CN106177906B - Application of Ddb1 protein - Google Patents

Abstract

The present invention provides the applications of Ddb1 albumen, belong to gene therapy technology field.The present invention is equivalent to the Ddb1 albumen of the mankind, it is found that it is able to suppress growth defect caused by Eco1 gene delection by being overexpressed Mms1 albumen in yeast.Therefore the application in genetic disease drug caused by being lacked the present invention provides Ddb1 albumen in preparation treatment ESCO2 (homologous protein of the Eco1 in people's cell is ESCO1 and ESCO2) gene mutation, missing or ESCO2 protein function, and then treat correlated inheritance disease caused by ESCO2 gene mutation, missing or ESCO2 protein function lack, such as Luo Baici syndrome, with good potential applicability in clinical practice, and significant social benefit can be obtained.

Description

Application of Ddb1 protein

technical field

The invention belongs to the technical field of gene therapy, in particular to Ddb1 protein and its application in treating growth defects caused by Eco1 gene deletion.

Background technique

DNA replication is the basis of all life activities. About 40 diseases are related to DNA replication. Currently, at least 14 drugs target proteins in the process of DNA replication. Studying the relationship between DNA replication and human diseases can excavate the mechanism of disease occurrence from a deep level, and provide a theoretical basis for new drug development and disease treatment. The correct delivery of genetic material is critical to the stability of the genome, in which sister chromatid adhesions play an integral role.

Sister chromatid cohesion (SCC) is a process in which two sister chromatids join together to establish a cohesion and dissociate, and the process is strictly regulated. Correct delivery is important. The function of SCC mainly depends on cohesin and its regulatory proteins. The cohesin is a quaternary ring complex composed of Smc1, Smc3, Scc1 and Scc3. In the G1 phase of the cell cycle, cohesin binds to the chromosome. At this time, under the action of negative regulatory factors, the binding is unstable, and the circular cohesin will slide on the chromosome; as the cell cycle progresses, the cell enters In S phase, acetyltransferase Eco1 acetylates the Smc3 subunit of cohesin, and the acetylated cohesin is stably bound to the chromosome; in G2 phase, sister chromatid adhesion is stably established and can antagonize the effect of negative regulators After the cell enters the late mitotic phase, the Scc1 subunit of cohesin is cleaved by separase, the cohesin is detached from the chromosome, the deacetylase Hos1 deacetylates Smc3, and the two sister chromatids are divided into two The poles of individual cells ensure the stability of genetic material. SCC is not only involved in DNA replication, but also in various biological processes such as DNA damage repair. It has been reported in the literature that when problems occur in the establishment of SCC, it will cause the occurrence of many serious diseases including melanoma, colon cancer, embryonic cancer, breast cancer, prostate cancer, etc. Studying the relationship between the establishment of SCC and disease will bring new hope for the treatment of various genetic diseases.

The establishment of sister chromatid adhesions requires the involvement of the acetyltransferase Eco1. Eco1 was first identified in a yeast mutant that displayed an increased rate of chromosome loss and was also defective in the process of heterochromatin recombination. Subsequently, Eco1 has been found in other organisms one after another, and all of them are essential genes. By aligning the sequences of Eco1 in different species, it is found that the N-terminal lengths of different species are different, but its C-terminal sequence is very conserved and is the active center of acetyltransferase. Cells deficient in acetyltransferase activity develop massive sister chromatid adhesion defects, which in turn lead to death, suggesting that the primary function of Eco1 is its acetyltransferase action.

In human cells, there are two homologous proteins of Eco1, ESCO1 and ESCO2, both of which can acetylate Smc3, but their functions are also different and not completely redundant. Regardless of which ESCO protein was missing, the other protein could not fully compensate for the defect it caused. Mutations in ESCO2 lead to a developmental disorder called Roberts syndrome (RBS). Roberts Syndrome (Roberts Syndrome) is a disease caused by and only by mutations in the ESCO2 gene. The symptoms include growth retardation, limb deformities, hand deformities, craniofacial bone deformities, etc. abnormal.

