Adeno-associated virus mediating trigenic cell expression system

An expression system and gene cell technology, which can be used in gene therapy, genetic engineering, plant genetic improvement, etc., and can solve the problems of reduced synthesis and the inability of TH to fully function.

Inactive Publication Date: 2006-06-07
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

intracellular BH 4 The levels of dopaminergic neurons limit the activity of TH, and the decrease of GCH

Method used

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  • Adeno-associated virus mediating trigenic cell expression system
  • Adeno-associated virus mediating trigenic cell expression system
  • Adeno-associated virus mediating trigenic cell expression system

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0059] 2.5 Preparation of target gene and vector fragment

[0060] 2.5.1 Restriction endonuclease digestion of target gene and vector fragment

[0061] pGEMT-AADC and pAAVMCS were double-digested with Cla I and Xba I respectively; pCIneo-GCHI and pAAVMCS were double-digested with EcoRI and Sal I respectively.

[0062] 2.5.2 Recovery and purification of target genes and vector fragments

[0063] After enzyme digestion, use 1% agarose gel electrophoresis at a voltage of 5v / cm for 1h. Cut out the 1.4kb fragment (AADC gene) from the plasmid pGEMT / AADC and the 0.9kb fragment (GCHI gene) from the plasmid pCIneo / GCHI and the pAAVMCS vector (4.7kb) linearized by different sets of double enzyme digestion, (refer to the agarose gel gel DNA fragment recovery and purification kit) to recover and purify the target gene and carrier DNA fragments, add distilled water or TE (10-30μl) to mix, 60 water bath for 5min, 12000rpm centrifuge for 1min, recover the supernatant and save it for later ...

Embodiment 2

[0183] Example 2 Dual gene cell expression system

[0184] The experimental method and reagents are the same as the three-gene cell expression system

[0185] result

[0186] 1. Construction of recombinant adeno-associated virus vector

[0187] After connecting the AADC fragment to pAAV-MCS, transforming, screening, and extracting the recombinant plasmid, the fragments of 1.2kb and 900bp were identified by enzyme digestion, which were completely consistent with the expected fragment size, indicating that the AADC gene was successfully recombined into the adeno-associated virus vector middle. At the same time, a 1.8kb TH fragment could be obtained by enzyme digestion and identification of rAAV-TH, which indicated that the sequence of rAAV-TH was correct.

[0188] 2. Results of HEK293 Transfection

[0189] Three days after transfection is the best time to prepare virus stock solution. At this time, HEK293 cells are close to 90% confluent. Compared with negative HEK293 cells ...

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Abstract

The present invention relates to a trigenic cell expression system, and includes constituting adeno-associated virus (AAV) vector rAAV/TH, rAAV/AADC and rAAV/GCH-I carrying tyrosine hydrozylase (TH), aromatic amino acid decarboxylase (AADC) and guanylyl cyclohydrolase-I (GCH-I); co-transfecting these three kinds of plasmid to packing cell by means of calcium phosphate mediation process to obtain AAV-TH, AAV-AADC and AAV-GCH-I virus grains with high titer and infection activity; collecting virus supernatant and infecting the expression cell. The present invention also relates to double gene cell expression system of TH and AADC.

Description

Technical field: [0001] The invention relates to an adeno-associated virus-mediated three-gene cell expression system, in particular to a transgenic cell expression system established through an adeno-associated virus vector system by using recombinant dopamine synthetase TH, AADC and GCH three genes. Background technique: [0002] Parkinson's Disease (PD) is an aging neurodegenerative disease. The main clinical manifestations are resting tremor, muscle stiffness, movement disorder and so on. The disease is mainly due to the progressive degeneration of dopaminergic neurons in the nigrostriatum, resulting in a severe decrease in dopamine content in the striatum. When the dopamine content in the nigrostriatum of the brain decreases by 60-80%, the patient will appear the above clinical symptoms. Dopamine synthesized in the midbrain is first converted from tyrosine to L-dopa (L-dihydroxy-phenylalanine, L-dopa) by Tyrosine Hydroxylase (TH), and then L-dopa is decarboxylated by ...

Claims

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Application Information

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IPC IPC(8): C12N15/861A61K48/00A61P25/16C12N5/10C12N9/00C12N15/52
Inventor 杨慧徐群渊段春礼段得义赵春礼鲁玲玲苏玉金赵焕英
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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