Protein Analysis of Royal Jelly Bee Apis Mellifera Carpatica as Candidates for Immunotherapy in Reproductive Disorders

Background: The honey bee of the Apis mellifera species is the main type of bee cultivated in almost all countries in the world, including Indonesia. Honey has benefits in various aspects, including food, health, and beauty. Royal jelly is a daily food menu as a food supplement to maintain and increase health and vitality. Purpose: Analyzing the immunogenic and toxinogenic proteins of Royal jelly Apis mellifera carpatica as immunotherapy. Method: Conversion of nucleotides into amino acids. Royal jelly Apis mellifera carpatica , analysis of the three-dimensional structure of Protein of Royal jelly Apis mellifera carpatica , Analysis of Ramachandran Plots of Royal jelly Apis mellifera carpatica , Analysis of epitope and allergen proteins, Analysis of antigens and toxins. Research result: The research results found that six proteins of Royal jelly Apis mellifera carpatica were epitope, antigenic, and non-toxic. Allergens and three Royal jelly proteins, Apis mellifera carpatica , are non-allergenic. In this study, the three-dimensional structure has not been found and opens opportunities for proteomic studies of the Royal jelly Apis mellifera carpatica protein, including protein isolation. Conclusion: The findings of this study can be used as a basis for the use of immunotherapy materials against the protein Royal jelly Apis mellifera carpatica against reproductive disorders. This study conducted an immunoinformatics analysis of the protein Royal jelly Apis mellifera carpatica . This study found six proteins of Royal jelly Apis mellifera carpatica: epitope, antigenic and non-antigenic proteins, allergen and non-allergenic proteins, toxin, and non-toxic proteins. Proteins that are antigenic in minimal portions (8-15 amino acids) can induce the immune system. 18,19 Water-soluble glycoproteins having molecular weights between 10-70 KD are frequently allergenic. 20 Allergies can trigger mild symptoms such as itching, runny nose and eyes, and swelling. Allergies can also cause severe reactions such as anaphylaxis, leading to death. for


INTRODUCTION
With its diverse climate, the Indonesian region allows for the growth of various plants, including various commodities from fruits, vegetables, and ornamental plants that produce nectar or pollen, which is a source of food for honey bees. Honey bee cultivation activities have existed and have been known to the Indonesian people since ancient times; only these activities were not properly cultivated at that time but were carried out with makeshift equipment; thus, results were not optimal. The honey bee of the Apis mellifera species is the main type of bee cultivated in almost all countries in the world, including Indonesia. These bees are found in many European countries (France, Greece, Italy, Spain, and Yogoslavia). [1][2][3] Health is one of the basic human needs besides food, clothing, and shelter. The Law of the Republic of Indonesia Number 36 of 2009 defines health as a physically, psychologically, spiritually, and socially sound condition that enables everyone to lead a socially and economically useful life. Therefore, everyone tries to maintain his health. One way to maintain health is to take preventive measures by consuming honey. Honey is a sweet liquid produced by bees from plant nectar and kept in the cells of a beehive. Honey has benefits in various aspects, including food, health, and beauty. The honey bee business has great potential to be developed in Indonesia. With 193 million hectares of agricultural and plantation land and 143 million hectares of forest area, Indonesia has vast natural resources for developing the honey industry. The benefits of honey are not just to overcome various kinds of diseases. Many bee products are no less useful, including royal jelly, pollen (bee pollen), and bee propolis. 4,5 In Indonesia, Royal jelly is better known as the queen bee's milk. Royal jelly is food for the queen bee and the larvae (prospective) bees whose age is 1 to 3 days; in one bee colony, there is one queen bee, hundreds of male bees, and tens of thousands of worker bees (99% of the number of bees). Secretions from the hypopharyngeal glands of worker bees aged 5-15 days. Royal jelly is food from the queen, supplied by larval and adult bees, young worker bees, and male bee larvae. Public knowledge about the Efficacy of Royal jelly has been known since the beginning of human civilization. Historical records of ancient Egypt inform about the efficacy of this queen bee milk, even though it is said that the beauty of Queen Cleopatra's face cannot be separated from the efficacy of the queen bee's milk in the cosmetic ingredients she uses. The nobility of Europe, the Middle East, and Asia put Royal jelly on a daily food menu as a food supplement to maintain and increase the vitality of their health; even in Islamic civilization, the role of bees and the efficacy of honey has a special place in the world of medicine/medicine. 6,7 This study aimed to analyze the immunogenic and toxinogenic proteins of Royal jelly Apis mellifera carpatica as immunotherapy.

