Method and kit for diagnosing systemic lupus erythematosus

Abstract

The invention relates to a method and a kit for diagnosing systemic lupus erythematosus. Specifically, the invention provides a reagent for detecting tryptophan in a blood-derived sample or a reagent for detecting tryptophan in a blood-derived sample and a tryptophan standard substance, and application of the reagent and the tryptophan standard substance in preparation of a kit, the kit is used for diagnosing the systemic lupus erythematosus and / or identifying the disease activity of a patient with the systemic lupus erythematosus and / or identifying the kidney fatigue degree of the patient with the systemic lupus erythematosus and / or judging the prognosis of the lupus nephritis. The method is simple and rapid, results are accurate, the SLE patient can be diagnosed in real time, and the disease activity, kidney fatigue degree and lupus nephritis prognosis of the SLE patient can be judged in an assisted mode.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method and a kit for diagnosing systemic lupus erythematosus. Background Art

[0002] Systemic lupus erythematosus (SLE) is a potentially fatal, chronic multi-system autoimmune inflammatory disease that usually affects women, especially women of childbearing age. Within 5 years after the diagnosis of SLE, approximately 40 - 80% of SLE patients will develop lupus nephritis (LN) during the course of the disease. LN is one of the most severe complications of SLE and is also a major driving factor for its morbidity and mortality. Despite aggressive immunosuppressive therapy, 10 - 30% of severe LN patients will progress to end-stage renal disease within 15 years.

[0003] Currently, the "gold standard" for diagnosing and monitoring LN is renal biopsy, but this technique is invasive, has potential risks, and is expensive for patients. Conventional biomarkers, such as dsDNA antibodies, complements, etc., lack sensitivity and specificity in diagnosing LN or evaluating renal pathology. Therefore, finding new non-invasive biomarkers for diagnosing, predicting renal prognosis, and guiding the treatment of LN remains one of the main goals and challenges in SLE research. Tryptophan is an essential amino acid for the human body. Some studies have shown that the IDO1-mediated degradation of Trp is regarded as a key feedback mechanism for regulating overactive immune responses and may be closely related to the occurrence and progression of autoimmune diseases such as rheumatoid arthritis and SLE. Summary of the Invention

[0004] Based on the deficiencies of the prior art, in the first aspect of the present invention, there is provided the use of a reagent for detecting tryptophan derived from a blood sample, or a reagent for detecting tryptophan derived from a blood sample and a tryptophan standard in the preparation of a kit for one or more of the following: diagnosing systemic lupus erythematosus, identifying the disease activity of systemic lupus erythematosus patients, identifying the degree of kidney involvement in systemic lupus erythematosus patients, and judging the prognosis of lupus nephritis; wherein, the disease activity of the systemic lupus erythematosus patients includes active systemic lupus erythematosus and inactive systemic lupus erythematosus, and the degree of kidney involvement includes systemic lupus erythematosus with kidney involvement and systemic lupus erythematosus without kidney involvement.

[0005] In one or more embodiments, the prognosis is the prognosis of immunotherapy. Preferably, the immunotherapy is carried out for 4 - 8 months.

[0006] In one or more embodiments, the sample is a blood sample.

[0007] In one or more embodiments, the blood sample is whole blood, serum, and / or plasma.

[0008] In one or more embodiments, the reagent for detecting tryptophan includes the reagents required for detecting tryptophan using one or more methods selected from the following: visible spectrophotometry, acidic ninhydrin method, chromatography, surface-enhanced Raman spectroscopy, and optical probe method.

[0009] In one or more embodiments, the reagent for detecting tryptophan includes reagents for transforming, enriching, separating, or identifying tryptophan.

[0010] In one or more embodiments, the reagent for detecting tryptophan includes one or more selected from the following: tryptophan-specific antibody, tryptophanase, hydroxylamine, sulfuric acid, TCEP, formic acid, hydrochloric acid, ninhydrin, ethanol, formic acid, acetonitrile, isopropanol, DEAM, sodium nitrite, tryptophan-binding protein, or functional variants thereof.

[0011] In one or more embodiments, the functional variant of the tryptophan-binding protein includes a tryptophan optical probe.

[0012] In one or more embodiments, the tryptophan optical probe comprises a tryptophan-binding protein and an optically active polypeptide.

[0013] In one or more embodiments, the tryptophan-binding protein has the sequence shown in SEQ ID NO:1 or a functional fragment thereof, or a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity therewith.

[0014] In one or more embodiments, the optically active polypeptide is located at one or more sites selected from the following of the tryptophan-binding protein: 114 / 115, 114 / 116, 114 / 117, 114 / 118, 115 / 116, 115 / 117, 115 / 118, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 233 / 235, 234 / 235, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 264 / 268, 265 / 266, 265 / 267, 265 / 268, 266 / 267, 266 / 268, and / or 267 / 268.

[0015] In one or more embodiments, the tryptophan-sensitive polypeptide contains mutations at the following selected sites: H13, T71, L84, D93, K105, N117, T130, V170, V192, F202, K214, Y217, K255, T259, I283, and the amino acid mutations include modifications, substitutions, or deletions of amino acids.

[0016] In one or more embodiments, the mutation is selected from one or more of H13F, H13W, T71F, T71W, L84F, L84W, D93F, D93W, K105F, K105W, N117F, N117W, T130F, T130W, V170F, V170W, V192F, V192W, F202W, K214F, K214W, Y217F, Y217W, K255F, K255W, T259F, T259W, I283F and / or I283W.

[0017] A second aspect of the present invention provides a method for diagnosing systemic lupus erythematosus, identifying the disease activity of a patient with systemic lupus erythematosus, identifying the degree of renal involvement in a patient with systemic lupus erythematosus, and judging the prognosis of lupus nephritis, comprising: (1) detecting tryptophan in a sample derived from blood, (2) comparing with a control level, wherein the tryptophan level in an active systemic lupus erythematosus patient is lower than the control level, the tryptophan level in a systemic lupus erythematosus patient with renal involvement is lower than the control level, the tryptophan level in an active lupus nephritis patient is lower than the control level, the tryptophan level in a non-complete remission group of lupus nephritis is lower than the control level, and the tryptophan level in a complete remission group is lower than that in a healthy subject.

[0018] In one or more embodiments, compared with the control level, the tryptophan level in a sample of an active systemic lupus erythematosus patient (aSLE) is significantly reduced. The control level is derived from the tryptophan level in a corresponding sample of a non-active systemic lupus erythematosus patient (iSLE) or a healthy subject. In an exemplary embodiment, the control level is derived from the tryptophan level in a corresponding sample of a healthy subject, and the control level is about 47±9 μM to 52±7 μM. In an exemplary embodiment, the control level is derived from the tryptophan level in a corresponding sample of a non-active systemic lupus erythematosus patient, and the control level is about 43±12 μM to 45±13 μM.

[0019] In one or more embodiments, compared with the control level, the tryptophan level in a sample of a systemic lupus erythematosus patient with renal involvement (LN) is significantly reduced. The control level is derived from the tryptophan level in a corresponding sample of a non-renal-involved systemic lupus erythematosus patient (NR) or a healthy subject. In an exemplary embodiment, the control level is derived from the tryptophan level in a corresponding sample of a healthy subject, and the control level is about 49±6 μM to 51±8 μM. In an exemplary embodiment, the control level is derived from the tryptophan level in a corresponding sample of a non-renal-involved systemic lupus erythematosus patient, and the control level is about 42±11 μM to 43±10 μM.

[0020] In one or more embodiments, compared to a control level, the tryptophan level in samples from patients with active lupus nephritis (aLN) is significantly decreased. The control level is derived from the tryptophan level in corresponding samples from healthy subjects, patients with active systemic lupus erythematosus without renal involvement (aNR), or patients with inactive systemic lupus erythematosus. In an exemplary embodiment, the control level is derived from the tryptophan level in corresponding samples from healthy subjects, and the control level is about 49 ± 6 μM to 51 ± 8 μM. In an exemplary embodiment, the control level is derived from the tryptophan level in corresponding samples from patients with active systemic lupus erythematosus without renal involvement, and the control level is about 40 ± 10 μM to 43 ± 7 μM. In an exemplary embodiment, the control level is derived from the tryptophan level in corresponding samples from patients with inactive systemic lupus erythematosus, and the control level is about 43 ± 12 μM to 45 ± 13 μM.

