CN115558670A

CN115558670A - Nucleic acid molecules, expression vectors and uses thereof

Info

Publication number: CN115558670A
Application number: CN202110752425.8A
Authority: CN
Inventors: 李斌; 任盛
Original assignee: Wuhan Niufusi Biological Technology Co ltd
Current assignee: Wuhan Niufusi Biological Technology Co ltd
Priority date: 2021-07-02
Filing date: 2021-07-02
Publication date: 2023-01-03

Abstract

The invention relates to the field of biomedicine, in particular to a nucleic acid molecule, an expression vector and application thereof. The invention proves that the AAV-CYP4V2 medicament treatment can obviously improve the lipid metabolism dysfunction lesion caused by CYP4V2 mutation. CYP4V2 can be expressed in 293 cells treated by the medicine with high efficiency. Meanwhile, after the RPE cells with the CYP4V2 mutation are treated by the medicament provided by the invention, the fatty acid metabolism capability of the RPE cells is improved, and the fact that the protein expressed by the medicament has normal catalytic activity is proved, so that the function of the mutant can be compensated. Therefore, the AAV-CYP4V2 medicine has the effect of preventing or treating the paraanorthite crystal malnutrition.

Description

Nucleic acid molecules, expression vectors and uses thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a nucleic acid molecule, an expression vector and application thereof.

Background

Bietti Crystalline Dystrophy (BCD) is a progressive chorioretinal degenerative disease. The disease accounts for 10 percent of the total quantity of autosomal recessive genetic retinopathy, the global morbidity is about 1/67000, the morbidity of people in Asian regions is high, and the morbidity of China and Japan is particularly prominent. BCD patients begin to develop signs of visual deterioration and visual field loss around the age of twenty, with the majority of patients developing a state of legal blindness around the age of fifty-six as the course of the disease progresses. Clinical features of BCD include the appearance of yellowish white crystals in the cornea and fundus, which, upon microstructural observation, are found to be mainly concentrated at the boundaries of Retinal Pigment Epithelium (RPE) cells; in addition, the RPE cells and visual cells of the patient gradually shrink as the disease progresses. Genetic linkage analysis first established that the BCD locus is located on human chromosome 4q35, and subsequent fine localization of this locus in subsequent studies confirmed that CYP4V2 gene mutations are responsible for BCD lesions.

The CYP4V2 gene encodes a heme protein and belongs to one of the 57 functional cytochrome P450 genes in the human genome. The protein participates in the metabolism of eye fatty acid, catalyzes the omega-hydroxylation of polyunsaturated fatty acid, and maintains the homeostasis of eye polyunsaturated fatty acid. The efficient lipid metabolism circulating system has important significance for maintaining normal cell membrane renewal of RPE cells and visual cells, and usually, the outer segment of the visual cell shedding is phagocytized by the RPE cells and catalyzes lipid metabolism renewal therein. CYP4V2 gene mutation causes protein function loss, so that the phagocytosis of RPE cells is defective, and the functional disorder of the RPE cells and cell atrophy are caused. Biochemical tracer studies have shown defects in the anabolism of omega-3 polyunsaturated fatty acids in BCD patients, and this finding also evidences the effect of mutations in the CYP4V2 gene on protein function. There is currently no effective therapeutic regimen for BCD, and AAV-mediated gene therapy has shown great potential in the treatment of genetic diseases caused by single gene mutations, given that BCD is such a disease.

Disclosure of Invention

In view of the above, the present invention provides cDNA nucleotide sequences, expression vectors and uses thereof. The AAV-CYP4V2 medicine provided by the invention has the function of preventing or treating Bietti Crystal Dystrophy (BCD).

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides a nucleic acid molecule having:

(I) a nucleotide sequence shown as SEQ ID No.1 or 2; or

(II) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID No.1 or 2; or

(III) a nucleotide sequence having at least 90% sequence identity, preferably at least 95%, 96%, 97%, 98%, 99% sequence identity to the nucleotide sequence of (I) or (II).

The nucleic acid molecule provided by the invention codes the protein shown in SEQ ID No. 5.

Importantly, the invention also provides expression vectors comprising the nucleic acid molecules.

In some embodiments of the invention, the expression vector further comprises a constitutive promoter and a polyA element.

In some embodiments of the invention, the expression vector comprises an expression vector plasmid pAAV-CAG-CYP4V2opt1-bGHpolyA and/or pAAV-CAG-CYP4V2opt2-bGHpolyA;

wherein the CYP4V2opt1 nucleotide sequence has:

(I) a nucleotide sequence shown as SEQ ID No. 1; or

(II) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID No. 1; or

(iii) a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of (i) or (ii), preferably at least 95%, 96%, 97%, 98%, 99% sequence identity.

Wherein the CYP4V2opt2 nucleotide sequence has:

(I) a nucleotide sequence shown as SEQ ID No. 2; or

(II) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID No. 2; or

Preferably, the promoter is a CAG promoter shown as SEQ ID No.3 or a TRPM1 promoter shown as SEQ ID No. 7; the bGH polyA is shown as SEQ ID No. 4;

the polyA element is a bGH polyA element or an SV40 polyA element; more preferably, the polyA element is a bGH polyA element as shown in SEQ ID No. 4.

Preferably, the expression vector is a viral vector, the viral vector being one of an adeno-associated virus, a lentivirus, a retrovirus, or an adenovirus;

preferably, the viral vector is an adeno-associated viral vector;

preferably, the serotype of the adeno-associated virus is selected from one or more of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 wild type or 7M8, TYF mutant.

The invention also provides a host comprising the cDNA, the protein or the expression vector.

More importantly, the invention also provides application of the cDNA or the expression vector in preparing a medicament for preventing and/or treating the paradolite crystal malnutrition.

In addition, the invention also provides a medicament which comprises the cDNA or the expression vector and pharmaceutically acceptable auxiliary materials.

The invention also provides a drug delivery method, wherein the drug preparation is applied to eyes, and the administration mode is subretinal administration or vitreous cavity administration.

The invention also provides application of the repaired CYP4V2 gene expression in preparing a medicament for preventing and/or treating the paradolite crystal malnutrition.

The invention proves that the AAV-CYP4V2 medicament treatment can obviously improve the lipid metabolism dysfunction lesion caused by CYP4V2 mutation. The AAV-CYP4V2 drug is used for treating 293 cells, and the CYP4V2 can be efficiently expressed in the cell line. Meanwhile, after the RPE cell with CYP4V2 mutation is treated by AAV-CYP4V2 drugs, the fatty acid metabolism capability of the RPE cell is improved, and the fact that the protein expressed by the drugs has normal catalytic activity is proved, and the function of the mutants can be compensated. Therefore, the AAV-CYP4V2 drug has the effect of preventing or treating Bietticystalline dystrophy (BCD).

