL. Put the culture flask steadily at 37°C, 5% CO2 cultured in an incubator. Cell viability was observed the next day, and the medium was changed. The cell growth was observed every day ther...
Embodiment 3
[0055] Example 3 Modification of CD34 by recombinant lentivirus + Preparation of cells
[0056] 1. CD34 + Cell sorting and culture
[0057] Before single-cell collection, patients took the drug Filgrastim, which stimulates the production of cells in the body, and single cells were collected on day 5. The collected cells were sorted by magnetic beads using the CD34 MicroBead Kit provided by Miltenyi to sort CD34 + cells, for cell counting, the concentration was 5 x 10 5 cells / mL of cell culture medium were inoculated into six-well plates, supplemented with 2 mL of CD34 expansion medium in each well, and placed at 37°C in 5% CO. 2 Incubator for 2 days. Six-well plate pre-used with 5µg / cm 2 The Fibronectin (purchased from Gibco) was placed in a 37°C incubator for 6 h, and the CD34 expansion medium was StemSpan SFEMMedium (purchased from STEMCELL Technologies), containing 20ng / mL SCF, 200ng / mL Flt-3L and 100ng / mL TPO ( were purchased from MCE Corporation).
[0058] 2. Infe...
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