Marker for diagnosing and treating bone diseases and application thereof

Abstract

The invention discloses a marker for diagnosing and treating bone diseases and application of the marker, belongs to the technical field of biology, and provides a marker of which the amino acid sequence comprises LVTGG, VESS, SATVYG, AGFIGSH, LLRYFNPIGA and THDGTGVRDY. The kit prepared according to the marker can be used for detecting and diagnosing bone diseases such as osteoarthritis, rheumatoid arthritis, osteoporosis, fracture and femoral head necrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a marker for diagnosing and treating bone diseases and application thereof. BACKGROUND

[0002] Bone diseases are a class of diseases that seriously affect human health and quality of life. Chronic diseases such as osteoarthritis and rheumatoid arthritis often cause long-term pain and joint dysfunction in patients. If osteoporosis, fractures and femoral head necrosis are not treated in time or improperly, they can also cause serious complications and even disability. Stem cell therapy, as a new and promising treatment, has shown unique advantages in the treatment of bone diseases. However, the prerequisite for precise and effective stem cell therapy is early and accurate diagnosis of the disease.

[0003] We found that the galE polypeptide of the conditional pathogenic bacteria has a certain potential role in the diagnosis of bone diseases, and it is closely related to the occurrence and development of bone diseases, and is expected to become a highly potential marker for diagnosing bone diseases.

[0004] The galE gene encodes UDP-galactose epimerase (hereinafter referred to as galE protein), which plays a key role in the virulence mechanism of many bacteria. In many gram-negative pathogens, they have been proved to be important virulence factors.

[0005] The galE polypeptide is produced by conditional pathogenic bacteria and is one of the key factors of bacterial virulence expression. Its structure is complex and contains multiple special functional domains, which endow the galE polypeptide with diverse biological activities. In the process of bacterial survival and pathogenesis, galE polypeptide plays an important role. It not only participates in the synthesis and modification of bacterial cell wall, maintains the structural integrity and stability of bacteria, but also can interfere with the normal physiological function of host cells through interaction with host cells, and induce a series of pathological reactions. In known bacterial metabolism research, galE protein is usually involved in galactose metabolic pathway. As uridine diphosphate-galactose 4-epimerase, it can catalyze specific chemical reactions, which is essential for bacteria to obtain and utilize galactose-related nutrients.

[0006] The present application provides a galE polypeptide as a marker for diagnosing bone diseases and provides a corresponding reagent, and further forms a technical solution of a detection kit. SUMMARY

[0007] To solve the problems in the background art, the present application aims to provide a galE polypeptide for diagnosing and treating bone diseases, and a bone disease detection kit based on galE polypeptide.

[0008] The technical scheme is specifically implemented as follows:

[0009] The application provides a marker for diagnosing and treating bone diseases, a galE polypeptide, and an amino acid sequence of the galE polypeptide comprises LVTGG, VESS, SATVYG, AGFIGSH, LLRYFNPIGA and THDGTGVRDY.

[0010] In order to meet the requirements of specificity and quantitative accuracy of clinical detection, the SDS-PAGE concentration of the marker is greater than or equal to 95%, the HPLC purity is greater than or equal to 98%, and 5 is less than or equal to 7.

[0011] The application of the galE polypeptide in a bone disease diagnosis kit.

[0012] The bone diseases include osteoarthritis, rheumatoid arthritis, osteoporosis, bone fracture and femoral head necrosis.

[0013] We screened bacteria containing galE gene sequences through bioinformatics tools such as BLAST (Basic Local Alignment Search Tool), and found that the following bacteria all contain galE proteins.

[0014]

[0015] By analyzing the amino acid sequence of the galE protein of Aggregatibacter lacticniger, it is found that the galE amino acid sequence of Aggregatibacter lacticniger contains common 20 kinds of amino acids, and contains more amino acids related to catalytic activity, such as serine (Ser), threonine (Thr) and histidine (His), which may play a key role in the active center of the enzyme.

