Nucleotides, recombinant vector comprising nucleotides, cell, composition, and application of recombinant vector, cell and composition
A technology of recombinant vectors and nucleotides, which can be applied to the introduction of foreign genetic material using vectors, cells modified by the introduction of foreign genetic material, and drug combinations, which can solve problems such as heavy burdens.
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Embodiment 1
[0050] Embodiment 1 chemical synthesis of nucleotides of the present invention
[0051] Handed over to Dalian Bao Biological Co., Ltd. to synthesize SEQ ID Nos.1-19 respectively, and add suitable restriction endonuclease sites Cla I (ATCGAT) and BglII (AGATCT) at both ends of them, and connect to pMD-18T vector above, respectively denoted as pMD-18T-S1, pMD-18T-S2, pMD-18T-S3, pMD-18T-S4, pMD-18T-S5, pMD-18T-S6, pMD-18T-S7, pMD-18T -S8, pMD-18T-S9, pMD-18T-S10, pMD-18T-S11, pMD-18T-S12, pMD-18T-S13, pMD-18T-S14, pMD-18T-S15, pMD-18T-S16 , pMD-18T-S17, pMD-18T-S18 and pMD-18T-S19. After sequencing, the synthetic sequence SEQ ID Nos.1-19 is correct.
Embodiment 2
[0052] Embodiment 2 Construction of recombinant adeno-associated gene transfer vector (hereinafter referred to as AAV)
[0053] 1) pMD-18T-S1 is transformed into competent Escherichia coli DH5a, cultivated overnight on an LB medium plate containing 100 micrograms / ml ampicillin, and the grown positive clones are cultured overnight in an LB liquid medium containing ampicillin, Collect the bacteria by centrifugation, extract pMD-18T-S1 with a plasmid extraction kit, and send the extracted product to the sequencing company for sequencing to confirm that the connected sequence is correct, and store the correct sequencing result at -80°C for future use;
[0054] 2) Use restriction enzymes Cla I (ATCGAT) and BglII (AGATCT) to cut out the fragment of SEQ ID No: 1 from the pMD-18T-S1 vector, and recover the target product by gel electrophoresis of 30ul of each fragment of SEQ ID No: 1 ;
[0055] 3) pAAV-hrGFP (Stratagene Company) was digested with restriction enzymes Cla I (ATCGAT) and ...
Embodiment 3
[0060] Example 3 Mass preparation and concentration of rAAV vectors carrying therapeutic genes
[0061] 3-1. Preparation of rAAV-S1 vector carrying a fragment of SEQ ID No: 1
[0062] Transformed gene transfer vector pAAV-S1, Cap / Rep helper plasmid R 2 C 2 AAV vectors were prepared on the basis of three plasmid systems (stratagene company, AAV Helper-Free System) and adenovirus helper virus (pAd helper). The vector carrying the therapeutic gene is produced in units of 20 15cm petri dishes. Inoculate approximately 10 7 The 293T cells were cultured for 24 hours, and the medium was replaced with OPTI-MEM medium, incubated at 37°C in a 5% carbon dioxide incubator, and prepared for transfection. Prepare the transfection complex for transfection with calcium phosphate transfection reagent. On the next day after transfection, exchange culture with 30 ml of fresh DMEM medium containing 10% fetal bovine serum. Two days after transfection, the supernatant and cells were recovered....
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