Nucleic acid for encoding human NADH dehydrogenase subunit 4 protein and application thereof
A dehydrogenase subunit and nucleic acid technology is applied to nucleic acid encoding human NADH dehydrogenase subunit 4 protein and its application field, and can solve the problem of low transfection efficiency and poor therapeutic effect of NADH dehydrogenase subunit 4 protein. and other problems, to achieve the effect of good hereditary optic neuropathy and high transfection efficiency
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Embodiment 1
[0044] Construction of Example 1 Plasmid and Preparation of Recombinant Adeno-Associated Virus
[0045] 1.1 Plasmid preparation: After obtaining the human ND4 nucleotide sequence (National Center for Biotechnology Information Reference Sequence: yp_003024035.1), the present invention optimizes the coding sequence of COX10 (as shown in SEQ ID NO: 2) and changes the mitochondria The ND4 nucleotide sequence is a nuclear coding sequence and is optimized (as shown in SEQ ID NO: 1, referred to as optimizing ND4 gene / nucleic acid in the present invention), the homology of optimized COX10+ND4 and unoptimized COX10+ND4 sequences Sex is 76.16%. And the UTR sequence (as shown in SEQ ID NO: 4) is connected to the 3' end of the optimized ND4 gene, and the sequence of its fusion gene (or fusion nucleic acid) is shown in SEQ ID NO: 5, provided by Chengdu Qingke Zixi Biotechnology Ltd Synthetics. The full-length gene was amplified by PCR ( figure 2 ), through EcoRI / SalI digestion to form ...
Embodiment 2
[0053] Example 2 rAAV2 Infection 293T Experiment
[0054] Resuscitate frozen 293T cells and carry out subculture, grow to about 90% of the T75 culture flask, digest the cells with trypsin, take the cell pellet, and resuspend the cells with DMEM complete medium at a density of 5×10 4 individual / mL. Cells were planted in a 96-well plate, and 100 μL of cell suspension was added to each well, about 5000 cells / well. 37°C, 5% CO 2 Cultivate in the environment until the cells are overgrown to about 50% of the well plate, according to MOI=10 4 Add virus liquid rAAV2-ND4-EGFP and rAAV2-optimized ND4-EGFP (2×10 10 vg / 0.02uL) and 0.02uL PBS, after 48h, the fluorescent microscope observation, RT-PCR detection and immunoblotting were performed.
[0055] After 48 h, the fluorescence microscope observation was carried out as follows: image 3 , Fluorescence photography showed that EGFP was successfully expressed, indicating that rAAV was used as the carrier, and the EGFP gene transfecte...
Embodiment 3
[0056] Example 3 Experiment of Intravitreal Injection of rAAV2 in Rabbit Eyes
[0057] 12 rabbits were divided into 3 groups equally, and the virus liquid rAAV2-ND4 and rAAV2-optimized ND4 (1×10 10 vg / 0.05mL) and PBS were punctured into the pars plana of the ciliary body 3mm away from the limbus into the vitreous cavity. After the vitreous cavity injection, slit lamp, fundus photographic examination and HE staining were performed. After 30 days of injection, each group underwent RT respectively. - PCR detection and immunoblotting.
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