[0016] Subsequently, we use bioinformatics tools to analyze the known amino acid sequences. With the help of multiple sequence alignment technology, the conserved regions and variation sites in the sequence are determined, and the conserved parts are often closely related to the key functions, providing important clues for subsequent prediction. At the same time, various features of the amino acid sequence are extracted, including amino acid composition, hydrophilicity and hydrophobicity, and secondary structure tendency. Through polypeptide synthesis technology, the polypeptides of LVTGG, VESS, SATVYG, AGFIGSH, LLRYFNPIGA and THDGTGVRDY are synthesized, which is a feasible technical scheme, and the sequence table is as follows:

[0017] 1 mankslakdh adicvlvtgg agfigshtcv elldqgyhvv vvddlsnsse laldrvrqit

[0018] 61 gla and drlk fyeanildra aldrvfsene vdaiihfagf kavgesvqkp leyywnnfag

[0019] 121 tlalcdvara hgvknlvfss satvygepef ipitedcpkh datnpygwtk smleqvltdl

[0020] 181 yvgddewnvv llryfnpiga hesgligedp kgipnnllpy vaqvavgkle svgvfgddyp

[0021] 241 thdgtgvrdy ihvvdlargh vaaldwmggk vgtgeaktag tmagepaadg trrgvgifnl

[0022] 301 gtgtgssvld vvhsferacg relpyqikpr ragdvavnya acdkardelg wvaqydldrm

[0023] 361 cadgwrwqsq npdgyatara

[0024] And the detection of a large number of clinical samples is verified.

[0025] By adopting the technical scheme, the beneficial effects of the present application are:

[0026] The toxicological effect of the toxicological factor galE polypeptide of the opportunistic pathogen is closely related to the occurrence and development of bone diseases, and it has important potential value in the diagnosis of bone diseases. Through in-depth study of the mechanism of action of galE polypeptide, optimization of detection method and development of large-scale clinical research, it is developed into an effective diagnostic marker for bone diseases, which provides new ideas and methods for early diagnosis, disease monitoring and stem cell treatment of bone diseases, and promotes the improvement of the diagnosis and treatment level of bone diseases. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The galE polypeptide content detected in the samples of different groups in Example 2;

[0028] Figure 2The relationship between the content of galE polypeptide in the joint fluid of the osteoarthritis patients in Example 2 and the degree of damage of the articular cartilage, wherein: the relationship between the content of galE polypeptide and the degree of damage of the articular cartilage of the osteoarthritis (A), the different response periods of rheumatoid arthritis (B) (*P<0.05, **P<0.01, ***P<0.001);

[0029] Figure 3 The content change of galE polypeptide in Example 2 in different periods of fracture (A) and different types of femoral head necrosis (B);

[0030] Figure 4 The curve obtained by the ROC curve analysis of the diseased and non-diseased individuals in Example 2;

[0031] Figure 5 Small animal live body five-dimensional awake imaging was performed on the model mice in Example 3;

[0032] Figure 6 The imaging of the polypeptide diffusing to the joints of the model mice and gathering;

[0033] The technical solutions of the present application are further described in detail through specific embodiments and examples. DETAILED DESCRIPTION

[0034] The technical solutions of the present application are further described in detail through specific embodiments and examples.

[0035] Example 1

[0036] Kit preparation:

[0037] The galE polypeptide is derived from Aggregatibacter lacticenidans and is a single-chain protein containing 371 amino acids (1-371 aa). Bioinformatics prediction combined with functional verification shows that its active center is composed of six conserved sequences: LVTGG, VESS, SATVYG, AGFIGSH, LLRYFNPIGA, and THDGTGVRDY. These segments mediate the interaction of the polypeptide with host cells and play a core role in pathological signal transduction.

[0038] To meet the requirements of specificity and quantitative accuracy of clinical detection, the galE polypeptide needs to meet the following purity specifications:

[0039] SDS-PAGE: ≥95 %

[0040] HPLC: ≥98 %

[0041] High purity can effectively exclude the interference of impurities and reduce the risk of false positive / negative in the detection of serum of patients with osteoarthritis and other bone diseases.

[0042] pH stability: The secondary structure can be maintained within the pH range of 5.0-7.0, with an optimal pH of 5.5-6.5. Irreversible denaturation occurs at pH < 4.0 or > 8.0.

[0043] Hydrophobicity: Overall moderate hydrophobicity, with the active center being more hydrophobic due to the enrichment of hydrophobic residues such as Leu and Val, which facilitates binding to the hydrophobic region of the cell membrane. Dissolution is recommended in PBS containing 0.01% Tween-20.

[0044] Molecular weight: Theoretical value 41.2 kDa; mass spectrometry measured deviation ≤ 0.5 kDa, which can be used as the basis for chromatographic identification and purification.

[0045] Thermal stability: No significant decrease in activity after 12 months of freezing at -20 °C; stable at 4 °C for 1 month; activity decreases by ≥ 30% after 48 h at 37 °C. Avoid repeated freeze-thaw cycles.

[0046] The specific composition of the kit is as follows:

[0047]

[0048] Example 2

[0049] Sample detection

[0050] We conducted a study to explore the differences in the content of galE polypeptide in blood samples of patients with osteoarthritis, rheumatoid arthritis, osteoporosis, bone fracture, and femoral head necrosis at different stages, and whether the content of galE polypeptide is significantly different from that of healthy people. During the research process, we strictly followed the standardized operation procedures to collect samples.

