CN116179597A - Preparation and Application of ND6 Protein Using Human Mitochondrial ND6 Gene Recombination Vector - Google Patents

Abstract

The invention discloses a ND6 protein prepared by utilizing a human mitochondrial ND6 gene recombinant vector and application thereof. The vector synthesizes and optimizes the ND6 nucleotide sequence encoded by the nuclear codon through a total gene synthesis method, and constructs the human ND6 gene reconstruction vector. The nucleotide sequence of the human ND6 gene recombinant vector is shown as SEQ ID NO. 5. The invention also discloses an amino acid sequence of the ND6 protein prepared by the method, a mitochondrial targeting sequence, a CMV promoter sequence and an expression vector sequence, and the ND6 protein preparation method and the preparation used by the ND6 protein preparation method. The human ND6 gene recombinant vector can be efficiently expressed in cells, and the prepared ND6 protein can target mitochondria, play a role of normal ND6 protein, and relieve the influence of ND6 protein abnormality caused by m.14484T > C mutation on the functions of the cells.

Description

Preparation and Application of ND6 Protein Using Human Mitochondrial ND6 Gene Recombination Vector

technical field

The invention belongs to the field of biological preparations, and relates to the preparation of ND6 protein using a human mitochondrial ND6 gene recombination carrier and its application. The application is the application of the human ND6 gene recombination carrier in the preparation of an ND6 protein kit.

Background technique

Leber hereditary optic neuropathy (LHON) is a degenerative mitochondrial genetic disease of the optic nerve caused by mutations in mitochondrial DNA (mtDNA). In the early stage, it can be manifested as changes in arterioles around the optic nerve head and swelling of the retinal nerve fiber layer. The disease is more common in young men, and the age of onset is concentrated in 20-30 years old. Most patients have bilateral or successive painless vision loss, and most patients have vision loss below 0.1.

Leber hereditary optic neuropathy is mainly related to the mutation of one of the three mitochondrial genes of NADH ubiquinone oxidoreductase, which are m. The ND6 amino acid sequence of respiratory chain complex Ⅰ changes, the activity of complex Ⅰ decreases, the oxidative phosphorylation process of optic nerve cells is affected, and the ATP production decreases. The patient develops LHON disease and the prognosis is poor.

] There is no effective clinical treatment for LHON at present, but with the advancement of gene therapy technology, by injecting recombinant adeno-associated virus agents into the vitreous cavity of the eye to infect the optic nerve cells, and introducing the exogenous wild-type normal protein into the mitochondria to play a role, it can reach The purpose of treating LHON, but this technology still has the disadvantages of low protein expression efficiency and low targeting of intracellular expression, which affects the therapeutic effect and needs to be improved.

Contents of the invention

The purpose of the present invention is to provide a human mitochondrial ND6 gene recombination vector to prepare ND6 protein, to solve the technical problems of low expression of ND6 protein targeting human mitochondria and low protein targeting. By optimizing the ND6 gene sequence and ND6 protein preparation system, the human ND6 gene recombinant vector was introduced into cells, encoding normal ND6 protein and localized to mitochondria, thereby alleviating Leber hereditary optic neuropathy caused by the m.14484T>C primary mutation.

One of the technical solutions of the present invention is to provide a human ND6 gene recombination vector, the gene sequence of the human ND6 gene recombination vector is the CMV promoter from the N-terminal to the C-terminal, the mitochondrial targeting sequence COX8A-MTS and the optimized The nuclear codon-encoded nucleotide sequence of human ND6.

Preferably, the amino acid sequence of the ND6 protein encoded by the nuclear codon is shown in SEQ ID NO:6.

Preferably, the mitochondrial targeting sequence COX8A-MTS is shown in SEQ ID NO:2.

Preferably, the nucleotide sequence of the CMV promoter is shown in SEQ ID NO:1.

Another object of the present invention is to provide the application of the human ND6 gene recombinant vector in the preparation of ND6 protein kit. The ND6 protein encoded by the human ND6 recombinant vector must be accurately targeted to human mitochondria to function correctly. The mitochondrial targeting sequence Mitochondrial Targeting Signal (MTS) can guide the ND6 protein encoded in the cell to the mitochondria. Therefore, the nuclear gene encoded The ND6 protein must be linked to a mitochondrial targeting sequence.