The transcript of ESCO2 gene has 3376 nucleotides, and the mature mRNA consists of 1806 nucleotides, encoding 601 amino acids. The N-terminus of ESCO2 protein contains a chromatin-binding domain, and the C-terminus has acetyltransferase activity. ESCO2 mutations cause Roberts syndrome. With two exceptions, most mutations are frameshifts or nonsense mutations, resulting in truncated or nonfunctional proteins. The other two cases were missense mutations, resulting in loss of acetyltransferase activity due to the substitution of highly conserved amino acid residues in the acetyltransferase domain. In human cells, ESCO2 is required for the establishment of chromatid adhesion.

DNA replication and repair processes are regulated by ubiquitination modifications, especially Cullin 4 (Cul4)-RINGE3 ubiquitin ligase. All Cullins interact with the RING finger protein Rbx1/Hrt1 via the C-terminal region and then recruit E2 ubiquitin-conjugating enzymes. The N-terminal domains of Cullin interact with different kinds of substrate-specific adaptors, respectively. The human genome expresses two Cul4 homologs: Cul4A protein and Cul4B protein, which interact through the N-terminal region with Ddb1 protein, which then interacts with various substrate-specific adaptor proteins. These adaptors are called DCAFs (Ddb1-Cul4associated factors). However, the regulatory mechanisms involved in ubiquitinated substrates and DCAFs in DNA replication and repair remain unclear.

There are two commonly used gene therapy methods at present: one is the "in vitro method", that is, the exogenous gene is introduced into the recipient cells cultured in vitro. Source gene expression plays a role in relieving patient symptoms. The other is called "in vivo method", which refers to the direct injection of exogenous DNA into the body. The DNA can be injected alone, or it can be injected together with auxiliary substances such as liposomes, so that the exogenous gene can be transcribed and translated in vivo to play a therapeutic role. effect. Compared with the in vitro method, the gene therapy method of the in vivo method is simple, direct and economical, and the curative effect is more accurate. Commonly used methods include virus-mediated, oligonucleotide direct injection, liposome-mediated and in vivo gene direct injection.

In the prior art, the research involving Ddb1 protein is mainly embodied in small molecule compounds that reduce the interaction between Cul4 and Ddb1, and are used to prepare or treat drugs related to DNA damage in animals (application number 200980115291.6). At present, there is no research report on Ddb1 in ESCO2-induced genetic diseases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide genes related to genetic diseases caused by ESCO2 mutation and their applications in view of the current gene therapy that the targeting is not accurate enough and more therapeutic genes need to be developed to obtain better curative effect.

In order to achieve the object of the present invention, the present invention overexpresses Mms1 (equivalent to human Ddb1 protein) in yeast, and finds that overexpression of Ddb1 protein can inhibit the growth defect caused by Eco1 gene deletion.

Furthermore, the present invention provides the application of Ddb1 protein in the preparation of a drug for treating diseases caused by ESCO2 gene mutation, deletion or ESCO2 protein function loss, wherein the Ddb1 protein contains the amino acid sequence shown in SEQ ID NO.1.

In the above application, the disease is Roberts syndrome.

The present invention provides the application of the gene encoding Ddb1 protein in the preparation of medicine for treating diseases caused by ESCO2 gene mutation, deletion or ESCO2 protein function loss, the gene encoding Ddb1 protein contains nucleotides as shown in SEQ ID NO.2 sequence.

In the above application, the disease is Roberts syndrome.

The present invention provides the application of a protein overexpression promoter in the preparation of a drug for treating diseases caused by ESCO2 gene mutation, deletion or ESCO2 protein function loss, the protein Ddb1 protein, which contains the amino acid shown in SEQ ID NO.1 sequence.

In the above application, the disease is Roberts syndrome.

The present invention provides the application of biological material containing Ddb1 protein encoding gene in the preparation of medicine for treating diseases caused by ESCO2 gene mutation, deletion or ESCO2 protein function loss, the Ddb1 protein encoding gene contains the nucleoside shown in SEQ ID NO.2 acid sequence.

In the above application, the biological material is an expression vector or a host cell. The disease in question is Roberts Syndrome.

The present invention also provides a medicine containing Ddb1 protein or its encoding gene, wherein the Ddb1 protein contains the amino acid sequence as shown in SEQ ID NO.1; the Ddb1 protein encoding gene contains the nucleotide sequence as shown in SEQ ID NO.2.