[0051] We used advanced immunoassay technology to accurately determine the galE polypeptide in the blood sample. This technology has high sensitivity and specificity, and can accurately detect the trace amount of galE polypeptide content in the sample. After a series of rigorous experimental operations and data analysis.

[0052] We found that: as the disease progresses in patients with bone diseases, the content of galE polypeptide detected in the blood is significantly higher.

[0053] 2. Test results

[0054] (1) The galE polypeptide content varies significantly among different groups. The galE polypeptide content in the blood samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA), fracture (Fx), and osteonecrosis of the femoral head (ONFH) is significantly higher than that in the healthy control group, as shown in Figure 1 .

[0055] In the joint fluid of patients with osteoarthritis, the galE polypeptide content increases significantly with the severity of joint cartilage damage ( Figure 2 .A ); the galE polypeptide content in the serum of patients with rheumatoid arthritis is significantly higher than that in healthy people, and is closely related to disease activity. The galE polypeptide level in patients with active disease is significantly higher than that in patients in remission ( Figure 2 .B ), indicating that the change in galE polypeptide content is closely related to the occurrence and development of bone diseases, supporting its potential as a diagnostic marker.

[0056] (2) Correlation with disease: The galE polypeptide content is significantly correlated with disease severity and disease progression. In patients with fractures, the galE polypeptide content in the blood gradually decreases as the fracture healing process progresses, with higher levels in the early stages of fracture and returning to near normal levels in the good fracture healing stage, indicating a close relationship between galE polypeptide content and fracture healing process. Figure 3 .A

[0057] According to the location and extent of necrosis, the current international standard is the Japanese Institute of Ceramics (JIC) classification, which divides femoral head necrosis into four groups: A, B, C1, and C2 (Table 1). We found that the galE polypeptide content in patients with femoral head necrosis was positively correlated with the extent of necrosis, with larger necrosis and more severe disease associated with higher galE polypeptide content. Figure 3 .B This indicates that galE polypeptide can not only be used for disease diagnosis, but also provide important evidence for disease monitoring and prognosis evaluation.

[0058]

[0059] Table 1: Classification of femoral head necrosis

[0060] 3. Diagnostic model performance

[0061] The diagnostic model based on galE polypeptide content exhibits good performance. The logistic regression model has high accuracy in diagnosing bone diseases and can effectively distinguish between diseased and non-diseased individuals.

[0062] The curve obtained through ROC curve analysis ( Figure 4 The area under the curve (AUC value of 0.996) is close to 1, indicating that the diagnostic model has strong discriminative ability. For example, in diagnosing rheumatoid arthritis, the model can accurately identify most patients with a low misdiagnosis rate. The determined optimal cutoff value provides a clear standard for clinical diagnosis. When the galE peptide content is higher than the cutoff value, the patient's likelihood of having bone diseases increases significantly, which helps doctors to conduct further examinations and diagnoses in a timely manner.

[0063] 4. Consistency and reliability of results

[0064] After cross-validation, bootstrap resampling, and repeated validation across different sample populations and research centers, the results showed a high degree of consistency. The diagnostic model performance indices obtained from different validation methods were similar, and similar conclusions were reached across samples from different regions and ethnicities: galE peptide levels differ significantly between patients with bone diseases and healthy individuals, and are correlated with disease severity and progression. This fully demonstrates the reliability of the research results and provides a solid theoretical and practical foundation for the widespread application of galE peptides in clinical diagnosis.

[0065] 5. Possibility of a negative result:

[0066] During the study, some negative results may also occur. Although the overall galE peptide level in the patient group was higher than that in the healthy control group, the galE peptide level in some patients may be within the normal range, leading to a certain rate of missed diagnosis when using it as a diagnostic marker.

[0067] In some patients with milder or specific types of bone diseases, galE peptide levels show little variation, making accurate diagnosis difficult based on differences in levels. Furthermore, the correlation between galE peptide levels and disease severity and progression may be insignificant in some patient populations, affecting its reliability as a disease monitoring indicator. When these negative results occur, further investigation is needed to determine the reasons, such as the representativeness of the sample selection and limitations of the detection method, to provide directions for improvement in future research.

[0068] Analysis of numerous clinical blood samples revealed a close correlation between changes in galE peptide levels and disease severity and progression. For example, in osteoarthritis patients, galE peptide levels in synovial fluid and blood gradually increased with the severity of articular cartilage damage; in rheumatoid arthritis patients, galE peptide levels were positively correlated with disease activity indicators. These findings suggest that galE peptides possess the potential to serve as diagnostic biomarkers for bone diseases.