In order to solve the above problems, one of the technical solutions of the present invention is to provide a Mitochondrial Targeting Signal (MTS) of COX8A as a specific mitochondrial protein guide sequence, so that the targeting effect of the fusion protein is better, and ND6 can be accurately and efficiently introduced into the mitochondria , the mitochondrial targeting sequence COX8A-MTS is shown in SEQ ID NO:2.

In order to solve the above problems, one of the technical solutions of the present invention is to provide a recombinant expression vector, which contains an optimized nucleotide sequence of the ND6 gene, which can be introduced into cells and normally express the ND6 protein targeting human mitochondria. It is pAAV-ZsGreen, and its nucleotide sequence is shown in SEQ ID NO:4.

Preferably, in the recombinant expression vector, the optimized nucleotide sequence of the ND6 gene targeting human mitochondrial ND6 protein is shown in SEQ ID NO:3.

A transformant capable of introducing a foreign recombinant vector into a host cell is provided.

The present invention provides mammalian cells capable of expressing ND6 protein, preferably, the mammalian cells are 293T cells (ie human embryonic kidney cells).

The present invention provides cells capable of detecting ND6 protein. The cells are preferably mammalian cells, and more preferably, m.14484T>C mutant cytoplasmic fusion cells. The cells use 143B cells purchased from Hangzhou Bajiao Biotechnology Co., Ltd. to remove Mitochondria were transformed into mitochondria containing the m.14484T>C mutation site to construct.

The present invention provides the recombinant carrier or protein preparation of the above-mentioned ND6 protein targeting human mitochondria. The sequence of the recombinant carrier is shown in SEQ ID NO:5, and the amino acid sequence of the protein preparation is shown in SEQ ID NO:6, which can be used in the preparation and treatment of Leber hereditary Use of drugs in optic neuropathy.

The reagents and raw materials used in the present invention are all commercially available. On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain the preferred examples of the present invention.

The present invention is the first to add COX8A Mitochondrial Targeting Signal (MTS) at the 5' end of ND6 as a specific mitochondrial protein guide sequence, so that the protein targeting effect is better, the ND6 protein can be accurately and efficiently expressed, and the ND6 protein is introduced into the mitochondria And exert its function, it provides a broad application prospect for the treatment of Leber hereditary optic neuropathy.

The positive progress effect of the present invention lies in: the present invention uses the method of whole gene synthesis to construct the ND6 nucleotide sequence encoded by the nuclear codon, so that after the recombinant expression vector is introduced into the cell, it can be correctly translated into a functional ND6 protein in the cell. At the same time, the mitochondrial positioning sequence is connected to the ND6 expression vector sequence, which can target and guide the ND6 protein to correctly locate in the mitochondria and perform functions, providing an application prospect for the treatment of Leber's hereditary optic neuropathy.

Description of drawings

Figure 1 is a schematic diagram of the structure of the recombinant pAAV-hrND6 expression vector.

Figure 2 is an immunofluorescence image.

Figure 3 is a diagram showing the expression of Flag protein in cells.

Fig. 4 is a diagram of ND6 protein expression in cells.

Fig. 5 is a graph showing the expression analysis of exogenous ND6 mRNA detected by qPCR.

Fig. 6 is an analysis chart of detection of cellular reactive oxygen species.

Detailed ways

The present invention will be further elaborated below in conjunction with the accompanying drawings and by way of the examples. Under the situation of conforming to the scope of the present invention, the optimization and modification of the technical solution of the present invention all belong to the protection scope of the present invention. No specific conditions and conditions are specified in the examples. For the test method, follow the routine test method and product instructions.

AgeI/BgIII enzyme (ThermoFisher).

AxyPrep ^™ PCR Cleanup Kit (AxyGEN).

transfection reagent(jetPRIME)。

transfection reagent (jetPRIME).

MT-ND6 Rabbit pAb (ABclonal A17991).