The present invention can solve the shortcomings of the current gene therapy: the targeting is not precise enough; more therapeutic genes need to be developed to obtain better curative effect. It is found in the present invention that the Ddb1 gene can be used as a new therapeutic gene and has broad prospects. Overexpression of Mms1 in yeast in the present invention can inhibit the growth defect caused by Eco1 gene deletion (Fig. 1), and can also compensate for the decreased Smc3 acetylation level caused by Eco1 gene deletion (Fig. 2). This mechanism is conserved in eukaryotes. Therefore, for patients with malignant Roberts syndrome caused by ESCO2 gene functional defect, those skilled in the art can achieve the treatment of the disease through the following technical solutions: (1) introducing the Ddb1 gene into somatic cells, so that the Ddb1 protein is overexpressed in the patient, Utilize one of two ways, viral or non-viral. Viral methods can be used to construct Ddb1 into adenovirus vectors, and non-viral methods can directly introduce exogenous DNA fragments into cells to inhibit the functional defect of ESCO2 gene and achieve the purpose of treatment. (2) Direct injection of Ddb1 recombinant protein to make up for the functional defect of ESCO2 gene. For example, by constructing and expressing Ddb1 recombinant protein particles, the Ddb1 recombinant protein can be expressed in large quantities, the expressed Ddb1 recombinant protein can be purified, and the purified Ddb1 protein can be directly injected into the body of patients with Roberts syndrome.

Description of drawings

Figure 1 is a schematic diagram showing that overexpression of the Mms1 subunit of the ubiquitin ligase complex Rtt101 ^Mms1 can inhibit the high-temperature growth defect of the eco1-1 mutant. The growth of each mutant strain at 25°C, 30°C, 34°C and 37°C was observed by gradient dilution experiments. The experimental results showed that overexpression of Mms1 allowed the normal growth of mutants lethal at 30℃, 34℃ and 37℃. AD-Eco1 is the positive control.

Figure 2 is a schematic diagram showing that overexpression of the Mms1 subunit of the ubiquitin ligase Rtt101 ^Mms1 can repair the acetylation defect of Smc3 in the eco1-1 mutant. The indicated antibodies were detected by Western Blot. The results show that when Mms1 is overexpressed in the eco1-1 mutant, the lower amount of Smc3 acetylation in the mutant can be complemented to the same level as the wild type. The Western Blot results used Tubulin as the sample loading control. AD is the abbreviation of vector pGADT7, which refers to the empty vector expressing pGADT7 in yeast cells as a control. AD-Mms1 is the abbreviation of the vector pGADT7-Mms1, and pGADT7-Mms1 was expressed in yeast cells as the experimental group. α-ac-Smc3 refers to an antibody that recognizes acetylation of Smc3, α-Smc3 refers to an antibody that recognizes Smc3, and α-Tubulin refers to an antibody that recognizes Tubulin.

Figure 3 shows the results of protein sequence alignment between human cell ESCO2 and yeast Eco1. The gi numbers of ESCO2 and Eco1 in NCBI are gi62899035 and gi14318550, respectively. It can be seen from the figure that the N-terminal of these two proteins has changed greatly, but the C-terminal acetyl transfer The enzymatic domains are very conserved (amino acids 534-601 of ESCO2, amino acids 211-281 of Eco1).

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art. In the following examples, Phusion high-fidelity polymerase was purchased from Thermo Fisher, Pfu was purchased from Quanjin Biological Company, and Tag polymerase was purchased from Purchased from Takara Company, restriction endonuclease was purchased from NEB Company, T4 ligase was purchased from NEB Company, pRS313, pRS316, pGADT7, pAG25 were purchased from Addgene, and primers were synthesized by Sangon Biotechnology Company. Unless otherwise specified, the raw materials used are all commercially available products.

Example 1 Construction of eco1-1 mutant strain

1. Construct the expression vector of acetyltransferase Eco1 in Saccharomyces cerevisiae: pRS316-Eco1, pRS313-Eco1

Note: pRS316 is a yeast expression vector with URA as nutritional screening marker,

pRS313 is a yeast expression vector with HIS as nutritional selection marker

(1) Using the genomic DNA of Saccharomyces cerevisiae BY4741 as a template, use Pfu polymerase PCR to amplify the Eco1 target fragment:

Eco1-F: 5'-CGCGGATCCCAATTTGATCTTTTTATAAA-3';

Eco1-R: 5'-CCGGAATTCTCATATGTATACCGGCAATA-3' and then use

Multifunctional DNA purification kit to recover PCR products.

