Technical Field

The invention relates to the production of specific antibodies for the DNMT3C protein, which is responsible for the methylation of retrotransposon elements during the spermatogenesis process and is important for male fertility, which will allow analysis at the protein level.

State of the Art

DNA methylation, an epigenetic mechanism, plays a role in the transcriptional activation or repression of genes that play a critical role in the spermatogenesis process in mammals. DNA methylation occurs in CpG islets or areas without CpG islets by attaching the methyl group to the fifth carbon atom of the cytosine nucleotide. These mechanisms, which take place in two forms as maintenance and de novo methylation, are catalyzed by DNA methyltransferase (DNMT) enzymes. Six different DNMT enzymes (DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3C ve DNMT3L) have been identified in mammals. Expression changes in DNMT genes cause significant changes in global DNA methylation levels, and this leads to developmental disorders.

A polyclonal antibody is a group of immunoglobulins secreted by plasma cells in the organism, which occurs during the immune reaction when stimulated by heterologous antigens (macromolecular antigen, hapten conjugates) in the body. Polyclonal antibodies are widely applied in research and diagnosis due to several advantages such as recognizing multiple antigen epitopes, causing precipitation reactions, short preparation time, and low cost.

In light of the information available in the literature, it is known that abnormal expression of DNMT genes and abnormal DNA methylation can also cause infertility. DNMT1 is responsible for maintenance methylation, while DNMT3A and DNMT3B are involved in de novo methylation. The DNMT3L enzyme participates in de novo methylation by inducing DNMT3A and DNMT3B. DNMT2, on the other hand, is responsible for the methylation of aspartic acid t-RNA instead of methylated DNA. It has been shown that the newly identified DNMT3C is responsible for the methylation of retrotransposon elements during spermatogenesis and is important for male fertility. When various somatic and gonadal tissues were analyzed, it was shown that DNMT3C was highest expressed as mRNA in testis tissue. However, analyzes are needed to determine the position of DNMT3C in the DNA methylation system and its role compared to other DNMTs during spermatogenesis, its protein partners and chromatin states, etc. At the same time, the relationship between DNMT3C and its paralog DNMT3B, and the relationships and uses between other proteins need to be determined. However, there are no antibodies specific to the DNMT3C protein in the world. The absence of this DNMT3C-specific antibody limits analysis at the protein level.

The present art does not detect the presence of Dnmt3c protein in cells. In 2016, Dnmt3c was defined at the mRNA level, and it was predicted that it could take part in many biological processes. Techniques that can measure the mRNAs of Dnmt3c have been developed in the course of time, but a method that allows it to be displayed as a protein has not been determined. Therefore, an antibody to be developed against the DNMT3C protein is needed to demonstrate the presence and level of the Dnmt3c protein.

As a result, due to the above-mentioned disadvantages and the inadequacy of the existing solutions on the subject, it has become necessary to make an improvement in the related technical field.

Brief Description of the Invention

The invention was inspired by the present situation and aims to eliminate the above- mentioned disadvantages.

The main object of the invention is to produce a specific antibody, specific to the DNMT3C protein, which is predicted to be involved in the control of DNA methylation.

An object of the invention is to produce antibodies specific to the Dnmt3c gene and to provide protein analysis. Another object of the invention is to produce antibodies specific to the Dnmt3c gene and to determine the expression level and expression distribution of DNMT3C in the ovary, testis, and somatic tissues at the protein level. In addition, to ensure the presence of the DNMT3C protein in reproductive cells and gonad tissues, as well as in other somatic cells and tissues.

Another object of the invention is to determine which sequences of the DNMT3C protein bind to these mRNAs after DNMT3C -related mRNAs are identified and how it affects the translational activation of this mRNA when bound to these sequences.

Another object of the invention is to investigate the effect of DNMT3C on male infertility by producing antibodies specific to the Dnmt3c gene and to evaluate the effects of this gene on infertility by evaluating the DNMT3C protein level in testes to be obtained from patients diagnosed with idiopathic infertility within the framework of ethical permissions.

In order to fulfill all the aforementioned objects and any other objects that may arise from the detailed description, the subject matter of the invention is a method of creating antibodies against the DNMT3C protein, and it comprises designing the peptide to include amino acid sequences in a certain region of the protein against the DNMT3C protein, wherein the said peptide is;

N- VMPQLFCETRIPSKTPAPLSWQANTSASTPWL-C (SEQ ID No: 1) or

EDRDGEVGGSSGSGTPVMPQLFCETRIPSKTPAPLSWQANTSASTPWLSP -C (SEQ ID No: 2) and comprises the steps of; inoculating the peptides into a non-human being, repeating the vaccination with 50-1000 pg peptide in each injection at different time periods until the antigen response (immunization) is achieved, injecting boosters to the being to increase the level of response to antigens, the being producing antibodies against the peptide whose B-lymphocytes have been given, taking the blood samples from the being and separating their serum containing polyclonal antibodies against DNMT3C protein, purifying the polyclonal antibodies with immunoglobulin structure. In order to fulfill all the aforementioned objects and any other objects that may arise from the detailed description, the subject matter of the invention is used to determine the expression level and expression distribution of the antibody DNMT3C against the DNMT3C protein in the ovary, testis and somatic tissues at the protein level and to determine its effect on infertility.