[0069] Example 3

[0070] In vivo experiment verification

[0071] With the deepening of the research on the pathogenesis of OA, targeted therapy has gradually become a research hotspot. The galE polypeptide, as a molecule with unique biological activity, has shown great potential in disease treatment because it can specifically bind to cell surface receptors or enter cells to play a role, which has prompted us to carry out a series of exploratory research on the application of polypeptides in OA treatment.

[0072] C57 osteoarthritis model mice were constructed. Model construction is an important basis for studying disease mechanisms and treatment methods. C57 mice have clear genetic background and high susceptibility to various diseases, making them an ideal animal for OA model construction. By transecting the anterior cruciate ligament, we successfully established an osteoarthritis model in C57 mice, simulating the pathogenesis of human OA and providing a reliable experimental object for subsequent research.

[0073] Previous studies have shown that 16S rRNA gene sequencing technology detected the presence of Aggregatibacter lacticola sequences in the synovial fluid of patients with arthritis. Based on this, we prefer to use Aggregatibacter lacticola extracellular vesicles to intervene in model mice. Since we used intravenous injection, we found that Aggregatibacter lacticola extracellular vesicles caused blockage at the tail root. Therefore, we used intramuscular injection, and the results showed that the fluorescence of Aggregatibacter lacticola extracellular vesicles in the muscle of model mice decreased over time. Figure 5

[0074] After intravenous injection for 6 hours, intramuscular injection was performed at 6 hours and 12 hours. Small animal live five-dimensional awake imaging was performed on model mice, and we found that the fluorescence of Aggregatibacter lacticola extracellular vesicles in the muscle of model mice decreased over time. Through Micro-CT and pathological sectioning, we found that the tibia of Aggregatibacter lacticola extracellular vesicle-intervened mice had obvious damage compared to model mice. This indicates that Aggregatibacter lacticola extracellular vesicles can accelerate the development of osteoarthritis.

[0075] We further analyzed Aggregatibacter lacticola extracellular vesicles and found that the galE protein, as a virulence factor, may induce or accelerate the occurrence and development of osteoarthritis.

[0076] As mentioned above, we found the functional region of galE and further verified it.

[0077] ​We will introduce the galE polypeptide into the model mice by intramuscular injection. (We choose intramuscular injection because it is relatively simple to operate and can make the drug enter the blood circulation system relatively quickly and then distribute to the joints.) During the injection process, we closely monitor the physiological indicators of the mice to ensure the safety and stability of the injection operation.

[0078] After a period of observation, we found that the polypeptide spread to the joints of the model mice and appeared to be aggregated. This phenomenon is of great significance. The joint is the main lesion site of OA, and the aggregation of polypeptide in the joint means that it may play a role in the local joint and directly affect the diseased joint. Figure 6

[0079] To further explore the mechanism of action of the polypeptide, we carried out a co-localization experiment of human cartilage cells and polypeptide.

[0080] Cartilage cells are the main component cells of articular cartilage and play a key role in the pathogenesis of OA. We co-cultured human cartilage cells with polypeptide and used laser confocal microscopy to observe the interaction between cells and polypeptide. This technology can clearly show the distribution of cell nuclei and polypeptide in cells.

[0081] After careful observation and analysis, we found that the polypeptide can enter the cartilage cells of osteoarthritis patients smoothly. This discovery provides an important theoretical basis for polypeptide-induced OA development, indicating that polypeptide may enter cartilage cells, regulate intracellular signaling pathways, affect cell metabolism and function, and thus achieve the purpose of exacerbating OA.

[0082] The purpose, technical solutions and beneficial effects of the present application are further described in detail. It should be understood that the above is only a specific embodiment of the present application and is not intended to limit the scope of protection of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application should be included within the scope of protection of the present application.​

Claims

1. A biomarker for diagnosing and treating bone diseases, characterized in that, The amino acid sequences include LVTGG, VESS, SATTYG, AGFIGSH, LLRYFNPIGA, and THDGTGVRDY.

2. The marker according to claim 1, characterized in that, The marker has an SDS-PAGE concentration ≥95%, an HPLC purity ≥98%, and a pH ≤7.

3. The application of the biomarker according to claim 1 or 2 in a diagnostic kit for bone diseases.

4. The application according to claim 3, characterized in that, The bone diseases mentioned include: osteoarthritis, rheumatoid arthritis, osteoporosis, fractures, and femoral head necrosis.

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