Anti-GAPDH (Proteintech Cat No. 60004-1-Ig).

Mouse anti DDDDK-Tag mAb (ABclonal AE005).

Example 1 Construction of recombinant expression vector of ND6 protein targeting human mitochondria.

The optimized ND6 nucleotide sequence shown in SEQ ID NO:3 and the COX8A-MTS sequence shown in SEQ ID NO:2 are in the order of COX8A-MTS and ND6 nucleotide sequence from N-terminal to C-terminal The sequence was obtained by whole gene synthesis method, and the sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd.

Add AgeI sequence to the 5' end of the obtained sequence, add BgIII sequence to the 3' end, digest the 5' end and 3' end with AgeI sequence and BgIII respectively, clone the gene into the pAAV-ZsGreen vector, and name the recombinant expression vector pAAV2 -hrND6, the nucleotide sequence is shown in SEQ ID NO: 5, and the schematic structure of the recombinant pAAV-hrND6 expression vector is shown in Figure 1 .

Example 2 Construction of recombinant expression vector containing ND6Flag gene targeting human mitochondria.

The optimized ND6 nucleotide sequence shown in SEQ ID NO: 3 and the COX8A-MTS sequence shown in SEQ ID NO: 2 and the nucleotide sequence of the Flag protein shown in SEQ ID NO: 7, according to the N-terminal To the C-terminus, the sequence of COX8A-MTS, ND6 nucleotide sequence, and Flag protein nucleotide sequence is sequentially connected, and the sequence is obtained by the whole gene synthesis method, and the sequence is synthesized by Beijing Qingke Biotechnology Co., Ltd. At the same time, add AgeI sequence to the 5' end of the obtained sequence, add BgIII sequence to the 3' end, digest the 5' end and 3' end with AgeI and BgIII respectively, clone the gene into the pAAV-ZsGreen vector, and name the recombinant expression vector It is pAAV2-hrND6Flag, and its nucleotide sequence is shown in SEQ ID NO:8.

Screening and Identification of Example 3 Recombinant Vector Positive Clones

The recombinant vectors prepared in Example 1 and Example 2 were respectively introduced into DH5αChemically CompetentCell, spread on the Amp ⁺ LB plate, cultured in a 37°C incubator for 20 hours, and then selected monoclonal colonies, and added to LB liquid culture containing 100mg/L Amp After incubating at 37°C and 240 rpm for 15 hours in the medium, the plasmid was extracted, digested with AgeⅠ/BgIII double enzymes, and the size of the DNA fragment was identified by agarose gel electrophoresis.

Sequencing identification of the plasmid (Hangzhou Qingke Biotechnology Co., Ltd.), the specific primers required for sequencing are:

1F: 5'-CGCAAATGGGCGGTAGGCGTG-3'

1R: 5'-CCAGCTTGGTTCCCAATAGA-3'

The plasmids whose sequencing results conform to the recombinant vector sequences of SEQ ID NO: 5 and SEQ ID NO: 8 are the target recombinant plasmids.

Amplification and plasmid extraction of embodiment 4 bacterium liquid

Take the identified bacterial solution, according to the ratio of bacterial solution:LB(Amp+) of 1:1000, after shaking the bacteria in a 240rpm incubator for 8 hours, mix the bacterial solution with sterilized 50% glycerol at a ratio of 1:1 (v/v) Mix well and store at -80°C.

Use omega bio-TEK Endo-free plasma Mini Kit II (REF D6950-20) to extract pAAV-hrND6 and pAAV-hrND6Flag plasmids from 15ml bacterial liquid respectively, determine the concentration of the plasmids, and determine the supercoiled state of the plasmids by agarose gel electrophoresis.