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIGS. 1 (A) and 1 (B) show the different codon sequences after optimization of the alignment of CYP4V2wt and CYP4V2opt1 sequences, which are bolded and underlined;

FIGS. 2 (A) and 2 (B) the sequences of CYP4V2wt and CYP4V2opt2 which are optimized for alignment and the differential codon sequences are bolded and underlined;

FIG. 3 shows a vector map of pAAV-CAG-CYP4V2opt1-bGHpolyA (A) a vector map of pAAV-CAG-CYP4V2opt2-bGHpolyA (B); the vector comprises AAV2 'ITR, a constitutive CAG promoter sequence, a CYP4V2opt1 cDNA sequence or a CYP4V2opt2cDNA sequence, a bGH polyA element sequence and AAV 2' ITR;

FIG. 4 expression assay of pAAV-CAG-CYP4V2opt1-bGHpolyA and pAAV-CAG-CYP4V2opt2-bGHpolyA plasmids in HEK293 cells. Respectively transfecting pAAV-CAG-CYP4V2opt1-bGHpolyA, pAAV-CAG-CYP4V2opt2-bGHpolyA plasmids and negative control plasmids in HEK293 cells, respectively extracting RNA and protein from lysed cells after 48 hours, detecting CYP4V2 mRNA (A) and protein expression level (B) by qPCR and WesternBlot, and finding that the two constructed expression vectors can efficiently express CYP4V2 in 293 cells;

FIG. 5AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA virus drug expression detection in 293 cells. AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA virus drugs are respectively infected in 293 cells, immunofluorescence staining is carried out after cell slide climbing, and the virus drugs of AAV5-CAG-CYP4V2opt1-bGHpolyA (A) AAV5-CAG-CYP4V2opt2-bGHpolyA (B) can be expressed in the 293 cells through microscopic observation, while protein expression (C) cannot be detected in a negative control group;

FIG. 6 assay of hydroxylated dodecanoic acid synthesis by RPE cells. AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA virus drugs are respectively sensed in the RPE cells with CYP4V2 mutation, the infected cells are incubated with substrates dodecanoic acid and GAPDH for 30 minutes, lipid components in cell culture are extracted by ethyl acetate, the content of hydroxylated dodecanoic acid in the cells is detected by liquid phase mass spectrometry, and the synthetic capacity of the hydroxylated dodecanoic acid of the RPE cells treated by the two virus drugs is higher than that of a control group not treated by the drugs.

Detailed Description

The invention discloses cDNA, expression vector and application thereof, and can be realized by appropriately improving process parameters by referring to the content in the text by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate variations and combinations of the methods and applications described herein may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention aims to prove that the AAV-CYP4V2 medicament has the effect of preventing or treating Bietti Crystalline Dystrophy (BCD) by in vitro experiments. The invention proves that the AAV-CYP4V2 drug treatment can obviously improve the lipid metabolism dysfunction lesion caused by CYP4V2 mutation. The AAV-CYP4V2 drug is used for treating 293 cells, and the CYP4V2 can be efficiently expressed in the cell line. Meanwhile, after the RPE cell with CYP4V2 mutation is treated by AAV-CYP4V2 drugs, the fatty acid metabolism capability of the RPE cell is improved, and the fact that the protein expressed by the drug has normal catalytic activity is proved, and the function of the mutant can be compensated is proved. Thus, the AAV-CYP4V2 drug has the effect of preventing or treating specific Epstein dystrophy (BCD).

In order to realize the task, the invention adopts the following technical scheme:

firstly, the CYP4V 2cDNA sequence is subjected to codon optimization (codon optimization) to obtain CYP4V2opt1 and CYP4V2opt2, then the CYP4V2opt1 and CYP4V2opt2 are placed between a constitutive CAG promoter and a bGH polyA element, and 2 different expression vector plasmids, namely pAAV-CAG-CYP4V2opt1-bGHpolyA and pAAV-CAG-CYP4V2opt2-bGHpolyA, are constructed. The two expression vector plasmids are transiently transfected into HEK293 cells, and the expression of CYP4V2 is detected by utilizing qPCR and Westernblot, so that the constructed two expression vectors can efficiently express the CYP4V2 in the 293 cells. After AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA virus drugs infect 293 cells, the expression of CYP4V2 is observed by immunofluorescence, and the fact that the two virus drugs can effectively express target proteins in the 293 cells is discovered. AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA virus drugs infect the RPE cells with CYP4V2 mutation, through incubating the infected RPE cells with dodecanoic acid and GAPDH, processing and detecting the content of dodecanoic acid hydroxylation products in the cells, the result shows that the capability of the CYP4V2 mutant RPE cells processed by the two virus drugs to synthesize hydroxylated dodecanoic acid is improved, which indicates that the lipid metabolism defect caused by CYP4V2 mutation is compensated. In conclusion, it is proved that AAV drug constructed by optimizing CYP4V2 gene has the function of preventing or treating specific Epstein dystrophy (BCD).

In the cDNA, the expression vector and the application thereof provided by the invention, the used raw materials and reagents can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 plasmid-transfected 293 cells and Virus-infected 293 cells construction of plasmid vectors capable of efficiently expressing CYP4V2 protein

pAAV-CAG-CYP4V2opt1-bGHpolyA and pAAV-CAG-CYP4V2opt2-bGHpolyA plasmids were synthesized commercially

Plasmid transfected cells

1. One day prior to transfection, HEK293 cells were trypsinized and counted, and the cells plated to achieve 70-80% confluence on the day of transfection.

2. For each well of cells, 0.8. Mu.g-1.0. Mu.g plasmid DNA was diluted with 50. Mu.l serum-free DMEM medium; mu.l to 3. Mu.l of LIPOFECTAMINE 2000 reagent was diluted with 50. Mu.l of DMEM medium.

4. The diluted DNA and diluted LIPOFECTAMINE 2000 were mixed and incubated at room temperature for 20 minutes.

5. Directly add the complex to each well, shake the plate, mix gently.

6. 5% CO at 37 deg.C ₂ And culturing for 48 hours.

7. The culture medium is discarded, washed by PBS, digested by pancreatin and centrifuged to collect cells for later use.

qPCR determination of mRNA content

1. Total RNA extraction

1) Harvesting 10 ⁵ 1ml of lysis solution was added to each cell to lyse the cells, and the cells were centrifuged at 13000g for 10min to obtain the supernatant.

3) Adding 250 μ l of chloroform, reversing the centrifuge tube for 15s, mixing well, standing for 3min; 13000g at 4 ℃ are centrifuged for 8min.

4) Transferring the supernatant into a new centrifuge tube, adding isopropanol with the volume of 0.8 times of that of the centrifuge tube, and reversing and uniformly mixing; standing at-20 deg.C for 15min; 13000g at 4 ℃ is centrifuged for 10min, and the white precipitate at the bottom of the tube is RNA.

5) Removing liquid by suction, adding 1.5ml of 75% ethanol, washing and precipitating; 13000g is centrifuged for 5min at 4 ℃; sucking off the liquid, and blowing the centrifugal tube on a super clean bench for 3min; adding 20 μ l of RNase-free water to dissolve the RNA; incubate at 55 ℃ for 5min.

2. Reverse transcription

1) Taking a PCR tube, and adding a solution containing 2 mu gRNA; add 1. Mu. Oligo (dT); make up to 12. Mu.l with ribonuclease-free deionized water.

2) Keeping the temperature on a PCR instrument at 70 ℃ for 5min, and quickly cooling on ice; sequentially adding 4. Mu.l of 5 XBuffer, 2. Mu.l of 10mM dNTPs, 1. Mu.l of RNA inhibitor and 1. Mu.l of reverse transcriptase, and uniformly mixing by using a gun to suck; keeping the temperature of the PCR sample at 42 ℃ for 60min, and keeping the temperature of the PCR sample at 80 ℃ for 5min to inactivate the reverse transcriptase.