[0021] In one or more embodiments, the lupus nephritis prognosis is an active lupus nephritis prognosis. In one or more embodiments, the prognosis is non-complete remission and / or complete remission.

[0022] In one or more embodiments, compared to a control level, the pre-treatment baseline serum tryptophan level in patients with lupus nephritis with a non-complete remission prognosis is significantly decreased. The control level is derived from the tryptophan level in corresponding samples from healthy subjects or the complete remission group. In an exemplary embodiment, the tryptophan level in corresponding samples from healthy subjects is about 48 ± 6 μM to 51 ± 9 μM. In an exemplary embodiment, the tryptophan level in corresponding samples from the complete remission group is about 38 ± 7 μM.

[0023] In one or more embodiments, compared to healthy subjects, the tryptophan level in patients with lupus nephritis with a complete remission prognosis is significantly decreased. In one or more embodiments, the tryptophan level in patients with lupus nephritis with a complete remission prognosis is lower than that in healthy subjects but higher than that in patients with lupus nephritis with a non-complete remission prognosis. In an exemplary embodiment, the tryptophan level in corresponding samples from healthy subjects is about 48 ± 6 μM to 51 ± 9 μM. In an exemplary embodiment, the tryptophan level in corresponding samples from the complete remission group is about 38 ± 7 μM. In an exemplary embodiment, the tryptophan level in corresponding samples from the non-complete remission group is about 31 ± 9 μM.

[0024] In one or more embodiments, the lower the tryptophan level in a patient with lupus nephritis, the worse the prognosis.

[0025] In one or more embodiments, determining the prognosis of lupus nephritis includes determining whether the tryptophan level in a sample from the patient is lower than 41.0 μM (incomplete remission) or 42.5 μM (complete remission). If it is lower than 41.0 μM, the prognosis of the patient is incomplete remission; if it is lower than 42.5 μM and higher than 41.0 μM, the prognosis of the patient is complete remission.

[0026] In one or more embodiments, the tryptophan level in a sample from a patient with lupus nephritis with an incomplete remission prognosis is at least 10% lower, preferably 24 - 66% lower, than that in a healthy subject.

[0027] In one or more embodiments, the tryptophan level in a sample from a patient with lupus nephritis with a complete remission prognosis is at least 10% lower, preferably 26 - 35% lower, than that in a healthy subject.

[0028] In one or more embodiments, the tryptophan level in a sample from a patient with lupus nephritis with an incomplete remission prognosis is at least 10% lower, preferably 11 - 57% lower, than that in a sample from a patient with lupus nephritis with a complete remission prognosis.

[0029] In one or more embodiments, the prognosis is the prognosis of immunotherapy.

[0030] In one or more embodiments, the immunotherapy is immunosuppressive therapy.

[0031] The third aspect of the present invention provides a kit for detecting tryptophan, which kit contains a reagent instruction for detecting tryptophan, and the instruction records methods for diagnosing systemic lupus erythematosus by detecting tryptophan, identifying the disease activity degree of patients with systemic lupus erythematosus, identifying the degree of kidney involvement in patients with systemic lupus erythematosus, or judging the prognosis status of lupus nephritis.

[0032] In one or more embodiments, the reagents for detecting tryptophan include the reagents required for detecting tryptophan using one or more methods selected from the following: visible light photometry, acidic ninhydrin method, chromatography, surface-enhanced Raman spectroscopy, and optical probe method.

[0033] In one or more embodiments, the reagents for detecting tryptophan include reagents for transforming, enriching, separating, or identifying tryptophan.

[0034] In one or more embodiments, the method is as described in any one of the embodiments of the second aspect of the present invention.

[0035] In one or more embodiments, the reagent for detecting tryptophan comprises one or more selected from the following: tryptophan-specific antibody, tryptophanase, hydroxylamine, sulfuric acid, TCEP, formic acid, hydrochloric acid, ninhydrin, ethanol, formic acid, acetonitrile, isopropanol, DEAM, sodium nitrite, tryptophan-binding protein, or functional variants thereof.

[0036] In one or more embodiments, the serum / plasma processing reagent comprises: 3NPH_HCl, EDC, buffer.

[0037] In one or more embodiments, the buffer is a phosphate-based buffer or Tris.

[0038] In one or more embodiments, the kit contains a tryptophan optical probe and a buffer, and the tryptophan optical probe comprises a tryptophan-binding protein and an optically active polypeptide.

[0039] In one or more embodiments, the optically active polypeptide is located within the sequence of the tryptophan-binding protein, at the N-terminus or C-terminus.

[0040] In one or more embodiments, the buffer is a phosphate-based buffer or Tris.

[0041] In one or more embodiments, the buffer is HEPES, PBS, etc.

[0042] In one or more embodiments, the tryptophan-binding protein has the sequence shown in SEQ ID NO:1 or a functional fragment thereof.

[0043] In one or more embodiments, the optically active polypeptide is located at one or more of the following sites of the tryptophan-binding protein: 114 / 115, 114 / 116, 114 / 117, 114 / 118, 115 / 116, 115 / 117, 115 / 118, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 233 / 235, 234 / 235, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 264 / 268, 265 / 266, 265 / 267, 265 / 268, 266 / 267, 266 / 268, and / or 267 / 268.

[0044] In one or more embodiments, the tryptophan-sensitive polypeptide comprises mutations at one, two, or three or more sites selected from the following: H13, T71, L84, D93, K105, N117, T130, V170, V192, F202, K214, Y217, K255, T259, I283, and the amino acid mutations include modifications, substitutions, or deletions of amino acids.

[0045] In one or more embodiments, the mutations are selected from one or more of H13F, H13W, T71F, T71W, L84F, L84W, D93F, D93W, K105F, K105W, N117F, N117W, T130F, T130W, V170F, V170W, V192F, V192W, F202W, K214F, K214W, Y217F, Y217W, K255F, K255W, T259F, T259W, I283F, and / or I283W.

[0046] In one or more embodiments, the kit further comprises: reagents for detecting systemic lupus erythematosus and / or lupus nephritis. Description of the Drawings

[0047] The present invention will be further described below in conjunction with the drawings and examples.

[0048] Figure 1 Clinical characteristics of healthy individuals, active lupus nephritis (aLN), active SLE without renal involvement (aNR), and inactive SLE (iSLE) in this article.

[0049] Figure 2 Flowchart of the point-of-care testing method for clinical samples based on probes.

[0050] Figure 3 Serum test results of probe STrp-H, comparison chart of tryptophan levels between three SLE disease classification samples and corresponding healthy samples.

[0051] Figure 4 ROC analysis chart of tryptophan levels in three SLE disease classification samples.

[0052] Figure 5 Serum validation results of probe STrp-H.

[0053] Figure 6 ROC analysis chart of tryptophan levels in three SLE disease classification samples in the serum validation results.

[0054] Figure 7Comparison of baseline serum tryptophan levels between the complete remission (CR) group and the non-complete remission (Non-CR) group in aLN patients after 6 months of immunosuppressive induction therapy. Detailed implementation mode

[0055] When giving a numerical value or range, the term "about" as used herein means within 20%, within 10%, and within 5% of the given numerical value or range.

[0056] The terms "comprising", "including" and their equivalent forms include the meanings of "containing" and "consisting of", for example, a composition "comprising" X may consist only of X or may contain other substances, such as X + Y.

[0057] In this article, "decrease", "low", "lower", "significantly lower", "increase", "high", "higher", "significantly higher", etc. indicate a statistically significant change relative to the comparison value, including but not limited to a change of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200% or more.

[0058] Due to the lack of significant differentiation in the onset age and clinical manifestations of patients, the current conventional biomarkers for diagnosing LN, such as dsDNA antibodies, complements, etc., lack sensitivity and specificity in diagnosing LN or evaluating renal pathology. By detecting serum samples of SLE patients, including active lupus nephritis (aLN), active SLE without renal involvement (aNR), and inactive SLE (iSLE), and analyzing the differences in tryptophan levels in the sera of different disease populations, the present invention finds that the decrease in tryptophan in serum / plasma is closely related to SLE patients. Therefore, tryptophan can be used as a potential screening biomarker for this disease, re-identifying the clinical manifestations of SLE disease subtypes to facilitate the rapid screening of SLE. Thus, the present invention is completed.