Conversion of nucleotides to amino acids Royal jelly Apis mellifera carpatica
Royal jelly Apis mellifera carpatica nucleotides were taken from the NCBI gene bank and converted into amino acids using the Expasy Translate Tool software.

Analysis of the three-dimensional structure of protein Royal jelly Apis mellifera carpatica
The research steps were carried out to predict the three-dimensional structure of the Royal jelly protein Apis mellifera carpatica by homology according to the Protein Structure Homology Modeling Using SWISS-MODEL Workspace protocol. 8,9 Ramachandran plot analysis of Royal jelly Apis mellifera carpatica The results of the three-dimensional protein structure analysis in PDB were entered into the Ramachandran Plot Server software (https://zlab. umassmed.edu/bu/rama/).

Epitope and allergen protein analysis
The amino acids of Royal jelly Apis mellifera carpatica were included in the IEDB software to obtain epitopic proteins. The amino acids of Royal jelly Apis mellifera carpatica are included in the Allerton software to obtain allergens proteins.

Analysis of proteins that are antigenic and toxin
The amino acids of Royal jelly Apis mellifera carpatica were included in Vaxijen software to obtain antigenic proteins. The amino acids of Royal jelly Apis mellifera carpatica are included in the Toxinpred software to obtain Toxic and Non-Toxic proteins.

RESULTS
The concept of Central dogma flow of genetic information consists of three main processes of using the information in cells. The first process is replication, which copies parent DNA to generate daughter DNA molecules with identical nucleotide sequences. The second phase is transcription, which involves copying the genetic code contained in DNA into RNA molecules. The third process is translation, wherein the genetic message encoded in messenger RNA is translated on the ribosome into a polypeptide with a particular amino acid sequence. The amino acid composition of Royal jelly Apis mellifera carpatica can be shown in Table 1 due to the translation of nucleotides into amino acids yielding six amino acids.
According to the research findings conducted using the SWISS-MODEL, the three-dimensional structure of the protein Royal jelly Apis mellifera carpatica could not be determined. Based on the analysis results from the SWISS-MODEL, no templates or proteins were found that were homologous to the six proteins of Royal jelly Apis mellifera carpatica. In this study, analysis was also carried out using the Ramachandran plot. In the Ramachandran plot, clusters formed from several residues indicate the secondary structure formed. Through the Ramachandran plot, it can be seen whether a protein structure has good quality or not. The research results obtained an overview of the six proteins from Royal jelly Apis mellifera carpatica, as shown in Figure 1.
From the results of research conducted using IEDB software to analyze proteins that are epitopes in Royal jelly Apis mellifera carpatica. The research results found that Royal jelly Apis mellifera carpatica protein is an epitope, specifically proteins number 1, 5, and 6. In Royal jelly Apis mellifera carpatica protein number 2,3 and 4, no protein is epitope.
In this study, an analysis of the Royal jelly Apis mellifera carpatica protein was also carried out; According to theanalysis results using bioinformatics, it was found that six proteins of Royal jelly Apis mellifera carpatica were epitopes, specifically 1 protein from protein      number 1, 2 proteins from protein number 5 and 3 pieces of protein from protein number 6. In addition, antigenicity analysis was also carried out on the protein Royal jelly Apis mellifera carpatica; from the research results, four proteins were antigenic and two non-antigenic.
In this study, an analysis was also carried out on the protein of Royal jelly Apis mellifera carpatica, a non-toxic and toxin protein, and a protein of Royal jelly Apis mellifera carpatica, which is allergen and non-allergenic. The research results found that six proteins of Royal jelly Apis mellifera carpatica were non-toxic. In addition, based on the analysis of Royal jelly Apis mellifera carpatica protein on allergen and non-allergenic properties, it was found that three proteins of Royal jelly Apis mellifera carpatica were allergen and three proteins of Royal jelly Apis mellifera carpatica were non-allergenic.