The structural and characteristic features and all advantages of the invention will be more clearly understood with the detailed description below, and therefore the evaluation should be made by considering the detailed description.

Detailed Description of the Invention

In this detailed description, the preferred embodiments of the invention are explained only for a better understanding of the subject matter.

The subject matter of the invention is a method of creating antibodies against the DNMT3C protein, and it comprises designing the peptide to include amino acid sequences in a certain region of the protein against the DNMT3C protein, wherein said peptide is;

N- VMPQLFCETRIPSKTPAPLSWQANTSASTPWL-C (SEQ ID No: 1) or

EDRDGEVGGSSGSGTPVMPQLFCETRIPSKTPAPLSWQANTSASTPWLSP -C (SEQ ID No: 2) and comprises the steps of; inoculating the peptides into a non-human being, repeating the vaccination with 50-1000 pg peptide in each injection at different time periods until the antigen response (immunization) is achieved, injecting boosters to the being to increase the level of response to antigens, the being producing antibodies against the peptide whose B-lymphocytes have been given, taking the blood samples from the being and separating their serum containing polyclonal antibodies against DNMT3C protein, purifying the polyclonal antibodies with immunoglobulin structure. Designing the peptide against DNMT3C protein and checking its specificity is carried out as follows:

The DNMT3C protein shows high homology in certain domains with other DNMT protein family members, DNMT3B and DNMT3A. Therefore, DNMT3C peptide sequences are selected from the PWWP domain region, where it has less (1%) similarity to these proteins. The homology of these peptide sequences with other protein regions is checked by the Blast program. BLAST (Basic Local Alignment Search Tool) program is an alignment search engine for the analysis of nucleic acid and protein sequences of the GenBank database used in bioinformatics studies.

When the peptide designed for DNMT3C was analyzed, this peptide was found to be 100% specific for the DNMT3C gene. In addition, it was found that it did not show homology with other proteins. This shows that the antibody to be produced is specific.

The production of the polyclonal antibody of the invention is carried out by administering peptides containing the amino acid sequence in a certain region of the protein or purified protein to a rabbit or other experimental animal (such as goat, sheep, mouse, rat, pig, and chicken). Rabbit is preferably used in the production of antibodies. The rabbit is immunized (reacted to the antigen) with peptides (50-1000 pg of peptide in each injection) injected into the rabbit at different time periods. In addition, antigens are given with boosters such as BSA, Freund’s complete, and Freund’s incomplete to increase the rabbit's level of response to antigen. Thereby, rabbit B-lymphocytes produce large amounts of antibodies against the administered peptide (antigen). After a certain period of time, blood samples are taken from the rabbit and the serum is separated. This serum contains polyclonal antibodies against the target protein. The polyclonal antibody with immunoglobulin G or another immunoglobulin structure is purified by methods such as protein-A Sepharose affinity chromatography. This purified polyclonal antibody is used in research after quantification and safety analysis.

Peptides designed against DNMT3C protein and checked for specificity are listed below:

1. N- VMPQLFCETRIPSKTPAPLSWQANTSASTPWL-C (SEQ ID No: 1) or 2. EDRDGEVGGSSGSGTPVMPQLFCETRIPSKTPAPLSWQANTSASTPWLSP -C (SEQ ID No: 2) to be used if there is no efficient result from the first peptide sequence.

In the method of the invention, the said promoters injected into a non-human mammal to increase the level of response to the antigen are BSA (Bovine Serum Albumin), Freund’s complete (Freund's complete adjuvant) and Freund’s incomplete (Freund's incomplete adjuvant) alone or in combination.

Freund's incomplete adjuvant and Freund's complete adjuvant

Freund's complete adjuvant immunopotentiator is a solution used to increase the immune system's response to a foreign substance. It usually consists of inactivated or dried bacteria from Mycobacterium tuberculosis or Mycobacterium butyricum species. Thanks to these mycobacterial components, Freund's complete adjuvant antigen ingredients help to develop and prolong an immune response. There are two types of Freund adjuvants. The first is Freund's complete adjuvant (Freund’s complete) solution containing mineral oil. This type is a water-in-oil emulsion that localizes antigens for release for about 6 months. It also contains mannide monooleate and a surfactant called heat-killed mycobacteria. The second type is called Freund's incomplete adjuvant (Freund’s incomplete), it does not contain mycobacteria and is less effective. In general, Freund's complete adjuvant stimulates the immune system in two different ways. First, Freund's complete adjuvant acts as a surfactant, allowing protein antigens to concentrate over large surface areas. Second, it increases the number of antibodies produced in response to protein antigens by prolonging the time of antibody production and enabling B cells to become memory cells. When memory cells are present, a subsequent entry of a previously injected antigen will induce a similar immune response. These actions are poorly observed when the antigen used is not composed of proteins.