[0059] Therefore, the present invention provides a method for diagnosing systemic lupus erythematosus (SLE), identifying the disease activity of SLE patients, identifying the degree of renal involvement of SLE patients, and / or judging the prognosis of lupus nephritis, including: (1) detecting tryptophan in a sample, (2) comparing with a control level, wherein the tryptophan level in the serum of SLE patients is lower than the control level. In one or more embodiments, the sample is whole blood, serum, and / or plasma.

[0060] In this text, the diagnostic criteria for systemic lupus erythematosus, the disease activity of patients with systemic lupus erythematosus, the degree of kidney involvement in patients with systemic lupus erythematosus, and the judgment criteria for the prognosis of patients with lupus nephritis refer to the "Diagnosis and Treatment Guidelines for Systemic Lupus Erythematosus (2023 Edition)", the SLE classification criteria revised by the American College of Rheumatology (ACR) in 1997, the SLE classification criteria published by the Systemic Lupus International Collaborating Clinics (SLICC) in 2012, and the SLE classification criteria jointly published by the European League Against Rheumatism (EULAR) and ACR in 2019.

[0061] In this text, lupus nephritis (LN) refers to nephritis secondary to systemic lupus erythematosus. Lupus nephritis is a relatively common and serious complication of systemic lupus erythematosus. Lupus nephritis can occur simultaneously with other clinical manifestations of systemic lupus erythematosus or can be the initial manifestation. As mentioned above, the inventors found that the change in tryptophan level is one of the manifestations of lupus nephritis. Combining the symptoms of kidney damage and other symptoms such as whether there are rashes and joint swelling and pain to determine whether it is lupus nephritis.

[0062] In this text, the control level is the tryptophan level derived from a blood sample (such as whole blood, serum, and / or plasma) that can be used as a diagnostic reference. Such a level can be obtained by comparing the samples of systemic lupus erythematosus subjects with those of healthy subjects or non-systemic lupus erythematosus subjects. In addition, the control level can also be the level of healthy subjects or non-systemic lupus erythematosus subjects. The control level can be derived from one subject or a group of at least two subjects. Those skilled in the art can select the reference level according to the desired sensitivity and specificity.

[0063] In one or more embodiments, compared with the control level, the tryptophan level in the samples of patients with active systemic lupus erythematosus (aSLE) is significantly reduced. The control level is derived from the tryptophan level in the corresponding samples of patients with inactive systemic lupus erythematosus (iSLE) or healthy subjects. In an exemplary embodiment, the control level is derived from the tryptophan level in the corresponding samples of healthy subjects, and the control level is about 47 ± 9 μM to 52 ± 7 μM. In an exemplary embodiment, the control level is derived from the tryptophan level in the corresponding samples of patients with inactive systemic lupus erythematosus, and the control level is about 43 ± 12 μM to 45 ± 13 μM.

[0064] In one or more embodiments, compared to a control level, the tryptophan level in samples from patients with lupus nephritis (LN) involving the kidney is significantly reduced. The control level is derived from the tryptophan level in corresponding samples from patients with non-renal-involved systemic lupus erythematosus (NR) or healthy subjects. In an exemplary embodiment, the control level is derived from the tryptophan level in corresponding samples from healthy subjects, and the control level is about 49 ± 6 μM to 51 ± 8 μM. In an exemplary embodiment, the control level is derived from the tryptophan level in corresponding samples from patients with non-renal-involved systemic lupus erythematosus, and the control level is about 42 ± 11 μM to 43 ± 10 μM.

[0065] In one or more embodiments, compared to a control level, the tryptophan level in samples from patients with active lupus nephritis (aLN) is significantly reduced. The control level is derived from the tryptophan level in corresponding samples from healthy subjects, patients with active systemic lupus erythematosus without renal involvement (aNR), or patients with inactive systemic lupus erythematosus. In an exemplary embodiment, the control level is derived from the tryptophan level in corresponding samples from healthy subjects, and the control level is about 49 ± 6 μM to 51 ± 8 μM. In an exemplary embodiment, the control level is derived from the tryptophan level in corresponding samples from patients with active systemic lupus erythematosus without renal involvement, and the control level is about 40 ± 10 μM to 43 ± 7 μM. In an exemplary embodiment, the control level is derived from the tryptophan level in corresponding samples from patients with inactive systemic lupus erythematosus, and the control level is about 43 ± 12 μM to 45 ± 13 μM.

[0066] In one or more embodiments, the lupus nephritis prognosis is an active lupus nephritis prognosis. In one or more embodiments, the prognosis is non-complete remission and / or complete remission.

[0067] In one or more embodiments, compared to a control level, if the tryptophan level in a patient with active lupus nephritis before treatment is significantly reduced, the patient prognosis is non-complete remission. The control level is derived from the tryptophan level in corresponding samples from healthy subjects or the complete remission group. In an exemplary embodiment, the tryptophan level in corresponding samples from healthy subjects is about 48 ± 6 μM to 51 ± 9 μM. In an exemplary embodiment, the tryptophan level in corresponding samples from the complete remission group is about 38 ± 7 μM. In an exemplary embodiment, the tryptophan level in corresponding samples from the non-complete remission group is about 31 ± 9 μM.

[0068] In one or more embodiments, compared with healthy subjects, if the tryptophan level of a patient with lupus nephritis before treatment is decreased, the prognosis of the patient is complete remission. In one or more embodiments, in one or more embodiments, if the tryptophan level of a patient with lupus nephritis before treatment is lower than that of healthy subjects but higher than that of a patient with lupus nephritis whose prognosis is non-complete remission, the prognosis of the patient is complete remission. In an exemplary embodiment, the tryptophan level in the corresponding sample from a healthy subject is about 48±6 μM to 51±9 μM. In an exemplary embodiment, the tryptophan level in the corresponding sample from the complete remission group is about 38±7 μM. In an exemplary embodiment, the tryptophan level in the corresponding sample from the non-complete remission group is about 31±9 μM.

[0069] In one or more embodiments, the lower the tryptophan level of a patient with lupus nephritis, the worse the prognosis. For example, determining the prognosis of lupus nephritis may include determining whether the tryptophan level in a sample of the patient is lower than 41.0 μM or 42.5 μM. Generally, the tryptophan level in the sample of a patient with lupus nephritis whose prognosis is non-complete remission is at least 10% lower, preferably 24-66% lower, than that of healthy subjects; the tryptophan level in the sample of a patient with lupus nephritis whose prognosis is complete remission is at least 10% lower, preferably 26-35% lower, than that of healthy subjects. The tryptophan level in the sample of a patient with lupus nephritis whose prognosis is non-complete remission is at least 10% lower, preferably 11-57% lower, than that in the sample of a patient with lupus nephritis whose prognosis is complete remission.

[0070] In one or more embodiments, the prognosis is the prognosis of immunotherapy. In one or more embodiments, the immunotherapy is immunosuppressive therapy. In one or more embodiments, at the time of prognosis judgment, the lupus nephritis is active lupus nephritis.

[0071] This article includes any treatment regimens for lupus erythematosus or lupus nephritis, especially immunotherapy, which are common knowledge to those skilled in the art. For example, immunotherapy that inhibits T cell proliferation or activation, B cell proliferation or activation, and immunotherapy targeting BLyS. For example, the immunosuppressant is inosine monophosphate dehydrogenase inhibitor, calcineurin inhibitor, anti-BLyS antibody. Clinicians can adjust the dosage of these immunotherapies according to the patient's condition. The immunotherapy can last for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, for example, 4-8 months.

[0072] The immunosuppressants of the present invention may include, but are not limited to, one or more of DNA synthesis inhibitors, inosine monophosphate dehydrogenase inhibitors, anti-BLyS antibodies, calcineurin inhibitors, hypoxanthine-guanine phosphoribosyltransferase inhibitors, dihydroorotate dehydrogenase inhibitors, and lymphocyte inhibitors.

[0073] The immunosuppressants of the present invention may include, but are not limited to, one or more of the DNA synthesis inhibitor cyclophosphamide (CTX), the inosine monophosphate dehydrogenase inhibitor mycophenolate mofetil (MMF), the anti-BLyS antibody belimumab, the calcineurin inhibitors tacrolimus (TAC) or voclosporin, the hypoxanthine-guanine phosphoribosyltransferase inhibitor azathioprine (AZA), the dihydroorotate dehydrogenase inhibitor leflunomide (LEF), and the lymphocyte inhibitor hydroxychloroquine (HCQ).