(2) The target fragment of Eco1 and the vector pRS316 were digested with BamHI and EcoRI double enzymes. Reaction system: 5 μL of reaction buffer, 1.5 μL of BamHI and EcoRI, 2 μg of DNA were added, water was added to 50 μL, and digested at 37°C for 2 hours.

(3) Electrophoresis, and the digestion product was recovered with a gel recovery kit.

(4) Take 3 μL of the recovered product for electrophoresis to estimate the DNA concentration. Set up a ligation reaction system: 100 ng of vector, target fragment (the molar ratio of target fragment to vector is 3:1-10:1), 1 μL of T4 DNA ligase, add water to 10 μL, and ligate overnight at 16°C.

(5) Take 5 μL of the ligation product to transform E. coli DH5α, spread it on LB solid medium (containing ampicillin), and invert at 37° C. for 16 hours.

(6) Pick colonies into 5 mL of LB liquid medium (containing ampicillin), and culture at 37° C. and 220 rpm for 12 hours.

(7) Extract the plasmid by alkaline lysis method.

(8) The plasmid was identified by enzyme digestion. The reaction system was: 1 μL of reaction buffer, 0.5 μL of BamHI and EcoRI, 3 μL of plasmid, added water to 10 μL, digested at 37°C for 1 hour, and the size was detected by electrophoresis.

(9) Send the correct plasmid to be sequenced, and get the correct pRS316-Eco1 vector. The construction of the pRS313-Eco1 vector is the same as that of the pRS316-Eco1 vector.

2. Construction of expression vector of acetyltransferase Eco1 mutant eco1-1 in Saccharomyces cerevisiae

pRS313-eco1-1

(1) Design primers for mutation sites. The point mutation primers are:

eco1-1(G211D)-F:

5-CCCGATTTTAAGATTGACATATCGAGAATTTGGGTGTG-3';

eco1-1(G211D)-R:

5-CCAAATTCTCGATATGTCAATCTTAAAATCGGGGTAC-3'.

(2) PCR was performed using pRS316-Eco1 and pRS313-Eco1 as templates. Set up the PCR reaction system: 10 μL of 5×HFBuffer; 1 μL of dNTPs (10 mM); 1 μL of Template (5-50 ng); 0.5 μL of the above upstream and downstream primers; 0.5 μL of Phusion high-fidelity polymerase; 1.5 μL of DMSO plus H ₂ O to 50 μL. Note that Phusion polymerase requires a hot start and should be added last and react immediately after addition. Reaction program: 98°C for 5 minutes; 98°C for 10 seconds, 58°C for 30 seconds, 72°C for 4 minutes (extension time = template length/2kb/minute), 17 cycles; 72°C for 20 minutes; using the above PCR system and program PCR amplification.

(3) Take 10 μL of PCR product and add 1 μL of DpnI to digest the template at 37°C (because the plasmid DNA has methylation modification, but the PCR product has no methylation modification, and DpnI only acts on DNA with methylation modification).

(4) Take 5 μL of the digested system to transform DH5α, and extract plasmids from the transformants and sequence them. Obtain the correct pRS313-eco1-1 vector.

3. Using Plasmid Shuffling experiment to obtain eco1-1 mutant strain

Because acetyltransferase is necessary for the growth of Saccharomyces cerevisiae, when the ECO1 gene on the genome is modified, a plasmid capable of expressing the Eco1 protein needs to be transferred in advance.

(1) Yeast transformation of pRS316-Eco1 and pRS313-eco1-1 plasmids

1) Saccharomyces cerevisiae BY4741 was used as a background strain to be inserted into a liquid medium, and cultured at 30° C. to 2×10 ⁷ cells/mL (OD600=1.0).

2) 5 mL of the cultured bacterial liquid was collected into a sterile 1.5 mL centrifuge tube with centrifugation conditions of 2500 g for 30 s, and the upper liquid medium was removed.

3) Suspend the cells in 1 mL of sterile ultrapure water, and then collect the cells in a centrifuge tube under the conditions of centrifugation at 2500 g for 30 s, and remove the supernatant.

