A complement protein composition for assessing risk of aids and uses thereof

Abstract

This invention provides a complement protein composition for assessing the risk of HIV / AIDS and its application, belonging to the field of disease risk assessment technology. The complement protein composition includes C1RL, C4BP, C7-cDNAFLJ78207, C7-cDNAFLJ93143, CFB, and CFHR2. By detecting the expression levels of the above complement protein composition in test serum and healthy human serum, if the expression of C4BP, C7-cDNAFLJ78207, and C7-cDNAFLJ93143 is upregulated, and the expression of C1RL, CFB, and CFHR2 is downregulated, it indicates a high risk of HIV / AIDS. This invention, by detecting the expression levels of each complement protein in the above complement protein composition, can rapidly assess the level of HIV / AIDS risk, providing a new option for HIV / AIDS detection reagents.

Description

Technical Field

[0001] This invention belongs to the field of disease risk assessment technology, and in particular relates to a complement protein composition for assessing the risk of HIV / AIDS and its application. Background Technology

[0002] Traditional Chinese medicine believes that viral diseases are often caused by dampness in the body. Epidemiological surveys and clinical practice of AIDS have also shown that dampness is a primary pathological factor in AIDS patients. Dampness is a yin evil, easily stagnating in the internal organs and meridians, obstructing the flow of qi and depleting the body's yang qi. Dampness is often heavy and turbid, causing a feeling of heaviness or immobility. After dampness invades the body, patients experience heaviness in the limbs, numbness in the joints, or difficulty in flexion and extension. The sticky nature of dampness, besides manifesting as subjective clinical discomfort, also affects the prognosis of the disease, leading to recurrent attacks, prolonged illness, and even extended disease course. Damp-heat refers to the combination of dampness and heat, which can cause diseases of the spleen, stomach, liver, gallbladder, or skin and tendons. Records of this existed before the Sui and Tang dynasties, such as in the *Suwen* (Plain Questions) chapter "On the Circulation of Vital Energy": "If damp-heat is not dispelled, the large tendons become soft and short, the small tendons become lax and long; softness and shortness lead to stiffness, laxity and longness lead to weakness." Damp-heat syndrome is also described as a syndrome in ancient literature. For example, Wu Kun'an's *Treatise on Cold Damage* states: "A weak, thready, and rapid pulse, fever, body aches, and scanty, difficult urination; or yellowing of the skin and eyes, indicates damp-heat. Wu Ling San with added Gardenia, Phellodendron bark, Artemisia capillaris, and Gentiana macrophylla is appropriate." *Treatise on Cold Damage, Volume Seven, Xue Shengbai's Differentiation of Damp-Heat* states: "Damp-heat syndrome begins with aversion to cold, followed by fever without chills, sweating, chest tightness, white tongue coating, and thirst without a desire to drink." The above summarizes the clinical manifestations and treatments for damp-heat syndrome. Therefore, proteomic research on dampness and damp-heat has significant practical implications for the prevention and treatment of viral and infectious diseases.

[0003] Complement is a glycoprotein in serum with enzymatic activity. Normally existing in an inactive state, it can exert biological activity upon activation by antigen-antibody complexes or microorganisms, eliminating pathogens or neutralizing viruses through direct cleavage or promoting phagocytosis, leading to significant inflammatory responses. Complement promotes inflammatory and immune responses and may affect cardiometabolic risk; plasma C3 levels are positively correlated with obesity. C3a and C5a binding to receptors or damaging cell membranes can induce inflammatory responses. Studies perfusing human C5a into the renal arteries of rats with normal blood volume have shown increased resistance in the efferent arterioles, confirming that complement activation reduces nephron filtration rate and LpA by constricting glomerular arterioles and chemotactic PMNs. Research in a mouse model of non-alcoholic hepatitis has found that complement C5 promotes steatosis and inflammatory responses; C5a can synergistically participate in adipocyte inflammation regulation with TLR4, stimulating the secretion of inflammatory factors. Complement also plays a crucial role in ischemia-reperfusion injury. Complement C5a, upon binding to its receptor, induces ischemia-reperfusion autophagy and regulates the inflammatory response during injury. Plasma C5a expression is significantly elevated in non-small cell lung cancer, suggesting activation of the complement system in vivo.