[0074] In an exemplary embodiment, the probe method is used to detect tryptophan. However, those skilled in the art can understand that other methods for detecting tryptophan in the art can also be used in the present invention, such as visible light spectrophotometry, acidic ninhydrin method, chromatography, surface-enhanced Raman spectroscopy, and optical probe method. Other methods for detecting tryptophan are within the knowledge of those skilled in the art. Those skilled in the art know the steps and reagents for detecting tryptophan by these methods. Exemplary descriptions of the above methods can be referred to: The currently most commonly used methods for detecting tryptophan include visible light spectrophotometry (Friedman et al., 2004; Wu et al., 2018), acidic ninhydrin method (Bao et al., 2006), HPLC method (Sadok et al., 2017), chromatography (Chen et al., 2016), surface-enhanced Raman spectroscopy (Guan et al., 2024), etc. These detection and analysis methods either require professional analytical instrument equipment, or have cumbersome steps, are prone to human errors, and are only applicable to in vitro detection, and cannot monitor the change of tryptophan concentration in living cells in real time. Therefore, there is an urgent need to develop new detection methods to achieve simple, fast, highly specific real-time, localization, quantification, and high-throughput detection of tryptophan inside and outside cells.

[0075] Tryptophan Detection

[0076] Visible Light Spectrophotometry:

[0077] Using purified tryptophanase, tryptophan is promoted to be converted into indole, and then indole reacts with hydroxylamine in an alkaline environment. After adding sulfuric acid, the reaction product presents a pink color, and its absorption peak is located at 530 nm. By detecting the absorbance value at 530 nm, the content of tryptophan in the sample can be quantitatively calculated. The reagents required for visible light spectrophotometry include, but are not limited to, tryptophanase, hydroxylamine, sulfuric acid, TCEP, etc.

[0078] Acidic Ninhydrin Method:

[0079] Tryptophan reacts with ninhydrin in a mixture of formic acid and hydrochloric acid at 100 °C for 10 min. Tryptophan is converted into a yellow product with an absorption peak at 390 nm. By detecting the absorbance at 390 nm, the content of tryptophan in the sample can be quantitatively calculated. The reagents required for the acidic ninhydrin method include, but are not limited to, formic acid, hydrochloric acid, ninhydrin, ethanol, etc.

[0080] Chromatography:

[0081] Tryptophan can be detected by various chromatographic methods, such as HPLC, LC-MS, UHPLC-MS, etc. Exemplarily, the steps for detecting tryptophan by UHPLC-MS include: sequentially mixing the sample or standard solution with 3N PH_HCl solution and EDC solution. Freeze the mixture, then centrifuge to obtain the supernatant for quantitative analysis. Inject the supernatant into the UHPLC system, separate with water, and use a gradient elution of formic acid and acetonitrile / isopropanol. The ion pairs for tryptophan quantification are 205 / 118 or 205 / 188.

[0082] Surface-enhanced Raman spectroscopy (SERS):

[0083] This method is to react tryptophan with a specific derivative to generate a product with a strong SERS signal, thereby realizing the detection of tryptophan. The Schiff base reaction is carried out under dark and magnetic stirring conditions. React tryptophan with DEAM, then add sodium nitrite, and finally put the silver nanodendrite substrate into the reaction solution for solid-phase extraction. After drying, collect the SERS spectrum. Select the characteristic peak (1620 cm -1 ) for quantitative analysis. The intensity of the characteristic Raman spectral peak is linearly related to the tryptophan concentration (10 -8 –10 -4 mol / L). The reagents required for surface-enhanced Raman spectroscopy include, but are not limited to, DEAM, sodium nitrite, etc.

[0084] Optical probe method:

[0085] The present invention relates to a method for the rapid detection of tryptophan in clinical samples based on genetically encoded optical probes. The method includes: contacting an optionally diluted sample derived from blood with a tryptophan optical probe, and quantifying tryptophan by detecting the fluorescence change of the tryptophan optical probe.

[0086] In this article, a tryptophan optical probe refers to a polypeptide probe that quantitatively detects the tryptophan level by utilizing changes in optical properties. Such a probe generally comprises a protein that can recognize tryptophan (i.e., a tryptophan-binding protein) and a protein that can respond to the binding of this protein to tryptophan and undergo changes in optical properties (i.e., an optically active polypeptide, such as a fluorescent protein). Generally, a tryptophan optical probe comprises one or more tryptophan-binding proteins and one or more optically active polypeptides, wherein one or more optically active polypeptides are located within the sequence, at the N-terminus, or at the C-terminus of one or more tryptophan-binding proteins. A variety of optical probes for detecting tryptophan are known in the art, see, for example: CN113336856A, FLIPW-CTYT (PLoS Biology 2007, 5(10), e257), GRIT (Cell Discovery 2023, 9(1), 106). Those skilled in the art can understand that, in addition to the optical probes used in the examples, other tryptophan optical probes can also be used in the present invention for tryptophan level detection.

[0087] An exemplary tryptophan optical probe used in the present invention is the optical probe described in CN113336856A, which is incorporated herein by reference in its entirety. The tryptophan optical probe comprises a tryptophan-binding protein or its functional variant and an optically active polypeptide or its functional variant, wherein the optically active polypeptide or its functional variant is located within the sequence, at the N-terminus, or at the C-terminus of the tryptophan-binding protein or its functional variant. Preferably, the probe of the present invention comprises a probe with a response multiple higher than 1.2-fold, preferably higher than 1.5-fold, in CN113336856A.

[0088] In one or more embodiments, the tryptophan-binding protein has the sequence shown in SEQ ID NO:1 or its functional fragment, or a variant having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identity with it and retaining the tryptophan-binding function. In a preferred embodiment, the tryptophan-binding protein is as shown in this SEQ ID NO:1, or a sequence having at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identity with it and retaining tryptophan sensitivity.

[0089] In one embodiment, the optically active polypeptide is a fluorescent protein or its functional fragment or variant retaining the fluorescence response ability. In one embodiment, the fluorescent protein is selected from yellow fluorescent protein, green fluorescent protein, blue fluorescent protein, apple red fluorescent protein (such as cpYFP, cpGFP, cpBFP, and cpmApple shown in SEQ ID NOs: 2-5 of CN113336856A, SEQ ID NOs: 2-5 of the present application).

[0090] In one embodiment, the tryptophan optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the present invention can be any amino acid sequence of any length. In one embodiment, the optically active polypeptide is flanked by linkers of no more than 5 amino acids, such as linkers of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linker flanking the optically active polypeptide comprises the amino acid Y. In one embodiment, the linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the tryptophan optical probe is as follows: the first part B1 of the tryptophan-binding protein - Y - the optically active polypeptide A - the second part B2 of the tryptophan-binding protein. In one embodiment, the tryptophan optical probe does not comprise a linker.

[0091] In one embodiment, the optically active polypeptide is located between residues 114 - 118, 233 - 235, and / or 263 - 268 of the tryptophan-sensitive polypeptide, numbered corresponding to the full length of the tryptophan-binding protein. In one embodiment, the optically active polypeptide replaces one or more amino acids between residues 114 - 118, 233 - 235, and / or 263 - 268 of the tryptophan-sensitive polypeptide, numbered corresponding to the full length of the tryptophan-binding protein.

[0092] In one embodiment, the optically active polypeptide is located at one or more sites of the tryptophan-binding protein selected from the following: 114 / 115, 114 / 116, 114 / 117, 114 / 118, 115 / 116, 115 / 117, 115 / 118, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 233 / 235, 234 / 235, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 264 / 268, 265 / 266, 265 / 267, 265 / 268, 266 / 267, 266 / 268, and / or 267 / 268, numbered corresponding to the full length of the tryptophan-binding protein. Preferably, the optically active polypeptide is located at one or more sites of the tryptophan-binding protein selected from the following: 263 / 265, 263 / 266, 263 / 267, and 263 / 268.