4) The cells were suspended in 1 mL of 0.1 mol/L lithium acetate solution, and then the cells were collected in a centrifuge tube under the conditions of centrifugation at 2500 g for 30 s, and the supernatant was removed.

5) Boil 1 mL of the single-stranded vector DNA sample for 5 min, and quickly cool in ice water.

6) Add the transformation mixture to the upper layer of the centrifuged cells. The transformation mixture has the following composition and is added in the following order: 240 μL of PEG3350 (50% W/V), 36 μL of 1mol/L LiOAc (lithium acetate), single-stranded carrier DNA (2 mg /mL) 50 μL, 1 μL each of pRS316-Eco1 and pRS313-eco1-1 to be transformed, 32 μL sterile ultrapure water, and the total volume is 360 μL. After the addition is complete, shake vigorously until the entire system is completely mixed, and place on ice for 3 min.

7) Heat shock at 42°C for 40min.

8) Collect the cells in a centrifuge tube at 2500g for 30s, and remove the supernatant.

9) Suspend the cells in 1 mL of sterile ultrapure water, and then collect the cells in a centrifuge tube under the conditions of centrifugation at 2500 g for 30 s, and remove the supernatant.

10) Suspend the cells in 100 μL of sterile ultrapure water, spread them evenly on the SC-URA-HIS solid medium, and invert at 30°C for 24-72 hours to obtain pRS316-Eco1

(2) Knockout of ECO1 on the genome by homologous recombination

1) Using the pAG25 plasmid as a template, design a pair of primers containing 39nt homology arms upstream and downstream of the target sequence at the 5' end, and PCR amplify the replacement fragment. The primers used are as follows:

eco1-del-F:

5’-ACATATTAGGGTTCAAACAGAATATAAATCGTTGCACAAAACATGGAGGCCCCAGAATACCCT–3’;

eco1-del-R:

5’-ATTTTTCCAGTGTCCCTTCTCGCTGTCTTTTCGAAAGAGCAGTATAGCGACCAGCATTCAC–3’.

2) Using the pAG25 plasmid as a template, PCR amplification with Tag polymerase can homologously replace the target fragment with expression NAT resistance of ECO1 on the genome. PCR reaction system: 2×Tag polymerase 25 μL; Template (5-50 ng) 1 μL; Primer F ₂ μL; Primer R 2 μL; Reaction program: 95°C for 5 minutes; 95°C for 1 minute, 58°C for 30 seconds, 72°C for 2 minutes (extension time = template length/1 kb/minute), 30 cycles; 72°C for 20 minutes; using the above PCR system and program PCR amplification.

(3) The target fragment obtained by PCR was transformed into the BY4741 pRS316-Eco1 pRS313-eco1-1 strain using the above yeast transformation method. Smear evenly on the solid medium of SC-URA-HIS+NAT, and cultivate by inversion. A single colony of transformants was picked and streaked on a new solid plate. Pick the bacteria, quickly extract the yeast genome, and identify whether the DNA fragment has been replaced by PCR. Finally, the ECO1 knockout strain on the genome was obtained: BY4741 eco1Δ::NAT pRS316-Eco1 pRS313-eco1-1.

(4) The eco1-1 mutant strain was obtained by Plasmid Shuffling experiment. 5-fluoroorotic acid (5-FOA) is used as a negative screening drug. When yeast cells can express URA3, the enzyme encoded by the URA3 gene can turn 5-FOA into a toxic substance to cells, making yeast cells in the No growth on medium containing 5-FOA. The pRS316-Eco1 plasmid mentioned in the above experiment can express URA3, so strains with loss of pRS316-Eco1 can be screened by adding 5-FOA.

The strain obtained by the above steps: BY4741 eco1Δ::NAT pRS316-Eco1pRS313-eco1-1 was screened on the SC-HIS+5-FOA plate, and the obtained strain was the acetyltransferase-deficient mutant eco1-1: BY4741eco1Δ ::NAT pRS313-eco1-1.