3. Quantitative PCR

1) 0.2ml of PCR tube was used to prepare the following reaction system, and 3 tubes were prepared for each reverse transcription product. 2 XqPCR Mix12.5. Mu.l; 7.5. Mu.M gene primer; 2.0. Mu.l of reverse transcription product; 2.5 μ lddH2O; 8.0. Mu.l. 2) 0.2ml of PCR tube was used to prepare the following reaction system, and 3 tubes were prepared for each reverse transcription product. 2 × qPCRMix12.5 μ l; mu.l of 7.5. Mu.M internal reference primer 2.0. Mu.l of reverse transcription product 2.5. Mu.lddH 2O 8.0. Mu.l.

Target gene flag amplification primers:

forward primer 5 'AGACCATGACGGTGAT-3' (shown as SEQ ID No. 8):

reverse primer 5-:

amplification primers of an internal reference gene actin:

forward primer 5 'and GGACTTCGAGCAAGAGATGG-3' (shown as SEQ ID No. 10):

reverse primer 5-:

3) PCR amplification

Pre-denaturation at 95 ℃ for 5min,

40 cycles of 95 ℃,15s → 60 ℃,60s,

the dissolution curve is 60 ℃ and about 95 ℃, and the temperature is raised by 1 ℃ every 20s

4) Results processing Δ Δ CT method: a = CT (target gene, sample to be tested) -CT (internal standard gene, sample to be tested); b = CT (gene of interest, control sample) -CT (internal standard gene, control sample); k = a-B; expression fold =2 ^-K 。

Western Blot

1. Protein samples were prepared by adding PMSF to the lysate in a ratio of 1.

2. Cells were lysed using a strong lysate for 30min on ice; centrifuging at 12000rpm for 15min at 4 deg.C, and collecting supernatant.

3. Protein concentration was determined using BCA method.

4. Electrophoresis

a. Preparing corresponding separation gel (5 ml/block) according to the size of the detected protein, and solidifying the separation gel.

b. 5% concentrated gum (2 ml/block) was prepared, filled on a glass plate, and a comb was inserted.

c. Mu.l of pre-stained protein molecule marker SDS-PAGE was added to the wells and 10. Mu.l of SDS-PAGE protein loading buffer at 1 Xwas added to the blank wells at the sides of the sample wells.

5. Rotary film

Placing a wet cushion layer on the film transferring white clamp, laying three pieces of wet filter paper which are overlapped together on the cushion layer, sequentially placing a wet pvdf film, glue, the filter paper, the cushion layer and a black splint on the filter paper, placing the splint into an electrophoresis tank filled with a film transferring buffer solution, and placing the film transferring tank in an ice bath for film transferring for 2 hours.

6. Sealing of

After the membrane is completely transferred, rinsing for 1-2 minutes, sucking up the buffer solution by a dropper, adding 5% of skimmed milk powder, slowly shaking on a side-swinging shaking table, and sealing at room temperature for 15-60min. TBS washing was added and the reaction solution was washed for 5 minutes. A total of 3 washes were performed.

7. Primary antibody incubation

The appropriate amount of primary antibody was diluted with 5% skim milk/PBS +2% BSA at the ratio and incubated overnight with slow shaking at 4 ℃ or for 2h on a side-shaking shaker at room temperature. After incubation, washing is carried out.

8. Incubation with secondary antibody

Adding diluted secondary antibody, and slowly shaking and incubating for 40min-1h on a side shaking table at room temperature. After incubation, washing is carried out.

9. Protein detection

And (3) detecting the protein by using ECL reagents, uniformly mixing 1ml of each ECL reagent, dripping the mixture on the surface of the protein membrane, and incubating for 1-2min in a dark place. The protein film was placed neatly on plastic paper with tweezers and exposed on a gel imager.

Virus package

1. HEK293 cells with a degree of polymerization of more than 90% are prepared according to the following steps of 1: and 3, a ratio transmission disc.

2. The serum-free culture medium is changed about 1-2h before plasmid transformation, and the target gene plasmid and the auxiliary plasmid are transformed into HEK293 by using a transfection reagent.

3. After 24h of plasmid transformation, the medium was replaced with new serum-free medium.

4. And (5) performing transfection for 72h for virus recovery. Blowing down cells with the culture medium, and centrifuging; the culture supernatant and the cell pellet were then harvested separately. The virus in the culture supernatant was precipitated with PEG8000, and the virus precipitate was collected overnight.

5. The virus mixture was purified by iodixanol density gradient centrifugation and then concentrated using an ultrafiltration tube.

Cell slide polylysine coating

1. Sterile 10. Mu.g/ml polylysine was added to each slide and incubated in an incubator at 37 ℃ for 2-3h.

2. After incubation, discard polylysine, weather polylysine on the slide in clean bench, 4 degrees preserves can be put to the slide, and PBS washs 1 times before the use, and the culture medium washs 2 times.

Virus-infected cells

1. HEK293 cells were digested and plated in 6-well plates as required for 1.5-4.

2. After overnight culture, a portion of the wells were plated at MOI =2 × 10 ⁴ Infecting the virus of interest and its control virus.

3. After 36-48h of infection, the infected group was re-seeded into 6-well plates with cell crawlers at a ratio of 1.

4. After 12-24h of inoculation, the growth state and expression condition of the reptile cells are observed.

5. The medium was discarded, and 1ml of 4-vol PFA fixative (37 ℃ incubation) was added to each well and fixed at room temperature for 10min.

6. The fixative was discarded, washed 2 times with PBS, and a small portion of PBS was left in the six-well plate and stored temporarily at 4 ℃.

Immunofluorescent staining

1. The cell slide was transferred to a 12-well plate and washed 2 times with PBS for 5min each time. The shaker is required to shake rapidly.

2. PBS was discarded and 200. Mu.l of 1% was added per well, triton was punched for 15min.

3. The 1-percent Triton-containing punch solution was discarded, and 1ml of 0.05-percent Triton was added to each well, and washed 3 times for 5min.

4. Add 200. Mu.l of Biyunyan sealing solution to each well, and seal for 2 hours at room temperature.

5. The cell climbing sheets were taken out, placed on a sealing membrane, and 70-90 μ l of primary antibody (1. The whole sealing film is put into a wet box and kept stand overnight at 4 ℃.

6. The climber was returned to the 12-well plate, 1.5ml of 0.05% Triton was added per well, and washed 3 times for 5min each time with shaking.

7. The cell slide was taken out, placed on a sealing film, and 40 to 50 μ l of a secondary antibody (1. The whole sealing film is placed in a wet box and kept stand for 1 hour at room temperature.

8. Placing the slide back into a 12-well plate, adding 1ml of 0.05% Triton per well, and washing 3 times for 5min each time with shaking.

9. Add 200. Mu.l of DAPI stain to each well and stain for 15min.

10. Add 1ml PBS per well and wash 4 times with shaking for 1-2min each time.

11. And (3) dropwise adding 15 mu l of the anti-fluorescence quenching mounting solution on the glass slide, carefully sealing the cover glass on the glass slide, and sealing with the nail polish.