[0093] In one or more embodiments, the tryptophan optical probe of the present invention in the B1-A-B2 form may be a probe in which cpYFP is located at positions 114 / 115, 114 / 116, 114 / 117, 114 / 118, 115 / 116, 115 / 117, 115 / 118, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 233 / 235, 234 / 235, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 264 / 268, 265 / 266, 265 / 267, 265 / 268, 266 / 267, 266 / 268 or 267 / 268 of a tryptophan-binding protein or a functional fragment thereof. In an exemplary embodiment, the tryptophan optical probe of the B1-A-B2 form may be a probe in which cpYFP is located at positions 114 / 115, 114 / 116, 114 / 118, 115 / 118, 116 / 118, 117 / 118, 233 / 235, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 265 / 266, 266 / 267 or 266 / 268 of a tryptophan-binding protein or a functional fragment thereof. In a preferred embodiment, the optical probe of the present invention may be a probe in which cpYFP is located at one or more sites selected from the following positions 263 / 265, 263 / 266, 263 / 267 and 263 / 268 of a tryptophan-binding protein.

[0094] In one or more embodiments, the tryptophan optical probe of the present invention in the B1-A-B2 form can be a probe where cpGFP is located at positions 114 / 115, 114 / 116, 114 / 117, 114 / 118, 115 / 116, 115 / 117, 115 / 118, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 233 / 235, 234 / 235, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 264 / 268, 265 / 266, 265 / 267, 265 / 268, 266 / 267, 266 / 268 or 267 / 268 of a tryptophan-binding protein or a functional fragment thereof. In an exemplary embodiment, the tryptophan optical probe in the B1-A-B2 form can be a probe where cpGFP is located at positions 114 / 117, 114 / 118, 115 / 117, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 265 / 267 or 266 / 267 of a tryptophan-binding protein or a functional fragment thereof. In a preferred embodiment, the optical probe of the present invention can be a probe where cpGFP is located at one or more sites selected from the following of a tryptophan-binding protein: 263 / 265, 263 / 266, 263 / 267 and 263 / 268.

[0095] In one or more embodiments, the B1-A-B2 type tryptophan optical probe of the present invention can be a probe where cpBFP is located at positions 114 / 115, 114 / 116, 114 / 117, 114 / 118, 115 / 116, 115 / 117, 115 / 118, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 233 / 235, 234 / 235, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 264 / 268, 265 / 266, 265 / 267, 265 / 268, 266 / 267, 266 / 268 or 267 / 268 of a tryptophan-binding protein or a functional fragment thereof. In an exemplary embodiment, the B1-A-B2 type tryptophan optical probe can be a probe where cpBFP is located at positions 114 / 115, 114 / 117, 114 / 118, 115 / 118, 116 / 118, 233 / 235, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 265 / 266, or 266 / 267 of a tryptophan-binding protein or a functional fragment thereof. In a preferred embodiment, the optical probe of the present invention can be a probe where cpBFP is located at one or more sites selected from the following of a tryptophan-binding protein: 263 / 265, 263 / 266, 263 / 267 and 263 / 268.

[0096] In one or more embodiments, the tryptophan optical probe of the present invention of the B1-A-B2 type may be a probe in which cpmApple is located at one or more sites selected from the following sites of the tryptophan-binding protein: 114 / 115, 114 / 116, 114 / 117, 114 / 118, 115 / 116, 115 / 117, 115 / 118, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 233 / 235, 234 / 235, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 264 / 268, 265 / 266, 265 / 267, 265 / 268, 266 / 267, 266 / 268 or 267 / 268. In an exemplary embodiment, the tryptophan optical probe of the B1-A-B2 type may be when cpmApple is located at: 114 / 115, 117 / 118, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 264 / 268 or 265 / 266 of the tryptophan-binding protein or a functional fragment thereof. In a preferred embodiment, the optical probe of the present invention may be a probe in which cpmApple is located at one or more sites selected from the following sites of the tryptophan-binding protein: 263 / 264, 263 / 265, 263 / 266.

[0097] In one embodiment, the optical probe of the present invention has the sequence shown in SEQ ID NO: 6-9 or consists of the same.

[0098] The tryptophan-binding protein in the tryptophan optical probe may have one or more mutations. The tryptophan optical probe containing the mutant tryptophan-binding protein can be used to detect tryptophan whether its response to tryptophan is higher or lower than that of the unmutated counterpart. Preferably, the tryptophan optical probe whose response to tryptophan (see CN113336856A) exceeds 1.5 times (such as 2 times, 2.2 times, 3 times, 4.3 times, 8.6 times, 9 times, 11 times, 15 times, etc.) that of the control fluorescent protein can be used to detect tryptophan. In one embodiment, the mutation is located at the 13th (H), 71st (T), 84th (L), 93rd (D), 105th (K), 117th (N), 130th (T), 170th (V), 192nd (V), 202nd (F), 214th (K), 217th (Y), 255th (K), 259th (T), and / or 283rd (I) position of the tryptophan-binding protein or its functional fragment, and is one, two, three, four, five or more of them. Exemplarily, in one or more embodiments, the mutation is selected from one or more of H13F, H13W, T71F, T71W, L84F, L84W, D93F, D93W, K105F, K105W, N117F, N117W, T130F, T130W, V170F, V170W, V192F, V192W, F202W, K214F, K214W, Y217F, Y217W, K255F, K255W, T259F, T259W, I283F and / or I283W. In one or more embodiments, the mutation is selected from one or more of H13F, L84F, L84W, D93F, D93W, K105F, K105W, N117F, N117W, V170F, V170W, V192F, F202W, K214F, K214W, Y217F, Y217W, K255F, K255W, T259F, T259W, I283F and I283W. In a preferred embodiment, the mutation is selected from one or more of L84F, K105F, K105W, N117F, V170F, F202W, K214F, K214W, Y217F, Y217W, K255F, K255W, T259F, T259W, I283F and / or I283W.

[0099] In an exemplary embodiment, the tryptophan optical probe may be a functional fragment of a tryptophan-binding protein with cpYFP inserted at one or more sites selected from 114 / 115, 114 / 116, 114 / 117, 114 / 118, 115 / 116, 115 / 117, 115 / 118, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 233 / 235, 234 / 235, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 264 / 268, 265 / 266, 265 / 267, 265 / 268, 266 / 267, 266 / 268 or 267 / 268 and having one or more mutations selected from the following: H13F, L84F, L84W, D93F, D93W, K105F, K105W, N117F, N117W, V170F, V170W, V192F, F202W, K214F, K214W, Y217F, Y217W, K255F, K255W, T259F, T259W, I283F and I283W. Preferably, the optical probe is a probe with cpYFP inserted at one or more sites selected from 263 / 265, 263 / 266 or 263 / 267 of a functional fragment of a tryptophan-binding protein and having one or more mutations selected from the following: L84F, K105F, K105W, N117F, V170F, F202W, K214F, K214W, Y217F, Y217W, K255F, K255W, T259F, T259W, I283F, I283W, and / or I283W. Exemplary sequences are shown in SEQ ID NO: 10-39.

[0100] The optical probe provided by the present invention comprises any one of the amino acid sequences SEQ ID NO: 6-39 or a variant thereof. In one embodiment, the optical probe provided by the present invention comprises a sequence having at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of the amino acid sequences SEQ ID NO: 6-39. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence that is substantially similar or identical to any one of the amino acid sequences SEQ ID NO: 6-39. In a more preferred embodiment, the optical probe provided by the present invention comprises any one of SEQ ID NO: 10-39 or consists of the same.

[0101] In a specific embodiment, the tryptophan optical probe includes the tryptophan optical probe having the sequences shown in SEQ ID NOs: 6-39 in CN113336856A.

[0102] The tryptophan optical probe can be fused with other functional polypeptides. For example, the functional polypeptide is located at the N-terminus and / or C-terminus of the tryptophan optical probe. In some embodiments, the functional polypeptide includes a tag for purification or a tag for immunoblotting. There may be a linker between the optical probe and the other functional polypeptide.

[0103] As used herein, the terms "functional variant", "derivative", and "analogue" refer to proteins that substantially retain the same biological function or activity as the original polypeptide or protein (such as a tryptophan-binding protein or a fluorescent protein). Functional variants, derivatives, or analogues of the polypeptides or proteins of the present invention can be (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are replaced, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins having a substituent group in one or more amino acid residues, or (iii) proteins formed by fusing the mature protein with another compound (such as a compound that prolongs the protein half-life, such as polyethylene glycol), or (iv) proteins formed by fusing an additional amino acid sequence to this protein sequence (such as a secretion sequence or a sequence used to purify this protein or a proprotein sequence, or a fusion protein formed with an antigen IgG fragment). According to the teachings herein, these functional variants, derivatives, and analogues are within the scope well known to those skilled in the art.

