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Recombinant human NADH (nicotinamide-adenine dinucleotide) dehydrogenase subunit-4 gene and constructing method of expression vector thereof

A technology for the construction of dehydrogenase subunit 4, which is applied in the field of recombinant human NADH dehydrogenase subunit 4 gene and its expression vector construction, which can solve the problems that human treatment cannot be used

Active Publication Date: 2013-11-06
WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Some people also use this technology in foreign research, but the nucleic acid sequences designed in foreign research are all added with green fluorescent protein (GFP), such nucleic acid sequences can be used in experimental research, but cannot be used in human treatment

Method used

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  • Recombinant human NADH (nicotinamide-adenine dinucleotide) dehydrogenase subunit-4 gene and constructing method of expression vector thereof
  • Recombinant human NADH (nicotinamide-adenine dinucleotide) dehydrogenase subunit-4 gene and constructing method of expression vector thereof
  • Recombinant human NADH (nicotinamide-adenine dinucleotide) dehydrogenase subunit-4 gene and constructing method of expression vector thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] 1. Test materials

[0052] Table 1

[0053]

[0054] Two experimental steps

[0055]The new gene sequence of the recombinant human NADH dehydrogenase subunit 4 (ND4) of the present invention is redesigned according to the ND4 gene sequence and the cox10 gene sequence provided by the genbank database. The gene sequence of the present invention is designed in two parts ( figure 1 ). The first part includes the mitochondrial targeting sequence (mitochondroitin-targeting sequence MTS) of cox10 and the coding sequence of ND4. Among them, since the genetic codes of mtDNA and nuclear genome are not completely the same, the present invention modifies its gene sequence according to the amino acid sequence of ND4 protein, so that the amino acid sequence of the modified new gene sequence translated in the cytosol is the same as the ND4 protein sequence provided by the genbank database. The amino acid sequence translated in the mitochondria of the gene sequence is exactly th...

Embodiment 2

[0096] Example 2: Effect experiment of rAAV2 / 2-ND4 recombinant adeno-associated virus on Leber hereditary optic neuropathy

[0097] 1. Intravitreal Injection of Rabbit Eyes

[0098] Take 24 rabbits and divide them into two groups, the experimental group and the control group, respectively. 0.1% rAAV-ND4 and 0.1% rAAV-GFP were used to puncture the pars plana of the ciliary body 3 mm away from the corneal limbus into the vitreous cavity, and perform vitrectomy. Body cavity injection.

[0099] 2. Slit lamp, intraocular pressure, fundus photography examination

[0100] The rabbits in the two groups were examined with slit lamp and intraocular pressure at 1, 3, 7, and 30 days after operation respectively. All rabbits had no obvious abnormalities, no conjunctival hyperemia, discharge, endophthalmitis, and no increase in intraocular pressure. Fundus photography one month after operation showed that there were no obvious complications or damage to the retinal blood vessels and opti...

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Abstract

The invention discloses a recombinant human NADH (nicotinamide-adenine dinucleotid) dehydrogenase subunit-4 gene and a constructing method of an expression vector thereof. The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the size of the nucleotide sequence is 2889 bp. The constructing steps of adeno-associate virus vectors are as follows: firstly constructing a recombinant adeno-associated virus vector containing the human NADH dehydrogenase subunit-4 gene, and then coating, infecting, purifying, concentrating and identifying the recombinant adeno-associated virus. According to the method, the recombinant adeno-associated virus vector with the recombinant human NADH dehydrogenase subunit-4 gene can be quickly and simply constructed, and is packaged to obtain the adeno-associated virus vector with complex defects. The gene can be used for gene therapy of LHON (leber's hereditary optic neuropathy).

Description

technical field [0001] The invention relates to a recombinant human NADH dehydrogenase subunit 4 gene and a method for constructing an expression vector thereof, belonging to the technical field of genetic engineering. technical background [0002] Leber's hereditary optic neuropathy (LHON) is a mitochondrial hereditary disease that mainly affects the fibers of the macular papillary tract, leading to degeneration of the optic nerve. The disease usually occurs in young and middle-aged men, and the clinical manifestations are acute or subacute painless vision loss in both eyes at the same time or successively, accompanied by central visual field defect and color vision disturbance. [0003] LHON is caused by mutations in mitochondrial sites. Chinese scholars have initially confirmed that more than 95% of LHON patients in China are related to site mutations in mitochondrial DNA 11778, 14484, and 3460, among which site 111778 is the most common mutation, accounting for 89.2% of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/66C12N15/864C12N7/01C12N1/15C12N1/19C12N1/21
Inventor 李斌裴晗万幸
Owner WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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