Example 5 Cell Culture and Plasmid Transfection

The 293T cells were inoculated into 75cm ² cell culture dishes according to the number of 2x10 ⁶ , and inoculated into 3 groups, which were labeled as group A, group B and group C respectively. 10% FBS high-sugar DMEM medium, cultured overnight in an incubator containing 5% _CO2 at 37°C, and when the cell density reached 60%-80%, transfection was performed using the jetPRIME transfection reagent transfection kit, Specific steps are as follows:

Dissolve 10ug of plasmid in 500ul jetPRIME buffer, vortex for 10s and then centrifuge quickly. Add 20uljetPRIME reagent, vortex 1s and centrifuge quickly. Incubate at room temperature for 10 minutes and add to the culture medium. Among them, 293T in group A were not transfected with plasmid, 293T in group B were transfected with pAAV2-ND6 recombinant plasmid, and 293T in group C were transfected with pAAV2-ND6Flag recombinant plasmid.

Place the transfected cells in a 5% CO ₂ incubator at 37°C for 6 hours, replace with fresh 10% FBS high-glucose DMEM medium, and continue to culture for 48 hours. After the cell density reaches 80%-90%, perform immunofluorescence , western blot and qPCR detection.

Example 6 Immunofluorescence

293T cells transfected with pAAV2-ND6Flag plasmid in group C for 48 hours were fixed with formalin at room temperature for 10 minutes, rinsed with 1xPBS for 5 minutes three times, and permeabilized with 0.1% TritonX-100 for 10 minutes at room temperature. After rinsing with 1xPBS for 5min for 3 times, block with 1% BSA-PBS at room temperature for 1h, add diluted Mouse anti DDDDK-Tag mAb and incubate overnight at 4°C; after discarding the primary antibody, rinse with 1xPBS for 3 times for 5min, and protect the secondary antibody from light at room temperature Incubate for 1 hour, discard the secondary antibody, rinse with 1xPBS for 3 times for 5 minutes; counterstain with DAPI (1ug/ml), incubate at room temperature for 5 minutes, wash with 1xPBS for 5 minutes twice; add 1X PBS, and observe the fluorescent parts with a fluorescence microscope.

Since Flag protein and ND6 protein are located in the same reading frame, Flag protein and ND6 protein are co-expressed, Flag can reflect the expression and localization of ND6 protein, and Flag shows green fluorescence. DAPI stained the nucleus and showed blue fluorescence, and the immunofluorescence results of intracellular protein expression are shown in Figure 2. The results showed that the Flag protein was normally expressed in the mitochondria of the cells, so the ND6 protein was also normally expressed in the mitochondria.

Example 7 Western blotting

The cell pellet was obtained after trypsin digestion, lysed with RIPA lysate, and the total protein of the cell was separated and then subjected to SDS polyacrylamide gel electrophoresis. After incubation overnight, the membrane was washed and incubated with a secondary antibody labeled with horseradish peroxidase corresponding to the species, and then developed by HRP-ECL luminescence.

Following the same steps, MT-ND6 Rabbit pAb and Anti-GAPDH (Proteintech CatNo. 60004-1-Ig) were used to detect ND6 protein and GAPDH protein in the sample, respectively.

The results of Flag protein immunoblotting are shown in Figure 3. The results showed that the expression of Flag protein in the control group was obvious, and the expression of Flag protein in the experimental group was obvious. Since the fusion of Flag protein and ND6 protein indicated that the recombinant expression vector of ND6 protein could successfully express protein in cells.

Using GAPDH as an internal reference, the results of intracellular ND6 protein immunoblotting are shown in Figure 4. The results showed that, compared with the control group, the protein expression was significantly increased in the pAAV-ND6 and pAAV-ND6Flag transfection groups.

Example 8 qPCR detection of exogenous ND6 mRNA expression in cells

The total RNA of 293T cells in the control group, pAAV2-ND6 group and pAAV2-ND6Flag group was extracted by TRIZOL kit, and after reverse transcription to synthesize cDNA template, Real-time PCR was carried out with GAPDH as the internal reference. The specific primers required for PCR sequencing were :

GAPDH-F: 5'-CCATGGGTGGAATCATATTGGA-3'

GAPDH-R: 5'-TCAACGGATTTGGTCGTATTGG-3'

pND6-F: 5'-ATTCCCCCGAGCAATCTCAA-3'

pND6-R: 5'-GGAGGATCCTATTGGTGCGG-3'

Add 10ul of SYBR Green Mix, 8ul of ddH2O, 0.5ul of each primer, and 1ul of cDNA sample into a 0.2ml PCR reaction tube, for a total of 20ul. Each sample was tested for GAPDH internal reference gene and target gene at the same time, and the amplification of each gene in each sample was repeated 3 times.