[0004] When HIV enters the human body and causes infection, the complement system is activated, a fundamental characteristic of the AIDS immune response. Complement proteins play a prominent role in the host response to HIV infection. C1 is the first component of the complement complex, including C1q, C1r, and C1s proteins. C1q can regulate the capacity of immune cells to respond. Researchers who treated 96 HIV / AIDS patients with HAART for 6 months found significant differences in serum C1q expression levels before and after treatment. Decreased C1q expression was observed in patients with kidney damage caused by HAART, while decreased, increased, or unchanged C1q expression was observed in patients without kidney damage caused by HAART. Therefore, detecting serum C1q levels can infer the inflammatory state in HIV / AIDS patients. C1q interacts with the HIV transmembrane protein gp41, thereby activating the classical complement pathway and causing C3 deposition on the viral surface. Some studies have inferred an association between C1RL and iatrogenic calcification (IAGP). C1s, an α2-globulin, possesses the physicochemical properties of both proteins and enzymes. C1s deficiency is closely associated with the development of systemic lupus erythematosus (SLE). Researchers have detected significantly increased expression of complement components such as C3 in the brain tissue of patients with HIV-associated neurocognitive impairment (HAND), suggesting that C3 and IL-6 induction may cause brain inflammation and cognitive dysfunction in HIV-infected individuals. Other data indicate a positive correlation between plasma C3 levels and cardiovascular disease risk. Downregulation of C3 and CFB expression inhibits the proliferation and migration of cutaneous squamous cell carcinoma (cSCC) cells and suppresses the growth of xenografts. Complement C3b and C4b can affect HIV infection and replication. C7 exerts its activity by forming a membrane attack complex, which can disrupt mitochondrial structure, promote apoptosis, and is closely related to tumorigenesis, potentially serving as a tumor suppressor. Researchers, by knocking out the CFB gene in spontaneously hypertensive rats, discovered that CFB plays a crucial role in metabolic syndrome in spontaneously hypertensive rats, potentially becoming a new target for the treatment of cardiometabolic diseases. Recent discoveries of CFH gene polymorphisms and rare mutations have been shown to be closely associated with age-related macular degeneration (AMD). Therefore, the activation effect of complement can reflect the viral activity and immune level in HIV-infected individuals.

[0005] Proteomics has made significant strides in clinical diagnosis, drug screening and development, personalized medicine, biomarker and pathological mechanism research. Mass spectrometry-based quantitative proteomics can precisely measure changes in the expression of multiple proteins, making it possible to objectively quantify TCM syndromes at protein levels. However, there are currently no reports on which serum complement protein expression levels can serve as important reference indicators for detecting immune homeostasis, disease progression, and assessing clinical drug efficacy in HIV / AIDS patients with damp-heat syndrome. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a complement protein composition for assessing the risk of HIV / AIDS and its application.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] The present invention provides a complement protein composition for assessing the risk of HIV infection, the complement protein composition comprising C1RL, C4BP, C7-cDNAFLJ78207, C7-cDNAFLJ93143, CFB and CFHR2.

[0009] The present invention also provides the use of the above complement protein composition in the preparation of products for assessing the risk of HIV infection.

[0010] Preferably, the AIDS is of the damp-heat type.

[0011] Preferably, the product includes a reagent kit.

[0012] Preferably, the kit further includes a negative control, which is serum from healthy individuals.

[0013] The present invention also provides a method for assessing the risk of HIV infection for non-diagnostic purposes, comprising the step of detecting the expression level of the above-mentioned complement protein composition in test serum and serum of healthy individuals.

[0014] Preferably, if the expression of C4BP, C7-cDNAFLJ78207, and C7-cDNAFLJ93143 is upregulated, and the expression of C1RL, CFB, and CFHR2 is downregulated, it indicates a high risk of HIV infection.

[0015] Preferably, when the ratio of test serum to healthy serum is ≥1.20 and p<0.05, it indicates that complement protein expression is upregulated; when the ratio of test serum to healthy serum is ≤0.83 and p<0.05, it indicates that complement protein expression is downregulated.

[0016] In the above method, the AIDS is the damp-heat type of AIDS.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides a complement protein composition for assessing the risk of HIV / AIDS and its application. The complement protein composition includes C1RL, C4BP, C7-cDNA FLJ78207, C7-cDNA FLJ93143, CFB, and CFHR2. By detecting the expression levels of the above complement protein composition in test serum and healthy human serum, if the expression of C4BP, C7-cDNA FLJ78207, and C7-cDNA FLJ93143 is upregulated, and the expression of C1RL, CFB, and CFHR2 is downregulated, it indicates a high risk of HIV / AIDS. This invention, by detecting the expression levels of each complement protein in the above complement protein composition, can rapidly assess the level of HIV / AIDS risk, providing a new option for HIV / AIDS detection reagents. Attached Figure Description

[0019] Figure 1 The image shows the secondary mass spectra of each peptide labeled by the iTRAQ eight-labeling method obtained in Example 1. Detailed Implementation

[0020] The present invention provides a complement protein composition for assessing the risk of HIV infection, the complement protein composition comprising C1RL, C4BP, C7-cDNAFLJ78207, C7-cDNAFLJ93143, CFB and CFHR2.