Experimental results and discussion

By optimizing multiple parameters such as codon usage bias, DNA repetitive sequences, mRNA secondary structure, GC content and the like, optimized sequences CYP4V2opt1 (figure 1) and CYP4V2opt2 (figure 2) which are obviously different from CYP4V2wt sequences are obtained. Subsequently, we constructed the CYP4V2opt1 expression vector and CYP4V2opt2 (FIG. 3) driven by the constitutive promoter CAG promoter, and then transfected the same amount of plasmid in HEK293 cells to detect the expression of CYP4V2. From the results of the measurement of the mRNA level and the protein level, it was found that: both codon-optimized CYP4V2opt1 and CYP4V2opt2 were expressed in HEK293 cells. The mRNA level of the target gene in 293 cells transfected with pAAV-CAG-CYP4V2opt1-bGHpolyA and pAAV-CAG-CYP4V2opt2-bGHpolyA far exceeded the basal level (FIG. 4A), and the specific values are shown in Table 1; no signal was detected for the CYP4V2 protein in lysates from control 293 cells, whereas significant signals were detected at the predicted size of the CYP4V2 protein in 293 cells transfected with pAAV-CAG-CYP4V2opt1-bGHpolyA and pAAV-CAG-CYP4V2opt2-bGHpolyA (FIG. 4B). The above results indicate that the plasmids pAAV-CAG-CYP4V2opt1-bGHpolyA and pAAV-CAG-CYP4V2opt2-bGHpolyA can efficiently and stably express CYP4V2 target protein in 293 cells.

TABLE 1 relative CYP4V2 mRNA expression levels in 293 cells transfected with plasmids

Then, the two plasmids are respectively packaged into AAV5-CAG-CYP4V2opt1-bGHpolyA virus medicaments and AAV5-CAG-CYP4V2opt2-bGHpolyA virus medicaments, the two virus medicaments are respectively infected into 293 cells, and the expression condition of the CYP4V2 protein in the cells is detected by immunofluorescence. After incubation of the infected 293 cells with antibodies, they were observed under a fluorescence microscope: no green fluorescence signal was detected in 293 cells of the control group (FIG. 5C), whereas 293 cells infected with AAV5-CAG-CYP4V2opt1-bGHpolyA (FIG. 5A) and AAV5-CAG-CYP4V2opt2-bGHpolyA (FIG. 5B) virus drugs were able to show green fluorescence signals under specific excitation light irradiation, and the fluorescence signals were located around the nucleus, indicating that the signals were specific and located in the cells. Therefore, the result shows that the 293 cells infected by AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA virus medicaments can efficiently express the CYP4V2 protein.

The specific sequence related by the invention is as follows:

CYP4V2 protein sequence (as shown in SEQ ID No. 5):

<xnotran> MAGLWLGLVWQKLLLWGAASALSLAGASLVLSLLQRVASYARKWQQMRPIPTVARAYPLVGHALLMKPDGREFFQQIIEYTEEYRHMPLLKLWVGPVPMVALYNAENVEVILTSSKQIDKSSMYKFLEPWLGLGLLTSTGNKWRSRRKMLTPTFHFTILEDFLDIMNEQANILVKKLEKHINQEAFNCFFYITLCALDIICETAMGKNIGAQSNDDSEYVRAVYRMSEMIFRRIKMPWLWLDLWYLMFKEGWEHKKSLQILHTFTNSVIAERANEMNANEDCRGDGRGSAPSKNKRRAFLDLLLSVTDDEGNRLSHEDIREEVDTFMFEGHDTTAAAINWSLYLLGSNPEVQKKVDHELDDVFGKSDRPATVEDLKKLRYLECVIKETLRLFPSVPLFARSVSEDCEVAGYRVLKGTEAVIIPYALHRDPRYFPNPEEFQPERFFPENAQGRHPYAYVPFSAGPRNCIGQKFAVMEEKTILSCILRHFWIESNQKREELGLEGQLILRPSNGIWIKLKRRNADERCYP4V2wt cDNA ( SEQ ID No.6 ): </xnotran>

ATGGCGGGGCTCTGGCTGGGGCTCGTGTGGCAGAAGCTGCTGCTGTGGGGCGCGGCGAGTGCCCTTTCCCTGGCCGGCGCCAGTCTGGTCCTGAGCCTGCTGCAGAGGGTGGCGAGCTACGCGCGGAAATGGCAGCAGATGCGGCCCATCCCCACGGTGGCCCGCGCCTACCCACTGGTGGGCCACGCGCTGCTGATGAAGCCGGACGGGCGAGAATTTTTTCAGCAGATCATTGAGTACACAGAGGAATACCGCCACATGCCGCTGCTGAAGCTCTGGGTCGGGCCAGTGCCCATGGTGGCCCTTTATAATGCAGAAAATGTGGAGGTAATTTTAACTAGTTCAAAGCAAATTGACAAATCCTCTATGTACAAGTTTTTAGAACCATGGCTTGGCCTAGGACTTCTTACAAGTACTGGAAACAAATGGCGCTCCAGGAGAAAGATGTTAACACCCACTTTCCATTTTACCATTCTGGAAGATTTCTTAGATATCATGAATGAACAAGCAAATATATTGGTTAAGAAACTTGAAAAACACATTAACCAAGAAGCATTTAACTGCTTTTTTTACATCACTCTTTGTGCCTTAGATATCATCTGTGAAACAGCTATGGGGAAGAATATTGGTGCTCAAAGTAATGATGATTCCGAGTATGTCCGTGCAGTTTATAGAATGAGTGAGATGATATTTCGAAGAATAAAGATGCCCTGGCTTTGGCTTGATCTCTGGTACCTTATGTTTAAAGAAGGATGGGAACACAAAAAGAGCCTTCAGATCCTACATACTTTTACCAACAGTGTCATCGCTGAACGGGCCAATGAAATGAACGCCAATGAAGACTGTAGAGGTGATGGCAGGGGCTCTGCCCCCTCCAAAAATAAACGCAGGGCCTTTCTTGACTTGCTTTTAAGTGTGACTGATGACGAAGGGAACAGGCTAAGTCATGAAGATATTCGAGAAGAAGTTGACACCTTCATGTTTGAGGGGCACGATACAACTGCAGCTGCAATAAACTGGTCCTTATACCTGTTGGGTTCTAACCCAGAAGTCCAGAAAAAAGTGGATCATGAATTGGATGACGTGTTTGGGAAGTCTGACCGTCCCGCTACAGTAGAAGACCTGAAGAAACTTCGGTATCTGGAATGTGTTATTAAGGAGACCCTTCGCCTTTTTCCTTCTGTTCCTTTATTTGCCCGTAGTGTTAGTGAAGATTGTGAAGTGGCAGGTTACAGAGTTCTAAAAGGCACTGAAGCCGTCATCATTCCCTATGCATTGCACAGAGATCCGAGATACTTCCCCAACCCCGAGGAGTTCCAGCCTGAGCGGTTCTTCCCCGAGAATGCACAAGGGCGCCATCCATATGCCTACGTGCCCTTCTCTGCTGGCCCCAGGAACTGTATAGGTCAAAAGTTTGCTGTGATGGAAGAAAAGACCATTCTTTCGTGCATCCTGAGGCACTTTTGGATAGAATCCAACCAGAAAAGAGAAGAGCTTGGTCTAGAAGGACAGTTGATTCTTCGTCCAAGTAATGGCATCTGGATCAAGTTGAAGAGGAGAAATGCAGATGAACGC

CYP4V2opt1 cDNA sequence (shown as SEQ ID No. 1):