[0104] The difference between the analogue and the original polypeptide or protein can be a difference in the amino acid sequence, a difference in a modified form that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as by random mutagenesis through radiation or exposure to mutagens, and can also be obtained by site-directed mutagenesis or other known molecular biology techniques.

[0105] In some embodiments, the process of detecting tryptophan includes the step of establishing a tryptophan standard curve. The determination of the tryptophan standard curve is a conventional means in the art, which is a curve obtained by correspondingly detecting different known concentrations of tryptophan standards and correlating the concentration with the detection result or the calculation result derived from the detection result.

[0106] For the tryptophan analysis based on the optical probe, after the optical probe contacts tryptophan in the sample, appropriate excitation and emission wavelengths are selected according to the different fluorescent proteins to detect the fluorescence intensity, and quantitative analysis is performed according to the tryptophan standard curve.

[0107] In an exemplary embodiment, for fluorescent proteins with a single excitation wavelength (such as cpBFP and cpmApple), the data processing process includes:

[0108] F = F Sample - F BLK

[0109] F represents the actual fluorescence intensity of a single channel, F Sample represents the fluorescence intensity of the sample expressing the probe, F BLK represents the fluorescence intensity of the sample not expressing the probe. The standard curve is established using the fluorescence intensity of the standard product, and then the quantitative analysis of tryptophan in the sample is carried out according to the standard curve. In an exemplary embodiment, for cpBFP, the excitation wavelength is 360nm BP 10nm, and the emission wavelength is 450nm BP 10nm; for cpmApple, the excitation wavelength is 540nm BP 25nm, and the emission wavelength is 590nm BP20nm.

[0110] For fluorescent proteins with multiple excitation wavelengths (such as cpYFP and cpGFP), the data processing process includes:

[0111] F = F Sample - F BLK

[0112] R = F 激发波长1 / F 激发波长2

[0113] F represents the actual fluorescence intensity of a single channel, F Sample represents the fluorescence intensity of the sample expressing the probe, F BLK represents the fluorescence intensity of the sample not expressing the probe. F 激发波长1 represents the fluorescence intensity emitted at the emission wavelength after the probe is excited at the second excitation wavelength (420nm BP 20nm for cpYFP), and (for cpYFP or cpGFP is 528nm), F 激发波长2 represents the fluorescence intensity emitted at the emission wavelength after the probe is excited at the first excitation wavelength (485nm BP 20nm for cpYFP), and (for cpYFP or cpGFP is 528nm). The first and second excitation wavelengths can be determined according to the spectral properties of the fluorescent protein carried by the probe.

[0114] R represents the fluorescence ratio of the probe. The bandwidth BP (band pass) of the filter represents the total range on both sides of the median value. For example, 485BP 20nm means 475 - 495nm.

[0115] [Trp] = K d (R - R min ) / (R max )

[0116] [Trp] represents the tryptophan level; K d represents the dissociation constant of the probe; R min and R max represent the fluorescence ratio of the probe protein with or without the addition of saturated concentration of tryptophan, respectively; R represents the fluorescence ratio of the sample.

[0117] Generally, before the detection of tryptophan, the sample can be pretreated to remove substances that may affect the detection. Such pretreatment methods can be adjusted according to the specific detection method. Those skilled in the art are aware of such pretreatment processes and the required reagents. For example, for the method of UHPLC-MS analysis as described by Whiley et al., 2019, the sample pretreatment includes: successively mixing the sample (such as blood) with 3N PH_HCl solution and EDC solution, then freezing at -20 °C and taking the supernatant for analysis. Another example is when using an optical probe to detect tryptophan in a sample, the sample (such as blood) is first diluted with a buffer (such as HEPES), and then mixed with the optical probe to measure the fluorescence intensity.

[0118] The present invention also provides a method for preparing a tryptophan optical probe, comprising the following steps: 1) incorporating the nucleic acid sequence encoding the tryptophan optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for the expression of the expression vector; 4) isolating the tryptophan optical probe.

[0119] As used herein, the terms "nucleic acid", "nucleotide", "polynucleotide", or "nucleic acid sequence" of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. When referring to nucleic acids, the term "variant" as used herein may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants, and insertion variants. The nucleic acids of the present invention may comprise nucleotide sequences having a sequence identity of at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% with the nucleic acid sequence. The present invention also relates to nucleic acid fragments that hybridize to the above sequences. In an exemplary embodiment, the nucleic acid sequence is as shown in SEQ ID NO: 40, which represents the coding sequence of a probe with cpYFP inserted at positions 263 / 266 of the functional fragment of tryptophan-binding protein and having a T259F mutation. As used herein, the length of the "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. The nucleic acid fragment can be used in nucleic acid amplification techniques (such as PCR).

[0120] In addition, the present invention also provides a kit for detecting tryptophan, which comprises a reagent for detecting tryptophan and an instruction manual, and the instruction manual describes a method for diagnosing systemic lupus erythematosus (SLE) by detecting tryptophan, and / or identifying the disease activity of patients with systemic lupus erythematosus, and / or identifying the degree of kidney involvement of patients with systemic lupus erythematosus, and / or judging the prognosis of lupus nephritis. In one or more embodiments, the reagent for detecting tryptophan actually includes reagents required for detecting tryptophan using one or more methods selected from the following: visible light photometry, acidic ninhydrin method, chromatography, surface-enhanced Raman spectroscopy, and optical probe method.

[0121] As used herein, the terms "tryptophan detection substance", "detection reagent", and "reagent for detecting tryptophan" can be used interchangeably, and all refer to substances that are specific for tryptophan and can be used to directly or indirectly detect the presence and / or content of tryptophan. For the convenience of detection, the detection reagent of the present invention may also be provided with a detectable label, and the detectable label includes but is not limited to: radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (such as biotin or hapten), etc.

[0122] In some embodiments, the reagent for detecting tryptophan includes reagents for converting, enriching, separating, or identifying tryptophan. As described above, those skilled in the art can understand that methods for detecting tryptophan, including chromatography and probe methods, can be used in the present invention. For example, visible light spectrophotometry, acidic ninhydrin method, chromatography, surface-enhanced Raman spectroscopy, and optical probe method. Those skilled in the art are aware of the steps and reagents for detecting tryptophan by these methods. Therefore, by way of example, the reagent for detecting tryptophan in this article is the reagent used in the methods described below: visible light spectrophotometry (Friedman et al., 2004; Wu et al., 2018), acidic ninhydrin method (Bao et al., 2006), HPLC method (Sadok et al., 2017), chromatography (Chen et al., 2016), surface-enhanced Raman spectroscopy (Guan et al., 2024), and so on. The reagents involved in the above methods include but are not limited to: tryptophan-specific antibody (https: / / www.uscnk.cn / uscn / Antibody-to--Tryptophan-(Trp)-6500.htm), DEAM, sodium nitrite, tryptophanase, hydroxylamine, sulfuric acid, TCEP, formic acid, hydrochloric acid, ninhydrin, ethanol, formic acid, acetonitrile, isopropanol, tryptophan-binding protein, or their functional variants. In an exemplary embodiment, the kit further includes a reagent for processing the test sample, the test being for detecting tryptophan in a sample derived from blood, and the reagent for processing the test sample can also be one or more selected from the following: 3NPH_HCl, EDC, formic acid, acetonitrile, isopropanol.

[0123] After using the serum / plasma processing reagent, the tryptophan in the sample may be more suitable for subsequent detection. Such a pretreatment method can be adjusted according to the specific detection method. Those skilled in the art are aware of such pretreatment processes and the required reagents. For example, for the method of UHPLC-MS analysis as described by Whiley et al., 2019, the sample pretreatment includes: sequentially mixing the sample (such as blood) with 3NPH_HCl solution and EDC solution, then freezing at -20 °C and taking the supernatant for analysis. Another example is when using an optical probe to detect tryptophan in a sample, the sample (such as blood) is first diluted with a buffer (such as HEPES), then mixed with the optical probe, and the fluorescence intensity is measured. In one or more embodiments, the sample processing reagent includes but is not limited to: 3NPH_HCl, EDC, buffer.