[0021] In this invention, C7-cDNA FLJ78207 refers to the cDNA FLJ78207 gene fragment in C7; C7-cDNA FLJ93143 refers to the cDNA FLJ93143 gene fragment in C7.

[0022] The present invention also provides the use of the above complement protein composition in the preparation of products for assessing the risk of HIV infection.

[0023] In this invention, the AIDS is preferably of the damp-heat syndrome type. The product preferably includes a kit, which preferably includes antibodies, primer pairs, or fluorescent reagents for labeling complement proteins to detect the expression levels of the aforementioned complement proteins. The complement proteins are preferably C1RL, C4BP, C7-cDNAFLJ78207, C7-cDNAFLJ93143, CFB, and CFHR2. More preferably, the kit also includes a negative control, which is preferably serum from healthy individuals.

[0024] The present invention also provides a method for assessing the risk of HIV infection for non-diagnostic purposes, comprising the step of detecting the expression level of the above-mentioned complement protein composition in test serum and serum of healthy individuals.

[0025] In the above method for assessing the risk of HIV infection, if the expression of C4BP, C7-cDNAFLJ78207, and C7-cDNAFLJ93143 is upregulated, and the expression of C1RL, CFB, and CFHR2 is downregulated, it indicates a high risk of HIV infection. The preferred method for determining the upregulation or downregulation of these complement proteins is as follows: if the ratio of the test serum to the serum of a healthy person is ≥1.20 and p < 0.05, it indicates upregulation of complement proteins; if the ratio is ≤0.83 and p < 0.05, it indicates downregulation of complement proteins. In the above method, the serum is preferably peripheral blood serum, and the HIV infection is preferably of the damp-heat syndrome type.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] 1. Case Studies and Methods

[0029] 1.1 Screening criteria for the study cases: (1) The diagnostic criteria for AIDS were based on the "Guidelines for the Diagnosis and Treatment of AIDS (2011 Edition)" issued by the AIDS Group of the Infectious Diseases Branch of the Chinese Medical Association; (2) The total score of the "AIDS Damp-Heat Syndrome Diagnosis Scale" was ≥20 points, the HIV antibody in the negative control group was negative, and there was no high-risk sexual behavior for 3 consecutive months; (3) The age was 16 to 60 years old; (4) The informed consent form had been signed.

[0030] In the "Diagnostic Scale for Damp-Heat Syndrome in AIDS", the symptoms and signs such as fullness in the epigastrium, dark and scanty urine, loose stools, bitter and sticky mouth, low fever, dry mouth without thirst, dizziness and vertigo, and heaviness in the limbs are graded as follows: none (0 points), mild (1 point), moderate (2 points), and severe (3 points). The weight coefficients of the above symptoms and signs are 3, 2, 3, 2, 2, 2, 1, and 1, respectively. The presence or absence of symptoms and signs such as red tongue, greasy yellow coating, thick coating, yellow and white mixed coating, and soft and rapid / slippery pulse are also considered. The weight coefficients of red tongue, greasy yellow coating, thick coating, yellow and white mixed coating, and soft and rapid / slippery pulse are 2, 5, 4, 3, and 1, respectively. The symptom score = weight coefficient × symptom grade score, and the total score is the sum of the symptom scores of all the above symptoms and signs.

[0031] 1.2 Exclusion criteria for study cases: (1) non-cooperation with the investigation; (2) mental illness or major organic disease.

[0032] 1.3 Case Collection

[0033] The study cases were selected from HIV / AIDS patients in a high-incidence area. All HIV / AIDS patients in the area were included as the initial subjects. After obtaining informed consent from the initial subjects, they were screened according to the "AIDS Damp-Heat Syndrome Diagnostic Scale". Those who met the screening and exclusion criteria were included as study cases, and 23 cases of HIV / AIDS damp-heat syndrome were included. At the same time, healthy people in the same area were collected as negative control group, with 16 cases in the negative control group. The basic information of the participants is shown in Table 1.

[0034] Table 1 Basic Information of Enrolled Personnel

[0035]

[0036] As shown in Table 1, there were no statistically significant differences in the demographic data of the two groups in this embodiment, indicating that the baseline values ​​of the two groups were consistent and comparable, and could be used for subsequent analysis and comparison.

[0037] 1.4 Preparation of serum samples

[0038] In the early morning, fasting peripheral blood was collected from subjects in the HIV / AIDS damp-heat syndrome group and the negative control group in step 1.3 in non-anticoagulated blood collection tubes, transported to the laboratory at low temperature, centrifuged at 2500 r / min for 10 min, and the serum was extracted and frozen in an ultra-low temperature freezer at -80℃ for unified testing.