<xnotran> ATGGCTGGACTTTGGCTGGGGCTTGTATGGCAGAAGCTCCTGCTGTGGGGCGCCGCGTCCGCGTTGTCCTTGGCCGGCGCCAGTCTTGTTTTGAGCCTGCTTCAACGGGTAGCATCCTACGCGAGAAAGTGGCAACAGATGAGACCGATCCCGACAGTTGCTCGGGCATATCCGCTTGTAGGCCATGCGCTCCTCATGAAACCAGACGGGCGAGAGTTTTTTCAGCAAATTATCGAATATACGGAAGAGTATCGCCACATGCCACTCCTGAAACTTTGGGTCGGCCCCGTCCCGATGGTTGCATTGTATAACGCTGAGAACGTGGAGGTAATACTTACATCCAGCAAGCAAATAGATAAATCATCTATGTACAAATTTTTGGAACCTTGGCTCGGGCTCGGTCTGCTCACCTCAACTGGCAATAAATGGCGGAGCCGCCGCAAAATGCTGACACCTACCTTTCACTTCACAATTCTCGAGGATTTTTTGGATATTATGAACGAGCAGGCGAACATCCTCGTCAAAAAGCTTGAAAAGCACATCAACCAGGAGGCGTTTAACTGCTTTTTCTACATTACACTGTGCGCTCTTGATATAATATGCGAGACAGCTATGGGAAAAAATATAGGTGCCCAGTCCAACGACGATTCCGAGTATGTTCGAGCGGTTTACCGCATGTCAGAGATGATATTCAGGCGAATTAAAATGCCATGGCTCTGGCTTGATTTGTGGTATTTGATGTTCAAAGAAGGCTGGGAACACAAAAAGTCTCTCCAAATTTTGCATACTTTCACTAATTCAGTAATTGCTGAGAGAGCGAACGAAATGAACGCAAACGAGGACTGTCGGGGGGATGGGAGGGGGTCTGCTCCTTCCAAGAATAAGAGGAGGGCTTTCCTGGATCTCTTGTTGTCCGTAACGGATGACGAAGGCAATAGACTCTCTCATGAGGATATTCGAGAAGAGGTCGACACGTTTATGTTTGAAGGGCATGATACAACCGCTGCAGCCATAAACTGGTCACTCTACTTGCTCGGGTCCAATCCTGAGGTGCAGAAAAAAGTCGATCACGAGTTGGACGACGTATTTGGTAAAAGCGATCGACCCGCGACGGTCGAAGATCTGAAGAAGTTGCGATACCTTGAATGTGTTATTAAAGAGACTCTTCGCTTGTTTCCTAGCGTTCCGCTCTTCGCGAGAAGCGTCAGTGAGGACTGTGAAGTCGCTGGCTATAGGGTTCTCAAGGGAACCGAGGCAGTGATCATACCCTACGCTCTCCACCGGGATCCTCGATATTTTCCAAACCCTGAAGAGTTCCAACCGGAAAGGTTTTTTCCCGAAAACGCACAGGGGAGACATCCATACGCCTACGTTCCGTTCTCAGCCGGACCGAGGAATTGTATAGGTCAGAAATTTGCGGTAATGGAGGAAAAGACAATCCTCAGCTGTATTCTTCGCCACTTCTGGATCGAGTCCAATCAGAAACGCGAGGAGCTCGGCTTGGAAGGCCAACTTATACTGCGCCCTAGTAATGGCATCTGGATAAAACTCAAAAGACGCAACGCTGACGAAAGACYP4V2opt 2cDNA ( SEQ ID No.2 ): </xnotran>

<xnotran> ATGGCAGGATTGTGGCTTGGATTGGTGTGGCAGAAATTGTTGCTGTGGGGCGCCGCCTCTGCATTGTCACTGGCTGGAGCTAGCTTGGTGCTTAGCCTGCTGCAGCGCGTCGCAAGCTATGCCAGGAAATGGCAGCAGATGAGGCCGATACCAACCGTAGCACGGGCTTACCCACTCGTGGGCCATGCACTCCTGATGAAGCCCGACGGTCGCGAATTCTTTCAGCAGATTATCGAATACACCGAGGAGTATCGACACATGCCCCTGCTCAAGCTGTGGGTGGGACCTGTGCCCATGGTCGCACTGTATAATGCAGAGAATGTGGAGGTGATTCTCACATCAAGCAAGCAAATCGATAAATCATCAATGTACAAGTTTTTGGAACCATGGCTCGGACTGGGCCTGCTGACATCCACAGGGAACAAGTGGAGGAGCCGCAGAAAAATGTTGACGCCCACGTTCCACTTCACCATCCTTGAGGACTTCCTCGATATCATGAACGAGCAGGCCAATATTCTCGTTAAGAAACTGGAAAAGCATATTAACCAGGAAGCATTCAACTGCTTCTTTTACATCACACTGTGCGCCCTGGATATTATTTGCGAAACCGCCATGGGGAAGAACATCGGAGCACAATCTAATGATGATTCAGAATATGTGAGAGCTGTGTACCGGATGTCTGAAATGATCTTTAGGCGGATTAAAATGCCCTGGCTTTGGCTTGACCTGTGGTATTTGATGTTTAAGGAGGGATGGGAGCATAAAAAGTCACTTCAGATTCTGCACACTTTCACAAACAGCGTGATTGCTGAAAGAGCTAATGAGATGAACGCTAATGAAGACTGTAGAGGCGACGGAAGAGGCTCTGCCCCATCAAAGAATAAGAGACGCGCGTTCCTGGACCTGCTTTTGAGTGTGACCGACGACGAGGGCAACAGACTCAGTCACGAGGATATTCGGGAGGAAGTCGATACCTTCATGTTTGAGGGTCACGACACAACGGCCGCAGCCATCAACTGGAGCCTCTATCTCCTGGGCAGCAACCCCGAGGTGCAAAAGAAAGTCGACCATGAACTGGATGACGTTTTTGGCAAGAGTGACCGACCTGCAACCGTGGAGGATCTTAAGAAGCTCAGATATCTGGAGTGCGTGATTAAAGAAACCCTCAGACTGTTCCCCAGCGTTCCTTTGTTCGCTCGGTCAGTGTCCGAGGACTGTGAAGTGGCTGGCTATAGGGTCCTCAAAGGAACCGAGGCCGTCATTATACCATATGCGTTGCATCGCGACCCCAGGTATTTCCCAAACCCCGAGGAGTTTCAGCCTGAGCGATTTTTTCCAGAGAACGCTCAGGGAAGACACCCTTACGCGTATGTGCCCTTCTCTGCCGGTCCTCGAAACTGTATTGGACAGAAATTTGCTGTTATGGAGGAAAAGACAATCTTGTCCTGTATCCTGCGCCACTTCTGGATTGAGTCAAATCAGAAAAGGGAAGAGTTGGGCCTCGAGGGGCAACTGATCCTTCGCCCATCTAATGGGATTTGGATAAAGCTCAAACGGAGAAATGCCGACGAGCGACAG ( SEQ ID No.3 ): </xnotran>

GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAG

bGH poly A sequence (shown as SEQ ID No. 4):

CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGA

example 2 AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA drugs can effectively improve the lipid metabolism function deficiency pathological virus infected cells caused by CYP4V2 mutation

AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA viruses infect RPE cells with CYP4V2 mutation, and the specific steps are shown in the virus package, cell slide polylysine coating and virus infected cell part of example 1.

CYP4V2 enzyme activity assay

1. 72h after infection of the cells, dodecanoic acid and GAPDH were added to the medium at two concentrations, 125uM (Km) and 250uM (Vmax), respectively, and incubated for 30min.