[0124] The kit may further include a buffer. The role of the buffer is to provide a stable buffer environment for the reactions involved in tryptophan detection. Those skilled in the art can select a suitable buffer according to experience, such as phosphate-based buffers (HEPES, PBS) or Tris, etc.

[0125] The kit may further include reagents required for other auxiliary detections that may be involved in the diagnosis of systemic lupus erythematosus, such as reagents for detecting 24-hour urinary protein (such as Coomassie Brilliant Blue G-250), and reagents for detecting glomerular filtration rate (such as 99mTc-DTPA). These are all within the knowledge of those skilled in the art.

[0126] In some embodiments, in the kit of the present invention, the method is a method for diagnosing systemic lupus erythematosus, identifying the disease activity of patients with systemic lupus erythematosus, identifying the degree of kidney involvement in patients with systemic lupus erythematosus, and judging the prognosis of lupus nephritis as described in any embodiment herein.

[0127] The present invention also provides the use of the above reagent for detecting tryptophan in the preparation of a kit for diagnosing patients with systemic lupus erythematosus or for classifying and judging the prognosis of patients with lupus nephritis in patients with systemic lupus erythematosus.

[0128] In this article, concentrations, contents, percentages, and other numerical values can be expressed in the form of ranges. It should also be understood that the use of this range form is only for convenience and brevity and should be interpreted flexibly to include the numerical values explicitly mentioned in the upper and lower limits of the range, and should also include all individual numerical values or sub-ranges included in this range.

[0129] The present invention has the following beneficial effects:

[0130] The probe-based determination of body fluid samples does not require time-consuming sample preparation (i.e., pretreatment or purification), so it is very fast and convenient. Generally, the entire process from pipetting to measurement for one sample takes about 1 minute, and for 96 samples in an automatic microplate assay, it usually takes about 3 minutes. These advantages make it a promising technology for metabolic diagnosis and screening.

[0131] The present invention will be further described below by way of specific examples. It should be understood that these examples are merely illustrative and not intended to limit the scope of the present invention. The methods and reagents used in the examples are conventional methods and reagents in the art unless otherwise specified.

[0132] Examples

[0133] Detection method:

[0134] 1. Protein induction expression and purification

[0135] Transform the probe plasmid into an expression strain such as BL21(DE3) or JM109(DE3), then pick a single clone into a test tube for primary culture. The next day, inoculate the bacteria from the primary culture into a conical flask at a ratio of 1:100. When the OD 600 is 0.4 - 0.6, add IPTG with a final concentration of 1 mM, and place it in an 18°C incubator to induce the expression of the target protein.

[0136] (1) Collect the bacterial cells: After 24 - 48 h of protein expression, use a centrifuge to centrifuge at 4000 rpm for 10 min to collect the bacterial cells expressing the protein, and resuspend them with buffer A.

[0137] (2) Ultrasonic disruption: Set the ultrasonic disruptor program to ultrasonic for 1 s and pause for 3 s. The working cycle time is 300 s. Use a probe with a diameter of Φ15 and a power of 55%.

[0138] (3) Centrifugation: When the bacterial cells are ultrasonicated until clear, use a centrifuge to centrifuge at 9600 rpm at 4 °C for 30 min. Retain the supernatant and discard the precipitate.

[0139] (4) Column pretreatment: The self - packed nickel - ion affinity chromatography column is first rinsed with 5 column volumes (CV) of deionized water, and then equilibrated with 5 column volumes (CV) of buffer A.

[0140] (5) Protein loading onto the column: Add the supernatant after disruption and centrifugation to the pretreated nickel column.

[0141] (6) Washing away impurities: Add a washing buffer containing 50 mM imidazole to remove the impurity proteins.

[0142] (7) Elution: Add an elution buffer containing 300 mM imidazole to elute the target protein from the nickel column.

[0143] (8) Nickel column treatment: Treat the used nickel column with 5 column volumes (CV) of buffer B (buffer containing 500 mM imidazole), then add 5 column volumes (CV) of deionized water, and finally immerse the column packing in a 20% ethanol solution.

[0144] Buffer A: 20 mM phosphate, 0.5 M NaCl, 10 mM imidazole.

[0145] Buffer B: 20 mM phosphate, 0.5 M NaCl, 500 mM imidazole.

[0146] The protein is dissolved in the elution solution after purification by the nickel column. When detecting the properties of the protein or storing the protein, the solution needs to be changed. Therefore, the purified protein needs to be desalted. First, the desalting column needs to be pretreated, also rinsed with 5 column volumes (CV) of deionized water, and then treated with 5 column volumes (CV) of desalting buffer; second, add the protein to be desalted to the desalting column and collect the protein; finally, treat the desalting column with 10 column volumes (CV) of deionized water.

[0147] 2. Clinical sample collection

[0148] The serum samples of SLE and healthy groups were provided by Renji Hospital, Shanghai Jiao Tong University. The sample collection and subsequent experiments were approved by the institutional review board of this institution and informed consent was obtained from all participants.

[0149] 3. Sample detection method

[0150] For the tryptophan analysis based on fluorescent protein probes, the serum samples were diluted 100-fold in HEPES buffer. The assay was performed using a 96-well black plate, and different concentration gradients of tryptophan standards were set. 50 μL of the diluted sample and 50 μL of 0.4 μM probe protein solution were added. Alternatively, 1 μL of serum sample was directly mixed with 100 μL of the probe protein (0.2 μM) using an Echo 650 acoustic pipetting system combined with a BioTek MultifloFX automated dispenser. The fluorescence intensity was immediately measured through a filter with an excitation bandwidth of 485BP20nm or 420BP27nm and an emission bandwidth of 532BP40nm using a Synergy neo2 multimode microplate reader, and quantitative analysis was performed according to the tryptophan standard curve.

[0151] Fluorescence detection data processing (taking the FiLa probe as an example)

[0152] F = F Sample - F BLK

[0153] R (Ratio) = F 420 / F 485

[0154] F represents the actual fluorescence intensity of a single channel, F Sample represents the fluorescence intensity of the sample expressing the probe, F BLK represents the fluorescence intensity of the sample without the added probe. F 420 represents the fluorescence intensity of the fluorescent protein sample excited at 420 nm and emitted at 532 nm, F 485 represents the fluorescence intensity of the fluorescent protein sample excited at 485 nm and emitted at 532 nm. Ratio FiLa represents the fluorescence intensity ratio of the probe.

[0155] The bandwidth BP (band pass) of the filter represents the total range on both sides of the median. 485BP20nm means 475 - 495 nm.

[0156] [Trp] = K d (R - R min ) / (R max )

[0157] [Trp] represents the tryptophan level; K d represents the dissociation constant of the STrp - H probe; R minand R max respectively represent the fluorescence ratio of the probe protein before or after adding tryptophan at saturation concentration; R represents the fluorescence ratio of the probe protein after adding serum.

[0158] 4. UHPLC-MS analysis of tryptophan in blood-derived samples

[0159] Tryptophan in serum samples derived from blood was determined by UHPLC and triple quadrupole mass spectrometry as described previously (Whiley et al., 2019). 5 μL aliquots of serum or standard solution were successively mixed with 25 μL of 160 mM 3NPH_HCl solution and 25 μL of 120 mM EDC solution. The mixture was frozen at -20 °C for 20 minutes and then centrifuged to collect the supernatant for quantitative analysis.

[0160] Each 5 μL sample was injected into an Agilent 1290 UHPLC system and separated with water, using a gradient elution of 0.1% formic acid and acetonitrile / isopropanol (7:3, v / v). Mass analysis was performed by multiple reaction monitoring in ESI negative mode. The ion pair for tryptophan quantification was 205 / 118.

[0161] Example 1

[0162] Serum samples from adult SLE patients provided by Shanghai Renji Hospital were detected, including 21 cases of active lupus nephritis (aLN), 13 cases of active SLE without kidney involvement (aNR), and 13 cases of inactive SLE (iSLE) patients. To exclude metabolic differences caused by metabolism of indicators such as age and gender, in this example, according to the gender and age of SLE patients, corresponding healthy individuals were matched as controls for each patient. Their clinical characteristics and baseline parameters are shown in Table 1 Figure 1 as shown below.