Example 2 Identification of ubiquitin ligase ^Rtt101 - subunit Rtt101 of Mms1 complementing acetyltransferase-deficient mutant eco1-1

1. Construction of related vectors pGADT7-Eco1 and pGADT7-Mms1 for ubiquitin ligase Rtt101-Mms1 to ^complement acetyltransferase-deficient mutant eco1-1

pGADT7 is a yeast expression vector with LEU as a nutritional selection marker

(1) Using the genomic DNA of Saccharomyces cerevisiae BY4741 as the template, the target fragments of Eco1 and Mms1 were amplified by PCR with Pfu polymerase.

The primer sequences used are as follows:

Eco1-2-F: 5'-CCGGAATTCATGAAAGCTAGGAAATCGCA-3'

Eco1-2-R: 5'-CGCGGATCCTCATATGTATACCGGCAATA-3'

Mms1-F: 5'-TCCCCCCGGGTATGCTAGGTTTGCGAACTCAT-3'

Mms1-R: 5'-CCGCTCGAGGGGAATTATACTGTGTTCTTGC-3'

The PCR products were then recovered using a multifunctional DNA purification kit.

(2) The target fragment ECO1 and the vector pGADT7 were digested with BamHI and EcoRI double enzymes. Reaction system: 5 μL of reaction buffer, 1.5 μL of BamHI and EcoRI, 2 μg of DNA were added, water was added to 50 μL, and digested at 37°C for 2 hours.

(3) The target fragment MMS1 and the vector pGADT7 were digested with XmaI and XhoI double enzymes. Reaction system: 5 μL of reaction buffer, 1.5 μL of XmaI and XhoI, 2 μg of DNA, and 50 μL of water were added, respectively, and digested at 37°C for 2 hours.

(4) Electrophoresis, and the digestion product was recovered with a gel recovery kit.

(5) Take 3 μL of the recovered product for electrophoresis to estimate the DNA concentration. Set up a ligation reaction system: 100 ng of vector, target fragment (the molar ratio of target fragment to vector is 3:1-10:1), 1 μL of T4 DNA ligase, add water to 10 μL, and ligate overnight at 16°C.

(6) Take 5 μL of the ligation product to transform E. coli DH5α, spread it on LB solid medium (containing ampicillin), and invert at 37° C. for 16 hours.

(7) Pick colonies into 5 mL of LB liquid medium (containing ampicillin), and culture at 37° C. and 220 rpm for 12 hours.

(8) Extract the plasmid by alkaline lysis method.

(9) The plasmid was identified by enzyme digestion. 1) The pGADT7-Eco1 reaction system was: 1 μL of reaction buffer, 0.5 μL of BamHI and EcoRI, 3 μL of plasmid, added water to 10 μL, digested at 37°C for 1 hour, and the size was detected by electrophoresis. 2) The reaction system of pGADT7-Mms1 is: 1 μL of reaction buffer, 0.5 μL of XmaI and XhoI each, 3 μL of plasmid, add water to 10 μL, digest with enzyme at 37°C for 1 hour, and measure the size by electrophoresis.

(10) Send the correct plasmids digested by enzyme to sequencing to obtain correct pGADT7-Eco1 and pGADT7-Mms1 vectors. The related vectors pGADT7-Eco1 and pGADT7-Mms1 were constructed to obtain the ubiquitin ligase Rtt101-Mms1 ^{complementing} the acetyltransferase-deficient mutant eco1-1.

2. Construction of strains of ubiquitin ligase complex Rtt101-Mms1 ^{complementing} acetyltransferase-deficient mutant eco1-1

Using the yeast transformation method, the expression vector obtained in step 1 was transferred into the acetyltransferase-deficient mutant eco1-1 prepared in Example 1, namely: BY4741eco1Δ::NAT pRS313-eco1-1, and finally the ubiquitin ligase was obtained Overexpression of the complex subunit complements the strain.

1) The acetyltransferase-deficient mutant eco1-1 obtained in Example 1, namely: BY4741eco1Δ::NATpRS313-eco1-1, was used as the background strain to be inserted into the liquid medium, and cultured at 30°C to 2×10 ⁷ cells/ mL (OD600=1.0).

2) 5 mL of the cultured bacterial liquid was collected into a sterile 1.5 mL centrifuge tube with centrifugation conditions of 2500 g for 30 s, and the upper liquid medium was removed.

3) Suspend the cells in 1 mL of sterile ultrapure water, and then collect the cells in a centrifuge tube under the conditions of centrifugation at 2500 g for 30 s, and remove the supernatant.