2. The pH of the medium was adjusted to around 3-4 with 2M sulfuric acid solution and the metabolites in the cell culture were extracted with ethyl acetate solution.

3. After concentrating the solution in a vacuum concentrator, the remaining concentrate was dissolved in 100ul of ethanol.

4. The content of the hydroxylated dodecanoic acid in the solution was determined by LC-MS, 100pg/ml of 20-HETE-6 was used as an internal reference, the mobile phase was water and acetonitrile containing 0.1% formic acid, the chromatographic column was an Atlantis dC18 reverse phase column, the loading temperature was 4 ℃, the loading volume was 3ul, and the flow rate of the mobile phase was 0.25ml/min.

Experimental results and discussion

Previous experiments have confirmed the in vitro expression efficiency of CYP4V2 vectors and viral drugs, and in order to further test the function of AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA drug expression proteins, pharmacodynamically verified drug-treated CYP4V2 mutant RPE cells.

We infected CYP4V2 mutant RPE cells with different viral drugs and then assayed the concentration of the catalytic product hydroxylated dodecanoic acid after incubating the cells with the lipid metabolism substrate dodecanoic acid for a period of time. As a result, it was found that: the enzyme activity of CYP4V2 in the CYP4V2 mutant RPE cells which are not treated by the drug is obviously reduced, and products generated under the conditions of two substrate concentrations (125 uM and 250 uM) are obviously reduced compared with the wild RPE cells (figure 6); after AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA drug treatment, the enzyme activity of CYP4V2 in CYP4V2 mutant RPE cell is recovered to a certain degree, products generated under two substrate concentration conditions (125 uM and 250 uM) respectively reach 80-83% and 72-78% of wild RPE cell (figure 6), and specific values are shown in Table 2 and Table 3. When RPE cells are incubated with dodecanoic acid and GAPDH, the expressed CYP4V2 protein can catalyze dodecanoic acid to generate hydroxylated dodecanoic acid, and the concentration of the generated product reflects the enzyme activity of CYP4V2 under the condition that the number of cells and the concentration of a substrate are the same. The enzyme activity of CYP4V2 mutant RPE cells is reduced due to protein function deficiency, the fatty acid metabolism capability is recovered through two drug treatments, and a substrate is catalyzed to generate a corresponding hydroxylation product. The results prove that the AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA drugs can recover the lipid metabolism defect caused by CYP4V2 mutation.

TABLE 2 concentration of hydroxylated dodecanoic acid produced by drug-treated mutant RPE cells at 125uM substrate concentration (umol/min/mg)

TABLE 3 concentration of hydroxylated dodecanoic acid produced by drug-treated mutant RPE cells at 250uM substrate concentration (umol/min/mg)

By combining the results, we prove that the therapeutic effect of the AAV5-CAG-CYP4V2opt1-bGHpolyA and AAV5-CAG-CYP4V2opt2-bGHpolyA gene therapeutic drugs on Bietti Crystal Dystrophy (BCD) caused by CYP4V2 mutation lays a foundation for further clinical application and development.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Sequence listing