The Real-time PCR amplification program was set as denaturation at 95°C (30s), annealing at 55°C (30s), extension at 72°C (30s), and the preset number of cycles was 40, and fluorescent signals were collected after each cycle.

Relative quantitative method was used to study gene expression differences, GAPDH was used as an internal reference gene, and the expression difference was calculated. The results of qPCR detection of exogenous ND6 mRNA expression were shown in Figure 5. The results showed that the pAAV2-ND6 group and pAAV2-ND6Flag group exogenous ND6 mRNA The expression level was significantly increased, compared with the control group P<0.001, there was a significant difference.

Example 9 active oxygen detection

Prepare reagent 0.5mM MitoSox: 50μg MitoSox+130μl DMSO; 5μM MitoSox: 10μl 0.5μM MitoSox+990μl PBS;

1×10 ⁶ m.14484T>C mutant cytoplasmic fusion cells with the same nuclear gene background were collected, and the cells were divided into 3 groups, group A was cytoplasmic fusion cells without mutation, and group B was m.14484T>C mutant cells Plasma fusion cells, group C is pAAV-ND6 overexpression m.14484T>C mutation cytoplasm fusion cells, each group was resuspended with 1ml PBS.

Centrifuge the above three groups of samples at 1,000rpm for 5min, discard the supernatant, resuspend in 500μl containing 5μM MitoSox (diluted in PBS) and incubate at 37°C for 10min;

The flow cytometer was used to detect at 510nm/580nm, statistically sorted and analyzed the data, and calculated the difference in expression. The results of the analysis of cellular reactive oxygen species are shown in Figure 6. The results showed that the expression of ROS in the pAAV2-ND6 group was lower than that in the mutant group. It has statistical significance, but there is no significant difference compared with the control group, indicating that the overexpression of AAV2-ND6 can effectively restore partial function of mitochondria.

Claims (5)

1. The ND6 protein is prepared by utilizing a human mitochondrial ND6 gene recombinant vector, and is characterized in that the gene sequence of the human ND6 gene recombinant vector is a CMV promoter, a mitochondrial targeting sequence COX8A-MTS and an optimized nuclear codon encoded human mitochondrial ND6 nucleotide sequence from the N end to the C end.

2. The method for preparing ND6 protein by using the human mitochondrial ND6 gene recombinant vector according to claim 1, wherein the nucleotide sequence of the human ND6 gene recombinant vector is the optimized human ND6 gene encoded by a nuclear codon, the nucleotide sequence of the human ND6 gene recombinant vector is shown as SEQ ID NO. 3, the amino acid sequence of the ND6 protein encoded by the nuclear codon is shown as SEQ ID NO.6, and the vector can express wild type ND6 protein in mutant cells to replace ND6 abnormal protein caused by mutation, thereby relieving Leber hereditary optic neuropathy caused by m.144T > C mutation.

3. The method for preparing ND6 protein by using the recombinant vector of human mitochondrial ND6 gene according to claim 1, wherein the nuclear codon encodes a translated ND6 fusion protein of human ND6 gene, the amino acid sequence of which is shown in SEQ ID NO.6, the mitochondrial targeting sequence COX8A-MTS is shown in SEQ ID NO. 2, and the nucleotide sequence of the CMV promoter is shown in SEQ ID NO. 1.

4. The method for preparing ND6 protein by using human mitochondrial ND6 gene recombinant vector according to any one of claims 1-3, wherein the skeleton vector of the recombinant vector is pAAV-ZsGreen plasmid with the sequence shown in SEQ ID NO. 4, and the recombinant vector is pAAV-ND6 plasmid with the sequence shown in SEQ ID NO. 5.

5. Use of the recombinant vector of human ND6 gene as defined in claim 1 in the preparation of a ND6 protein kit.

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