4) The cells were suspended in 1 mL of 0.1 mol/L lithium acetate solution, and then the cells were collected in a centrifuge tube under the conditions of centrifugation at 2500 g for 30 s, and the supernatant was removed.

5) Boil 1 mL of the single-stranded vector DNA sample for 5 min, and quickly cool in ice water.

6) Add the transformation mixture to the upper layer of the centrifuged cells. The transformation mixture is composed of the following, and is added in the following order: 240 μL of PEG3350 (50% W/V), 36 μL of 1mol/L LiOAc (lithium acetate), single-stranded carrier DNA ( 2mg/mL) 50μL, pGADT7, pGADT7-Eco1, or pGADT7-Mms1 to be transformed 1μL each, sterile ultrapure water 32μL, total volume 360μL. After the addition is complete, shake vigorously until the entire system is completely mixed, and place on ice for 3 min.

7) Heat shock at 42°C for 40min.

8) Collect the cells in a centrifuge tube at 2500g for 30s, and remove the supernatant.

9) Suspend the cells in 1 mL of sterile ultrapure water, and then collect the cells in a centrifuge tube at 2500 g for 30 s, and remove the supernatant.

10) Suspend the cells in 100 μL of sterile ultrapure water, spread evenly on the SC-HIS-LEU solid medium, and invert at 30° C. for 24-72 hours.

11) Pick a single colony of the transformant and streak it on a new solid plate.

12) At the same time, in the cells with normal acetyltransferase Eco1 function (ie, the background strain BY4741), the empty vector of pGADT7 was transformed as a control.

13) Finally, an overexpressed complementing strain of the ubiquitin ligase complex subunit is obtained. which is

BY4741 eco1Δ::NAT pRS313-eco1-1 pGADT7;

BY4741 eco1Δ::NAT pRS313-eco1-1 pGADT7-ECO1;

BY4741 eco1Δ::NAT pRS313-eco1-1 pGADT7-MMS1;

3. The complementation of acetyltransferase-deficient mutant eco1-1 by ubiquitin ligase Mms1 was detected by western blotting.

(1) Extraction of yeast cell total protein by TCA-acetone method Collect 5OD yeast cells, resuspend the cells with 1mL 0.25mol/L NaOH, 1% β-mercaptoethanol, pipette evenly, and place on ice for 10min. 160 μL of 50% TCA solution was added, shaken and mixed, and then placed on ice for 10 min. Centrifuge at 15000g for 10 min at 4°C to remove the supernatant. The precipitate was resuspended with 1 mL of pre-cooled acetone, pipetted with a micropipette until the precipitate was fully dispersed, centrifuged at 15,000 g for 10 min at 4°C, and the supernatant was removed. Dry the pellet in a 60°C oven, suspend the pellet with 1×SDS protein loading buffer, and take a boiling water bath for 5 min. 1×SDS protein loading buffer components: Tris-Cl (pH6.8) 50mmol/L, SDS 2%, bromophenol 0.1%, glycerol 10%, β-mercaptoethanol 100mM.

(2) Western blotting The above samples were separated by 8% SDS-PAGE gel. The electrophoresed SDS-PAGE gel was equilibrated in transfer buffer. The PVDF membrane as large as the gel was cut out, soaked in methanol for 1 min to activate, and then placed in transfer buffer to equilibrate. Open the splint, soak one side of the black splint in transfer buffer, and place the soaked sponge pad and two filter papers of appropriate size from bottom to top. The entire section is submerged in transfer buffer to prevent air bubbles. On the filter paper, in bottom-to-top order, the gel, PVDF membrane, and two sheets of soaked filter paper. The entire movement is submerged in transfer buffer to prevent air bubbles. Finally, place the soaked sponge pad on the top layer, tighten and close the film transfer splint, align the electrode, and insert it into the film transfer groove. 250mA, 1h, 4 ℃ transfer membrane electrophoresis. After the membrane transfer was completed, the PVDF membrane was taken out, placed in 5% nonfat milk prepared with PBST, and blocked at room temperature for 1 h. Remove the blocking solution, dilute the antibody with 2% nonfat milk prepared in PBST to an appropriate concentration, and incubate at room temperature for 1 h. (anti-ac-Smc3, anti-Smc3, anti-Tubulin) remove the primary antibody, and wash the PVDF membrane 3 times with PBST at room temperature for 10 min each time. The corresponding secondary antibodies were diluted with 2% nonfat milk prepared in PBST to an appropriate concentration, and incubated at room temperature for 1 h. The secondary antibody incubation solution was removed, and the PVDF membrane was washed three times with PBST at room temperature for 10 min each time. After mixing equal amounts of ECL chromogenic solution A and B, react with PVDF membrane for 3 min at room temperature, remove excess chromogenic solution on the membrane, seal the PVDF membrane, fix it in a dark box, and expose and develop in a dark room. The results are shown in Figure 2.