[0039] 1.5 Detection of serum samples

[0040] The serum samples obtained in step 1.4 were thawed, and high-abundance proteins in the serum were discarded by filtering with an Agilent multiple affinity centrifuge column. Low-abundance proteins were concentrated and enzymatically digested. Peptide quantification was performed using the Bradford method to determine protein concentration. Approximately 20 μg of peptides from each group of samples were labeled and fractionated using the iTRAQ 8-labeling method, following the instructions of the iTRAQ Reagent-8plex Multiplex Kit (AB SCIEX). The internal control protein was REF. The labeled peptides were then separated and identified by LC-MS mass spectrometry (Q-Exactive mass spectrometer, Thermo Finnigan). The secondary mass spectra obtained from the separation and identification are shown in the figure. Figure 1 .

[0041] 1.6 Data Analysis and Statistics

[0042] Database identification and quantitative analysis were performed using the software Mascot2.2 and Proteome Discoverer1.4 (thermo).

[0043] The raw mass spectrometry data were identified and quantified using Mascot 2.2 and Proteome Discoverer 1.4 (thermo). A t-test was performed on the protein quantification data. Upregulation of protein expression was defined as a ratio ≥1.20 (p<0.05) between the HIV / AIDS damp-heat syndrome group and the negative control group, and downregulation as a ratio ≤0.83 (p<0.05). Basic information on the detected complement protein expression is shown in Table 2, where the fold change is the ratio of complement protein expression levels between the HIV / AIDS damp-heat syndrome group and the negative control group.

[0044] Table 2 Basic Information on Complement Proteins

[0045]

[0046] As shown in Table 2, a total of 22 complement proteins were identified in this embodiment, and 6 differentially expressed proteins were screened. Among them, 3 proteins were upregulated, namely C4BP, C7-cDNAFLJ78207, and C7-cDNAFLJ93143; and 3 proteins were downregulated, namely C1RL, CFB, and CFHR2.

[0047] Example 2

[0048] This embodiment included 11 cases of HIV / AIDS with damp-heat syndrome and 8 cases of negative control group, following the screening criteria, exclusion criteria, and case collection methods mentioned in Embodiment 1. The expression levels of six complement proteins (C4BP, C7-cDNAFLJ78207, C7-cDNAFLJ93143, C1RL, CFB, and CFHR2) in the HIV / AIDS with damp-heat syndrome and negative control groups were detected using the serum sample preparation, serum sample detection, data analysis, and statistical methods described in Embodiment 1. A t-test was performed on the protein quantification data. Upregulation of protein expression was defined as a ratio of complement protein expression ≥1.20 and p<0.05 between the HIV / AIDS with damp-heat syndrome and negative control groups, while downregulation was defined as a ratio ≤0.83 and p<0.05. If the expression of C4BP, C7-cDNAFLJ78207, and C7-cDNAFLJ93143 is upregulated, and the expression of C1RL, CFB, and CFHR2 is downregulated, it indicates a high risk of HIV / AIDS with damp-heat syndrome. The basic information on the expression of complement proteins detected is shown in Table 3, where the fold-over ratio is the ratio of the expression levels of each complement protein in the HIV / AIDS damp-heat syndrome group to the negative control group.

[0049] Table 3. Basic information on complement protein expression.

[0050]

[0051] As shown in Table 3, compared with the negative control group, the expression of C4BP, C7-cDNAFLJ78207, and C7-cDNAFLJ93143 in the serum of 11 HIV / AIDS patients with damp-heat syndrome was upregulated, while the expression of C1RL, CFB, and CFHR2 was downregulated. Therefore, this invention can rapidly and accurately determine the risk of HIV / AIDS with damp-heat syndrome by detecting the expression levels of six complement proteins: C4BP, C7-cDNAFLJ78207, C7-cDNAFLJ93143, C1RL, CFB, and CFHR2.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A complement protein composition for assessing the risk of AIDS, characterized by, The complement protein composition is C1RL, C4BP, C7-cDNA FLJ78207, C7-cDNA FLJ93143, CFB, and CFHR2; The AIDS mentioned is the damp-heat type of AIDS.

2. Use of a reagent for detecting the expression level of the complement protein composition according to claim 1 in the preparation of a product for assessing the risk of AIDS, characterized in that, The AIDS mentioned is the damp-heat type of AIDS.

3. Use according to claim 2, characterized in that, The product includes a reagent kit.

4. The application according to claim 3, characterized in that, The kit also includes a negative control, which is serum from healthy individuals.

5. The application according to claim 2, characterized in that, Upregulation of C4BP, C7-cDNA FLJ78207, and C7-cDNA FLJ93143 expression, and downregulation of C1RL, CFB, and CFHR2 expression, indicate a high risk of HIV infection.

6. The application according to claim 5, characterized in that, When the ratio of test serum to healthy serum is ≥1.20 and p<0.05, it indicates that complement protein expression is upregulated; when the ratio of test serum to healthy serum is ≤0.83 and p<0.05, it indicates that complement protein expression is downregulated.

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