Mini promoter pATP1B1 and application thereof
A promoter and mini technology, applied in the field of neural engineering, can solve the problems of limited functional protein packaging capacity, low rAAV targeting, limited packaging, etc., and achieve the effects of large packaging capacity, improved expression specificity, and high expression efficiency.
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Embodiment 1
[0033] Example 1 Synthesis of pATP1B1 gene fragment
[0034] The promoter pATP1B1 is a partial sequence of the human sodium potassium ATP transporter beta subunit 1 (ATPase Na+ / K+transportingsubunit beta 1) gene, and the sequence selects the -80 to +40 position of the transcription initiation site of the ATP1B1 gene The core promoter region sequence of 120 bp in total is used as the final promoter sequence, and the nucleotide sequence of the promoter pATP1B1 is shown in SEQ ID NO:1.
[0035] The two ends of the promoter pATP1B1 gene fragment were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. after adding restriction endonuclease MluI-HF and BamHI-HF recognition sites.
Embodiment 2
[0036] Example 2 Construction of recombinant adeno-associated virus vector pAAV-pATP1B1-eYFP
[0037] In this example, because YFP is used as the reporter gene of the promoter, when constructing the recombinant vector, the recombinant adeno-associated vector pAAV-hSyn-eYFP containing the YFP gene is selected as the carrier backbone to connect the pATP1B1 promoter, and the steps are as follows:
[0038] Such as figure 1 As shown, the pAAV-hSyn-eYFP vector was treated with restriction endonucleases MluI-HF and BamHI-HF, and the pATP1B1 fragment was treated with restriction endonucleases MluI-HF and BamHI-HF at the same time, digested at 37°C for 3 hours, The enzyme digestion system is shown in Table 1 and Table 2. After recovering the enzyme digestion product, the ligation premix (2x ligation premix, TAKARA) was used for ligation at 16°C for 30 minutes. The system was shown in Table 3, and the successfully ligated pAAV- pATP1B1-eYFP vector.
[0039] Table 1 pAAV-hSyn-eYFP vect...
Embodiment 3
[0045]Example 3 Preparation of recombinant adeno-associated virus AAV-PHP.B-pATP1B1-eYFP
[0046] In this example, the virus was prepared by three-plasmid co-transfection method. Before virus preparation, the plasmids required for packaging viruses need to be extracted using QIAGEN Plasmid Plus Midi Kit (QIAGEN Company, Cat. No. 12943), including recombinant adeno-associated virus vector pAAV-pATP1B1-eYFP, packaging plasmid AAV-PHP.B and helper plasmid pHelper. Specific steps are as follows:
[0047] Preparation of cells: spread 293T cells in a petri dish containing full medium (DMEM, 10% fetal calf serum, 1% double antibody), and culture in an incubator.
[0048] Prepare transfection reagent: pipette 5.25ml ultrapure water, 75μg packaging plasmid, 75μg recombinant plasmid, 75μg helper plasmid and 800μL 2M calcium chloride solution, and mix gently. Add an equal volume of 2x HBS to the aforementioned reagents, vortex and let stand for 30 minutes.
[0049] Transfected cells:...
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