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Coding sequence of retinal cleavage protein, and construction and application of expression vector of coding sequence

A technology of retina and carrier, applied in the field of coding sequence of retinoschisis protein, can solve problems such as vision loss and dissatisfaction of patients

Active Publication Date: 2020-09-04
WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, all kinds of treatment methods for XLRS have no satisfactory effect, and the final outcome of patients is mostly vision loss.

Method used

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  • Coding sequence of retinal cleavage protein, and construction and application of expression vector of coding sequence
  • Coding sequence of retinal cleavage protein, and construction and application of expression vector of coding sequence
  • Coding sequence of retinal cleavage protein, and construction and application of expression vector of coding sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0187] Example 1 Sequence Optimization

[0188] In this example, based on the amino acid sequence of RS1 protein (SEQ ID NO.:3) and the natural coding sequence (SEQ ID NO.:2), the inventors optimized the coding sequence. Specifically, the present invention optimizes sequence fragments that affect gene expression, and these sequence fragments include but are not limited to: codon usage bias, elimination of secondary structures (such as hairpin structures) that are not conducive to expression, changes in GC content, CpG dinuclear Nucleotide content, secondary structure of mRNA, cryptic splice sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, regions of RNA instability, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0189] In this example, dozens of optimized RS1 coding sequences were designed, and the optimized RS1 coding sequences were analyzed and tested to obtai...

Embodiment 2

[0190] Example 2 Construction of recombinant adeno-associated virus vector of recombinant human retinoschisis protein gene and its virus packaging and purification method, the steps of which include:

[0191] (1) Construction of recombinant adeno-associated virus vector containing human retinoschisis protein gene

[0192] 1) Vector construction

[0193] The natural unoptimized coding sequence (SEQ ID NO.: 2) and the optimized recombinant human retinoschisis protein gene (SEQ ID NO: 1) in Example 1 were added with two enzyme cutting sites Kpn I and Sal I or the Use the new gene to design primers, use PCR amplification product and pAAV-MCS plasmid vector, carry out Kpn I and Sal I double enzyme digestion respectively, recover the digestion products, T4DNALigase ligation overnight, and the ligation products are transformed into competent cells to obtain recombinant pAAV-MCS -RS1 (containing the coding sequence shown in SEQ ID NO.: 2) and pAAV-MCS-optimized RS1 (containing the co...

Embodiment 3

[0222] Example 3 RS1 original nucleic acid sequence and optimized sequence in vitro cell validity experiment

[0223] 1. Cell infection

[0224] Take 6-well plates and divide them into 7 groups, inoculate 5×10 5 Cells in each well, one day after seeding, the cell count was 1×10 6 About, inoculate 10 10 vg / 50ul rAAV2 / 2-EGFP (control group), rAAV2 / 2-optimized RS1 (group A), rAAV2 / 9-optimized RS1 (group B), rAAV2 / Rh10-optimized RS1 (group C), rAAV2 / 2- RS1 (group D), rAAV2 / 9-RS1 (group E) and rAAV2 / Rh10-RS1 (group F). After culturing for 48 hours, mRNA and total protein were extracted for detection and analysis.

[0225] 2. Real-Time PCR detection of RS1 gene expression

[0226] First, use NCBI's conserved domain analysis software to analyze the conserved structure of ND6 to ensure that the amplified fragment of the designed primer is located in the non-conserved region; then, according to the primer design principle of fluorescent quantitative PCR, use Premier 5 to design pr...

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Abstract

The invention provides a coding sequence of retinal cleavage protein, and construction and application of an expression vector of the coding sequence. Specifically, a coding sequence of the RS1 gene is specially optimized and designed in a targeted manner, so that a nucleotide sequence which is particularly suitable for efficiently expressing the RS1 protein in mammalian (such as human) cells (such as photoreceptor cells, bipolar cells and optic nerve cells) is obtained; and a recombinant AAV virus for expressing the normal humanized RS1 protein is constructed. Compared with an unoptimized coding sequence, the expression quantity of the specially optimized RS1 coding sequence (SEQ ID NO.: 1) is remarkably increased, and the RS1 coding sequence is very suitable for being expressed in mammalian (especially human) cells and can effectively treat X-linked retinal cleavage.

Description

technical field [0001] The invention relates to the field of biological preparations, in particular to the coding sequence of retinoschisis protein, the construction of its expression vector and its application. Background technique [0002] X-Linked juvenile Retinoschisis (XLRS) is a rare hereditary blinding eye disease with an incidence of about 1:5000-1:25000. XLRS mainly involves the bilateral retina, and a split cavity appears between the retinal nerve fiber layer and the ganglion cell layer. The disease occurs in males, and females have no characteristic clinical manifestations as carriers. XLRS is the leading cause of macular degeneration in male adolescents. At present, all kinds of treatment methods for XLRS have no satisfactory effect, and the final outcome of patients is mostly vision loss. [0003] Mutations in the RS1 gene are currently believed to be the main gene responsible for retinoschisis. Unlike a large number of pathogenic mutations, there are only 5...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/864A61K48/00A61P27/02
CPCC07K14/47C12N15/86A61K48/005A61P27/02C12N2750/14143
Inventor 李斌
Owner WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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