Example 3 Overexpression of Mms1 (equivalent to human Ddb1 protein) in yeast can suppress growth defects caused by deletion of Eco1 gene

Gradient sensitivity experiments were carried out with the strains obtained in Example 2. which is:

BY4741 eco1Δ::NAT pRS313-eco1-1 pGADT7;

BY4741 eco1Δ::NAT pRS313-eco1-1 pGADT7-ECO1;

BY4741 eco1Δ::NAT pRS313-eco1-1 pGADT7-MMS1

The specific operations are as follows:

1) The above strains were cultured overnight, the cells were counted on the day of the experiment, and the cell concentration of all experimental strains was adjusted to 2×10 ⁶ cells/mL with water.

2) Aspirate 250 microliters of cells of each experimental strain and add dropwise to the first row of the 96-well plate, and use a multi-channel micropipette to drop 200 microliters of sterile ultrapure water in the second to fifth columns of the 96-well plate.

3) A total of five dilutions are set from the first column to the sixth column of the 96-well plate. Use a multi-channel micropipette to suck 50 μl of cells from the first column to the second column to mix well, and then from the second column. Pipette 50 microliters of cells into the third column and perform a 5-fold serial dilution in this step.

4) Use a multi-channel micropipette to draw 5 microliters of cells from each dilution and drop them onto the -HIS-LEU plate, and the distance between the yeast spots dropped at each dilution is 1.5 cm. The temperature gradient plates were incubated at 25°C, 30°C, 34°C and 37°C, respectively.

5) After culturing for 48 hours, take pictures to record the experimental results, and the results are shown in Figure 1. Figure 1 is a schematic diagram showing that overexpression of the Mms1 subunit of the ubiquitin ligase complex Rtt101 ^Mms1 can inhibit the high-temperature growth defect of the eco1-1 mutant. The growth of each mutant strain at 25°C, 30°C, 34°C and 37°C was observed by gradient dilution experiments. The experimental results showed that overexpression of Mms1 allowed the normal growth of mutants lethal at 30℃, 34℃ and 37℃. AD-Eco1 is the positive control, AD is the negative control, and AD-Mms1 is the experimental group. Because the mechanism of chromatid adhesion (SCC) is conserved from yeast to humans, the acetylase ESCO2, which acetylates Smc3 in human, and Eco1 in yeast are also highly conserved (Fig. Treatment of diseases caused by ESCO2.

Although the present invention has been described in detail above with general description, specific embodiments and tests, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art . Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (7)

1. In disease medicament caused by 1.Ddb1 albumen is lacked in preparation treatment ESCO2 gene mutation, missing or ESCO2 protein function Application, the amino acid sequence of the Ddb1 albumen is as shown in SEQ ID NO.1.
2. 2. application as described in claim 1, the disease is Luo Baici syndrome.
3. 3. the gene of encoding D db1 albumen is caused by preparation treatment ESCO2 gene mutation, missing or ESCO2 protein function lack Application in disease medicament, the gene of the encoding D db1 albumen are the nucleotide sequence as shown in SEQ ID NO.2.
4. 4. application as claimed in claim 3, the disease is Luo Baici syndrome.
5. 5. the biomaterial containing Ddb1 protein coding gene is in preparation treatment ESCO2 gene mutation, missing or ESCO2 albumen function Application in disease medicament caused by capable of lacking, the Ddb1 protein coding gene are the nucleotide as shown in SEQ ID NO.2 Sequence.
6. 6. application as claimed in claim 5, the biomaterial is expression vector or host cell.
7. 7. application as claimed in claim 5, the disease is Luo Baici syndrome.