<110> Wuhan Newcastle Biotechnology Ltd

<120> nucleic acid molecule, expression vector and use thereof

<130> MP21009169

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1575

<212> DNA

<213> Artificial sequence

<400> 1

atggctggac tttggctggg gcttgtatgg cagaagctcc tgctgtgggg cgccgcgtcc 60

gcgttgtcct tggccggcgc cagtcttgtt ttgagcctgc ttcaacgggt agcatcctac 120

gcgagaaagt ggcaacagat gagaccgatc ccgacagttg ctcgggcata tccgcttgta 180

ggccatgcgc tcctcatgaa accagacggg cgagagtttt ttcagcaaat tatcgaatat 240

acggaagagt atcgccacat gccactcctg aaactttggg tcggccccgt cccgatggtt 300

gcattgtata acgctgagaa cgtggaggta atacttacat ccagcaagca aatagataaa 360

tcatctatgt acaaattttt ggaaccttgg ctcgggctcg gtctgctcac ctcaactggc 420

aataaatggc ggagccgccg caaaatgctg acacctacct ttcacttcac aattctcgag 480

gattttttgg atattatgaa cgagcaggcg aacatcctcg tcaaaaagct tgaaaagcac 540

atcaaccagg aggcgtttaa ctgctttttc tacattacac tgtgcgctct tgatataata 600

tgcgagacag ctatgggaaa aaatataggt gcccagtcca acgacgattc cgagtatgtt 660

cgagcggttt accgcatgtc agagatgata ttcaggcgaa ttaaaatgcc atggctctgg 720

cttgatttgt ggtatttgat gttcaaagaa ggctgggaac acaaaaagtc tctccaaatt 780

ttgcatactt tcactaattc agtaattgct gagagagcga acgaaatgaa cgcaaacgag 840

gactgtcggg gggatgggag ggggtctgct ccttccaaga ataagaggag ggctttcctg 900

gatctcttgt tgtccgtaac ggatgacgaa ggcaatagac tctctcatga ggatattcga 960

gaagaggtcg acacgtttat gtttgaaggg catgatacaa ccgctgcagc cataaactgg 1020

tcactctact tgctcgggtc caatcctgag gtgcagaaaa aagtcgatca cgagttggac 1080

gacgtatttg gtaaaagcga tcgacccgcg acggtcgaag atctgaagaa gttgcgatac 1140

cttgaatgtg ttattaaaga gactcttcgc ttgtttccta gcgttccgct cttcgcgaga 1200

agcgtcagtg aggactgtga agtcgctggc tatagggttc tcaagggaac cgaggcagtg 1260

atcataccct acgctctcca ccgggatcct cgatattttc caaaccctga agagttccaa 1320

ccggaaaggt tttttcccga aaacgcacag gggagacatc catacgccta cgttccgttc 1380

tcagccggac cgaggaattg tataggtcag aaatttgcgg taatggagga aaagacaatc 1440

ctcagctgta ttcttcgcca cttctggatc gagtccaatc agaaacgcga ggagctcggc 1500

ttggaaggcc aacttatact gcgccctagt aatggcatct ggataaaact caaaagacgc 1560

aacgctgacg aaaga 1575

<210> 2

<211> 1575

<212> DNA

<213> Artificial sequence

<400> 2

atggcaggat tgtggcttgg attggtgtgg cagaaattgt tgctgtgggg cgccgcctct 60

gcattgtcac tggctggagc tagcttggtg cttagcctgc tgcagcgcgt cgcaagctat 120

gccaggaaat ggcagcagat gaggccgata ccaaccgtag cacgggctta cccactcgtg 180

ggccatgcac tcctgatgaa gcccgacggt cgcgaattct ttcagcagat tatcgaatac 240

accgaggagt atcgacacat gcccctgctc aagctgtggg tgggacctgt gcccatggtc 300

gcactgtata atgcagagaa tgtggaggtg attctcacat caagcaagca aatcgataaa 360

tcatcaatgt acaagttttt ggaaccatgg ctcggactgg gcctgctgac atccacaggg 420

aacaagtgga ggagccgcag aaaaatgttg acgcccacgt tccacttcac catccttgag 480

gacttcctcg atatcatgaa cgagcaggcc aatattctcg ttaagaaact ggaaaagcat 540

attaaccagg aagcattcaa ctgcttcttt tacatcacac tgtgcgccct ggatattatt 600

tgcgaaaccg ccatggggaa gaacatcgga gcacaatcta atgatgattc agaatatgtg 660

agagctgtgt accggatgtc tgaaatgatc tttaggcgga ttaaaatgcc ctggctttgg 720

cttgacctgt ggtatttgat gtttaaggag ggatgggagc ataaaaagtc acttcagatt 780

ctgcacactt tcacaaacag cgtgattgct gaaagagcta atgagatgaa cgctaatgaa 840

gactgtagag gcgacggaag aggctctgcc ccatcaaaga ataagagacg cgcgttcctg 900

gacctgcttt tgagtgtgac cgacgacgag ggcaacagac tcagtcacga ggatattcgg 960

gaggaagtcg ataccttcat gtttgagggt cacgacacaa cggccgcagc catcaactgg 1020

agcctctatc tcctgggcag caaccccgag gtgcaaaaga aagtcgacca tgaactggat 1080

gacgtttttg gcaagagtga ccgacctgca accgtggagg atcttaagaa gctcagatat 1140

ctggagtgcg tgattaaaga aaccctcaga ctgttcccca gcgttccttt gttcgctcgg 1200

tcagtgtccg aggactgtga agtggctggc tatagggtcc tcaaaggaac cgaggccgtc 1260

attataccat atgcgttgca tcgcgacccc aggtatttcc caaaccccga ggagtttcag 1320

cctgagcgat tttttccaga gaacgctcag ggaagacacc cttacgcgta tgtgcccttc 1380

tctgccggtc ctcgaaactg tattggacag aaatttgctg ttatggagga aaagacaatc 1440

ttgtcctgta tcctgcgcca cttctggatt gagtcaaatc agaaaaggga agagttgggc 1500

ctcgaggggc aactgatcct tcgcccatct aatgggattt ggataaagct caaacggaga 1560

aatgccgacg agcga 1575

<210> 3

<211> 1676

<212> DNA

<213> Artificial sequence

<400> 3

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct 420

cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga 480

tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg 540

gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 600

cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 660

gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc 720

cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 780

cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa 840

gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg 900

tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct 960

gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg 1020

gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt 1080

gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc 1140

cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg 1200

gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg 1260

ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg 1320

gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc 1380

agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc 1440

cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg 1500

gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc 1560

ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga 1620

ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacag 1676

<210> 4

<211> 208

<212> DNA

<213> Artificial sequence

<400> 4

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180

gggaagagaa tagcaggcat gctgggga 208

<210> 5

<211> 525

<212> PRT

<213> Artificial sequence

<400> 5

Met Ala Gly Leu Trp Leu Gly Leu Val Trp Gln Lys Leu Leu Leu Trp

1 5 10 15

Gly Ala Ala Ser Ala Leu Ser Leu Ala Gly Ala Ser Leu Val Leu Ser

20 25 30

Leu Leu Gln Arg Val Ala Ser Tyr Ala Arg Lys Trp Gln Gln Met Arg

35 40 45

Pro Ile Pro Thr Val Ala Arg Ala Tyr Pro Leu Val Gly His Ala Leu

50 55 60

Leu Met Lys Pro Asp Gly Arg Glu Phe Phe Gln Gln Ile Ile Glu Tyr

65 70 75 80

Thr Glu Glu Tyr Arg His Met Pro Leu Leu Lys Leu Trp Val Gly Pro

85 90 95

Val Pro Met Val Ala Leu Tyr Asn Ala Glu Asn Val Glu Val Ile Leu

100 105 110

Thr Ser Ser Lys Gln Ile Asp Lys Ser Ser Met Tyr Lys Phe Leu Glu

115 120 125

Pro Trp Leu Gly Leu Gly Leu Leu Thr Ser Thr Gly Asn Lys Trp Arg

130 135 140

Ser Arg Arg Lys Met Leu Thr Pro Thr Phe His Phe Thr Ile Leu Glu

145 150 155 160

Asp Phe Leu Asp Ile Met Asn Glu Gln Ala Asn Ile Leu Val Lys Lys

165 170 175

Leu Glu Lys His Ile Asn Gln Glu Ala Phe Asn Cys Phe Phe Tyr Ile

180 185 190

Thr Leu Cys Ala Leu Asp Ile Ile Cys Glu Thr Ala Met Gly Lys Asn

195 200 205

Ile Gly Ala Gln Ser Asn Asp Asp Ser Glu Tyr Val Arg Ala Val Tyr

210 215 220

Arg Met Ser Glu Met Ile Phe Arg Arg Ile Lys Met Pro Trp Leu Trp

225 230 235 240

Leu Asp Leu Trp Tyr Leu Met Phe Lys Glu Gly Trp Glu His Lys Lys

245 250 255

Ser Leu Gln Ile Leu His Thr Phe Thr Asn Ser Val Ile Ala Glu Arg

260 265 270

Ala Asn Glu Met Asn Ala Asn Glu Asp Cys Arg Gly Asp Gly Arg Gly