DISCUSSION
Traditional medicine is still used and trusted by the community, one of which is honey. Since ancient times honey has been used for treatment such as wounds, fever, internal heat, and mixed with food ingredients to increase body fitness. Honey is generally adequate for creating energy, enhancing endurance, and enhancing stamina. The magnesium mineral content of honey is identical to the magnesium mineral content of blood serum. In addition, the Fe content in honey can increase the number of erythrocytes in human blood and can increase hemoglobin levels. 10 Royal jelly functions as a tonic to restore energy, get rid of pain, and improve appetite. 11 Royal jelly mostly contains protein, sugar, fat (fatty acid), and minerals. Royal jelly is the most effective for maintaining stamina when combined with honey; royal jelly is useful as an energy and stamina booster, boosts the immune system, and maintains overall health. Royal jelly relieves various problems such as fatigue, anxiety, mild depression, insomnia, and lack of energy and stamina. 12 Royal jelly also has the ability as a stimulant hormone to stimulate and regulate endocrine function and secretion of other hormones and its involvement in sexual manifestations and endocrine disorders. Royal jelly is associated with therapy to accelerate the restoration of disturbed normal functions through its action on the adrenal cortex. 13 Royal jelly is said to increase appetite, increase memory, treat diabetes, overcome infertility, and in people who are recovering, honey is used to accelerate healing and help form body cells. 14 Royal jelly has a high protein content obtained from pollen processing, although it is believed that honey is also a secretion. Royal jelly is a thick, milky white liquid with a strong sour taste, rich in nutrients, pungent taste, and slightly bitter taste. Scientists and nutritionists from various countries with sophisticated laboratories have repeatedly analyzed what is contained in this royal jelly. Investigations of the content of natural compounds were started in 1852 by a chemical analyst, LL Langstroth; the content of natural compounds is very complex. The most recent research conducted by scientists and nutritionists demonstrated that royal jelly has a perfect protein composed of 22 types of amino acids that are further split into two groups, necessary amino acids and nonessential amino acids. In detail royal jelly contains: -Protein: 12,50% (22 types of amino acids) -Carbohydrates: 12.50% -Fat (unsaturated fat): 6.00% -Water (H20): 65.00% -Minerals: 0.82% -Bio-Active Agent: 3-4%. [15][16][17] This study conducted an immunoinformatics analysis of the protein Royal jelly Apis mellifera carpatica. This study found six proteins of Royal jelly Apis mellifera carpatica: epitope, antigenic and nonantigenic proteins, allergen and non-allergenic proteins, toxin, and non-toxic proteins. Proteins that are antigenic in minimal portions (8-15 amino acids) can induce the immune system. 18,19 Water-soluble glycoproteins having molecular weights between 10-70 KD are frequently allergenic. 20 Allergies can trigger mild symptoms such as itching, runny nose and eyes, and swelling. Allergies can also cause severe reactions such as anaphylaxis, leading to death.

CONCLUSION
The research results found that six proteins of Royal jelly Apis mellifera carpatica were epitopes, specifically 1 protein from protein number 1, 2 proteins from protein number 5, and 3 proteins from protein number 6. In addition, antigenicity analysis was also carried out on Royal protein.
Jelly Apis mellifera carpatica, from the research results carried out, four proteins were antigenic, and two proteins were non-antigenic. This study also found that six proteins of Royal jelly Apis mellifera carpatica were non-toxic. In addition, based on the results of the analysis of the Royal jelly Apis mellifera carpatica protein on allergen and nonallergenic properties, it was found that three proteins of Royal jelly Apis mellifera carpatica were allergen and three proteins of Royal jelly Apis mellifera carpatica were non-allergenic. In this study, the threedimensional structure has not been found and opens opportunities for proteomic studies of the Royal jelly Apis mellifera carpatica protein, including protein isolation.