Freund's complete adjuvant is a very potent agent because it stimulates both cell-mediated and humoral immunity; this means that both antibodies and attack cells of the immune system called thymus-derived cells (T cells) are mobilized to destroy antigens. The solution is a toxic agent because mineral oil cannot be metabolized efficiently. Granuloma formation at injection sites can result in necrosis, inflammation, induration, and pain. Multiple exposures to the complete adjuvant can cause serious adverse reactions, decreased immunity, and even death. When using Freund's complete adjuvant, all materials must be sterile and injection sites must be thoroughly cleaned to prevent contamination and infection. The preferred route is subcutaneous or intraperitoneal. Other injection routes may cause complications. Ulcers and necrosis may result from intradermal administration, muscle death and lameness may result from intramuscular administration, and pulmonary embolism may result from intravenous administration. Freund's complete adjuvant is a hazardous substance and is not approved for human testing. It is harmful both by inhalation and ingestion and may cause skin tenderness on contact with the skin. When working with this substance, protective clothing should be worn, and the area of use should be well-lit and well-ventilated.

In the method of the invention, polyclonal antibodies with immunoglobulin structure are purified by protein-A Sepharose affinity chromatography method. Affinity chromatography is a separation method that depends on the molecular shape (conformation), and generally, a different chromatography resin is used for each protein. On the surface of these resins, specific ligands are attached to the proteins to be separated. Since these ligands, which work similarly to antibody-antigen binding, bind specifically to the target protein, the entire sample loaded on the column flows out without attaching to it, and only the protein to which the ligand is attached remains attached to the column.

Purified polyclonal antibodies obtained in the method of the invention are subjected to quantitative and reliability analyses using ELISA, Western blot, and immunohistochemistry methods.

ELISA TEST

Elisa (Enzyme-Linked Immunosorbent Assay) test is a quantitative measurement method based on investigating the antigen-antibody relationship and the activity of an enzyme bound to an antibody. It enables the search for antibodies against antigens or antigens against antibodies.

The following steps are generally followed to search for antibodies;

1. The known antigen adheres to a plastic surface. In the Micro-Elisa system, this antigen is coated on the surface of the wells made for each use. The serum to be searched for antibodies is placed in these wells and washed after a while. If the appropriate antibody is present in the serum, it combines with the antigen.

2. Human globulin antiserum labeled with an enzyme is added. Washed after a while. If there is an antibody suitable for the antigen in the serum under examination, since it will bind to the antigen, it will bind to the human antiglobulin labeled with the last added enzyme and will not be able to be removed by washing.

3. A suitable chromogen substrate is added to the enzyme. The color that appears when the enzyme bound to the system degrades this substrate is measured by the colorimetric methods to be performed and the antibody bound due to the bound enzyme provides information.

Western Blot

Western blot is a molecular biology technique that allows the analysis of a specific protein in the tissue. With this technique, the presence, dimension, concentration, and concentration changes of a protein in the tissue, and concentrations of the same between different groups are compared. The WB technique consists of three basic steps. These are, respectively: (1) discrimination based on the principle of having different dimensions, (2) transfer to a solid phase (membrane), (3) labeling the target protein using appropriate primary and secondary antibodies to make it visible.

Immunohistochemistry method

Immunohistochemistry is a technique of searching for a specific antigen (protein) or cell in a tissue. The review includes two different stages: preparation and interpretation-analysis. In the immunohistochemistry method, the antigens sought in a tissue can be identified by the application of an antibody and the specific binding of the antibody to the antigen. With the antigen-antibody binding, the searched cell forms a certain color and becomes visible under the microscope. The method is therefore used for diagnostic purposes in research or pathology laboratories.

The antibody against the DNMT3C protein created by the method of the invention is used to determine the expression level and expression distribution of DNMT3C in the ovary, testis, and somatic tissues at the protein level and to determine its effect on infertility.

The advantages of the method of the invention and the antibody against the DNMT3C protein are given below:

The production of antibodies specific to mouse DNMT3C protein, which is involved in DNA methylation in the spermatogenesis process, is performed for the first time. Thereby, this antibody produced against the DNMT3C protein can be purchased by different researchers and companies.

With the antibody against the DNMT3C protein created by the method of the invention, the expression level and expression distribution of DNMT3C in the ovary, testis, and somatic tissues allow determination at the protein level for the first time. In addition, it allows the investigation of the presence of the DNMT3C protein in reproductive cells and gonad tissues, as well as in other somatic cells and tissues.

After DNMT3C -related mRNAs are identified, new studies can be planned on which sequences of DNMT3C protein binds to these mRNAs and how it affects the translational activation of this mRNA when bound to these sequences.

Although the information in the literature has shown that the DNMT3C gene is important for the testis and especially for the spermatogenesis process, the relationship of DNMT3C with male infertility has not been explained. The antibody obtained with the method of the invention allows evaluations to be made in this regard.

In addition, DNMT3C protein level evaluations in testes to be taken from patients diagnosed with idiopathic infertility within the framework of ethical permissions, and evaluation of the effects of this gene on infertility will be possible with the antibody created.