275 280 285

Ser Ala Pro Ser Lys Asn Lys Arg Arg Ala Phe Leu Asp Leu Leu Leu

290 295 300

Ser Val Thr Asp Asp Glu Gly Asn Arg Leu Ser His Glu Asp Ile Arg

305 310 315 320

Glu Glu Val Asp Thr Phe Met Phe Glu Gly His Asp Thr Thr Ala Ala

325 330 335

Ala Ile Asn Trp Ser Leu Tyr Leu Leu Gly Ser Asn Pro Glu Val Gln

340 345 350

Lys Lys Val Asp His Glu Leu Asp Asp Val Phe Gly Lys Ser Asp Arg

355 360 365

Pro Ala Thr Val Glu Asp Leu Lys Lys Leu Arg Tyr Leu Glu Cys Val

370 375 380

Ile Lys Glu Thr Leu Arg Leu Phe Pro Ser Val Pro Leu Phe Ala Arg

385 390 395 400

Ser Val Ser Glu Asp Cys Glu Val Ala Gly Tyr Arg Val Leu Lys Gly

405 410 415

Thr Glu Ala Val Ile Ile Pro Tyr Ala Leu His Arg Asp Pro Arg Tyr

420 425 430

Phe Pro Asn Pro Glu Glu Phe Gln Pro Glu Arg Phe Phe Pro Glu Asn

435 440 445

Ala Gln Gly Arg His Pro Tyr Ala Tyr Val Pro Phe Ser Ala Gly Pro

450 455 460

Arg Asn Cys Ile Gly Gln Lys Phe Ala Val Met Glu Glu Lys Thr Ile

465 470 475 480

Leu Ser Cys Ile Leu Arg His Phe Trp Ile Glu Ser Asn Gln Lys Arg

485 490 495

Glu Glu Leu Gly Leu Glu Gly Gln Leu Ile Leu Arg Pro Ser Asn Gly

500 505 510

Ile Trp Ile Lys Leu Lys Arg Arg Asn Ala Asp Glu Arg

515 520 525

<210> 6

<211> 1575

<212> DNA

<213> Artificial sequence

<400> 6

atggcggggc tctggctggg gctcgtgtgg cagaagctgc tgctgtgggg cgcggcgagt 60

gccctttccc tggccggcgc cagtctggtc ctgagcctgc tgcagagggt ggcgagctac 120

gcgcggaaat ggcagcagat gcggcccatc cccacggtgg cccgcgccta cccactggtg 180

ggccacgcgc tgctgatgaa gccggacggg cgagaatttt ttcagcagat cattgagtac 240

acagaggaat accgccacat gccgctgctg aagctctggg tcgggccagt gcccatggtg 300

gccctttata atgcagaaaa tgtggaggta attttaacta gttcaaagca aattgacaaa 360

tcctctatgt acaagttttt agaaccatgg cttggcctag gacttcttac aagtactgga 420

aacaaatggc gctccaggag aaagatgtta acacccactt tccattttac cattctggaa 480

gatttcttag atatcatgaa tgaacaagca aatatattgg ttaagaaact tgaaaaacac 540

attaaccaag aagcatttaa ctgctttttt tacatcactc tttgtgcctt agatatcatc 600

tgtgaaacag ctatggggaa gaatattggt gctcaaagta atgatgattc cgagtatgtc 660

cgtgcagttt atagaatgag tgagatgata tttcgaagaa taaagatgcc ctggctttgg 720

cttgatctct ggtaccttat gtttaaagaa ggatgggaac acaaaaagag ccttcagatc 780

ctacatactt ttaccaacag tgtcatcgct gaacgggcca atgaaatgaa cgccaatgaa 840

gactgtagag gtgatggcag gggctctgcc ccctccaaaa ataaacgcag ggcctttctt 900

gacttgcttt taagtgtgac tgatgacgaa gggaacaggc taagtcatga agatattcga 960

gaagaagttg acaccttcat gtttgagggg cacgatacaa ctgcagctgc aataaactgg 1020

tccttatacc tgttgggttc taacccagaa gtccagaaaa aagtggatca tgaattggat 1080

gacgtgtttg ggaagtctga ccgtcccgct acagtagaag acctgaagaa acttcggtat 1140

ctggaatgtg ttattaagga gacccttcgc ctttttcctt ctgttccttt atttgcccgt 1200

agtgttagtg aagattgtga agtggcaggt tacagagttc taaaaggcac tgaagccgtc 1260

atcattccct atgcattgca cagagatccg agatacttcc ccaaccccga ggagttccag 1320

cctgagcggt tcttccccga gaatgcacaa gggcgccatc catatgccta cgtgcccttc 1380

tctgctggcc ccaggaactg tataggtcaa aagtttgctg tgatggaaga aaagaccatt 1440

ctttcgtgca tcctgaggca cttttggata gaatccaacc agaaaagaga agagcttggt 1500

ctagaaggac agttgattct tcgtccaagt aatggcatct ggatcaagtt gaagaggaga 1560

aatgcagatg aacgc 1575

<210> 7

<211> 1550

<212> DNA

<213> Artificial sequence

<400> 7

ggtcctgcac tattgcctct gcagctgccc tcatccctgc atcccctctc ccagccgcct 60

gtgccttctt ggtcttcctt gaatatgtca gccaggctcc agagtcaagg cctttggctg 120

ctgcctctgc ctggaaatcc tcctcccaca tccctgccca gtgggctcct tcaccagcca 180

cctgctccca gtctctgttc aaaagccatc ttctcagaga cctttccttt ttcttttttt 240

tttttgagac ggagtctcac tctgtcaccc aggctggagt gcagtggcac gatctcggct 300

cactgcaagc tccgcctccc gggtttacgc cattctcctg cctcagcttc ccgagtagct 360

gggactacag gtgcccgcca ccacgcctgg ctaatttttt gttttttagt acagacgggg 420

tttcaccgtg ttagccagga tggtcctcag agacctttcc tatttaaaat tgccaccgcc 480

cccagcccgg cctttctctc tgctttcttt ccctgcctgg ctcttacacc ctctggcatg 540

ctatatagtt tattgctcga ggcatctgcc tccccatgat aaggtcagtt ccaggaaggc 600

agggactctc ccctgctgtt cccagatgta tccctagcac ttgatgtata caatatgtgg 660

cccatagtac cagctcaaaa tatatcagtt gaattcactt caggggtgtg agagatagag 720

gggaacagag ggccggcccc gcaccataag agatgtgttg gttgtaagcc agaccccaag 780

aggtggaggt catgcctcca ccctccaaac ttccacagcc gacaggcctc ccagaggagg 840

tggtgggtcc tggcctcaca gctatgaggg ctccacagcc ttctattatt ttattttcag 900

cttgctggtc cttagataaa aatctatcta agacgatggg aatacatggc ctgttctctc 960

tgagctccag tctgttccct tgcatgtggg aggtgacatg catgtgcctg taagagacag 1020

aggagggtcg gcagcaaggg tggtacactt caggccaggc ttgtgctgca aagacccatg 1080

accccatctg cctttaaaag gggcaagaca ggcacccctg aaatcagaaa tgacacagag 1140

agccccacac acagagcagc acagaacaga cgggccaccg gccacaggat cttcagaaga 1200

aggcttgctg tgtcgctgcc agattaacgg ggcgaaaatg cgcactggca gctccaagga 1260

gagagctgag acccacagcc tgggcctgga tgcaaattct tctctgagac tcaggcggtt 1320

ggccaggtgg gcaccgggcc tctgggatct ggatgccacc ctccaagggg cctcagatgc 1380

cagcagcaag gggctgtctg ccctcgcaat gcgcctttcc agacgcccaa atccttccca 1440

tttgaccacc ctttgcagcc accactttct caaagtagat ttactttcaa tctccatttg 1500

ctaaatctct tccctctaaa ccttttgggg atggagggat taagcctgat 1550

<210> 8

<211> 16

<212> DNA

<213> Artificial sequence

<400> 8

agaccatgac ggtgat 16

<210> 9

<211> 17

<212> DNA

<213> Artificial sequence

<400> 9

cttgtcatcg tcatcct 17

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<400> 10

ggacttcgag caagagatgg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<400> 11

aggaaggaag gctggaagag 20

Claims

1. A nucleic acid molecule, characterized in that it has:

(I) a nucleotide sequence shown as SEQ ID No.1 or 2; or

(III) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of (I) or (II).

2. The nucleic acid molecule of claim 1, encoding a protein as set forth in SEQ ID No. 5.

3. An expression vector comprising the nucleic acid molecule of claim 1 or 2.

4. The expression vector of claim 3, further comprising a promoter and a polyA element.

5. The expression vector of claim 4, wherein the promoter is a CAG promoter as shown in SEQ ID No. 3; the polyA element is a bGH polyA element as shown in SEQ ID No. 4.

6. The expression vector of any one of claims 3-5, wherein the expression vector is a viral vector, and wherein the viral vector is one of an adeno-associated virus, a lentivirus, a retrovirus, or an adenovirus.

7. The expression vector of claim 6, wherein the viral vector is an adeno-associated viral vector and the serotype of the adeno-associated virus is selected from one or more of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 wild type or 7M8, TYF mutant.

8. Use of a nucleic acid molecule according to claim 1 or 2, an expression vector according to any one of claims 3 to 7 for the preparation of a medicament for the prevention and/or treatment of bithio crystal dystrophy.

9. Pharmaceutical preparation comprising a nucleotide sequence according to claim 1 or 2 or an expression vector according to any one of claims 3 to 7, and a pharmaceutically acceptable excipient.

10. A method of drug delivery comprising administering the pharmaceutical formulation of claim 9 to the eye by subretinal administration or intravitreal administration.