[0163] Table 1

[0164]

[0165] Data are mean±SD, n(%) or median(IQR)

[0166] First, an instant detection method for clinical samples based on probes was established. When using the STrp-H probe to detect tryptophan in serum, at room temperature, only 1 μL of serum and 0.2 μM of STrp-H protein are required for a 96-well plate, and the measurement time is less than 1 minute. The detection process is as Figure 2 shown below.

[0167] Example 2

[0168] In the random serum test of SLE patients, detected by the probe STrp-H, it was found that the serum tryptophan level in the aLN group was the lowest (35±7 μM), followed by the aNR group, the iSLE group, and the healthy group (51±8 μM); however, there was no significant difference between the serum tryptophan levels of the aNR group and the iSLE group (43±7 μM vs 43±12 μM, p>0.5) and those of the iSLE group and its healthy control group (43±12 μM vs 47±9 μM, p>0.5). In addition, as Figure 3 shown, the inventors observed that the serum tryptophan level in the active SLE group (aLN + aNR) (aSLE, 38±7 μM, p<0.001) was lower than that in the non-active SLE group (iSLE, 43±12 μM) and the matched healthy control group (52±7 μM; 47±9 μM), indicating that the serum tryptophan level may be related to disease activity. Moreover, the serum tryptophan level in SLE patients was also related to kidney involvement. For example, the kidney involvement (LN) group (35±7 μM) was lower than the non-kidney involvement (NR, aNR + iSLE) group (43±10 μM) and the matched healthy control group (51±8 μM, p<0.001; 49±8 μM).

[0169] Table 2

[0170]

[0171] To evaluate the diagnostic performance of the STrp-H probe in measuring serum tryptophan levels, a receiver operating characteristic (ROC) analysis was performed in this example. As Figure 4As shown, the serum tryptophan signature score could reasonably distinguish between the active SLE group (aSLE) and the healthy control group (AUC = 0.902, p < 0.001), or between the active SLE group and the inactive SLE group (iSLE) (AUC = 0.696, p < 0.05). However, this score could not significantly distinguish between inactive SLE patients (iSLE) and the healthy control group (AUC = 0.624, p > 0.05). In addition, the AUC value for kidney involvement (LN) compared to the healthy control group was 0.951 (p < 0.001), the AUC value for non-kidney involvement (NR) compared to the healthy control group was 0.735 (p < 0.01), and the AUC value between the kidney involvement group and the non-kidney involvement group was 0.778 (p < 0.01), indicating that serum tryptophan could very significantly distinguish the kidney involvement group from the healthy group and the non-kidney involvement group. Moreover, this example also found that the serum tryptophan signature score was helpful in distinguishing patients in the aLN group from those in the aNR or iSLE groups (aLN vs aNR, 0.778, p < 0.01; aLN vs iSLE, 0.782, p < 0.01), but was ineffective for aNR patients in the iSLE group (AUC = 0.556, p > 0.05). These results all demonstrated that the level of tryptophan in serum was closely related to the activity of SLE disease and the degree of kidney injury.

[0172] Example 3

[0173] To verify the above results, this example collaborated with Shanghai Renji Hospital again to recollect a batch of serum samples from patients with systemic lupus erythematosus and healthy controls for independent testing as a validation set. The validation set samples included 21 patients with active lupus nephritis (aLN), 14 patients with active SLE without kidney involvement (aNR), and 13 patients with inactive SLE (iSLE). Similarly, to exclude metabolic differences caused by metabolism of indicators such as age and gender, this example matched corresponding healthy individuals as controls for each patient according to the gender and age of SLE patients. Their clinical characteristics and baseline parameters are shown in Table 3 below.

[0174] Table 3

[0175]

[0176] Data are mean±SD,n(%)or median(IQR)

[0177] Example 4

[0178] Independent test results of the validation set samples showed that, similar to the training set, the serum tryptophan level in the aLN group was still the lowest (33 ± 9 μM), followed by the aNR group, the iSLE group, and the healthy group (49 ± 6 μM); there was no significant difference between the serum tryptophan levels in the aNR group and the iSLE group (40 ± 10 μM vs 45 ± 13 μM, p > 0.5). Similarly, there was no significant difference between the serum tryptophan levels in the iSLE group and its healthy control group (45 ± 13 μM vs 47 ± 6 μM, p > 0.5). Similarly, the serum tryptophan level in the active SLE (aLN + aNR) group (aSLE, 36 ± 10 μM) was lower than that in the inactive SLE group (iSLE, 45 ± 13 μM) and the matched healthy control group (49 ± 6 μM, p < 0.001; 47 ± 6 μM), indicating that the higher the disease activity of SLE patients, the lower the serum tryptophan level may be. In addition, the test results of the validation set also proved that the serum tryptophan level in SLE patients was related to kidney involvement. The kidney involvement (LN) group (33 ± 9 μM) was lower than the non-kidney involvement (NR, aNR + iSLE) group (42 ± 11 μM) and the matched healthy control group (49 ± 6 μM, p < 0.001; 48 ± 6 μM), as Figure 5 shown.

[0179] Table 4

[0180]

[0181] In this embodiment, the receiver operating characteristic (ROC) analysis was also performed on the test results of the validation set, as Figure 6 shown. Similar to the test results of the training set, the serum tryptophan characteristic score could reasonably distinguish the active SLE group from the healthy control group (AUC = 0.889, p < 0.001), or distinguish the active SLE group from the inactive SLE group (AUC = 0.726, p < 0.01), but could not significantly distinguish the inactive SLE patients from the healthy control group (AUC = 0.615, p > 0.5). Similarly, serum tryptophan could very significantly distinguish the kidney involvement group (LN) from the healthy group (AUC = 0.959, p < 0.001) and the non-kidney involvement group (NR, AUC = 0.747, p < 0.01). Slightly different from the training set, the validation set results only showed that serum tryptophan was helpful in distinguishing patients in the aLN group from those in the iSLE group (AUC = 0.800, p < 0.01), and was not significantly different from the aNR group (AUC = 0.697, p > 0.5). These results once again proved that the tryptophan level in serum could help evaluate the disease activity of SLE and the degree of renal injury in patients.

[0182] Example 5

[0183] In both of the two sampling cohorts, some aLN patients received 6 - month induction therapy with immunosuppressants (cyclophosphamide (CTX), mycophenolate mofetil (MMF), belimumab, tacrolimus (TAC) or voclosporin, azathioprine (AZA), leflunomide (LEF), hydroxychloroquine (HCQ)) and were followed up with sampling. Since the number of such patients in the two cohorts was small, in order to ensure the accuracy of the analysis, such samples in the training set and the validation set were analyzed together in this example. The baseline serum tryptophan level was detected before treatment. After 6 - month treatment, according to the latest KDIGO guidelines, this part of the patients was divided into a complete remission (CR) group and a non - complete remission (Non - CR) group. Interestingly, compared with the CR group (38 ± 7 μM), the baseline serum tryptophan level before treatment in the Non - CR group (31 ± 9 μM, p < 0.05) was significantly lower. The serum tryptophan signature score was helpful in predicting the treatment response of patients with lupus nephritis (CR vs Non - CR, AUC = 0.715, p < 0.05). The AUC value of the comparison between the Non - CR group and the healthy control group was 0.955 (p < 0.001), and the AUC value of the comparison between the CR group and the healthy control group was 0.891 (p < 0.001), as Figure 7 shown.

[0184] Table 5

[0185]

[0186] Therefore, the decrease in serum tryptophan level seems to be part of the clinical manifestations of patients with active lupus nephritis and is an important evaluation factor for disease severity and prognosis remission status. The status after immunotherapy can be predicted by comparing the serum Trp level before treatment.

[0187] The above results indicate that the decrease in serum tryptophan level is significantly correlated with the occurrence of lupus nephritis and the prediction of the response after induction therapy. Therefore, serum tryptophan has broad potential in the precise diagnosis of lupus nephritis and avoiding invasive renal biopsy.

[0188] SEQ ID NO:1

[0189] Tryptophan - binding protein

[0190] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVFSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0191] SEQ ID NO:2cpYFP

[0192] YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0193] SEQ ID NO:3cpGFP

[0194] NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0195] SEQ ID NO:4 cpBFP

[0196] NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0197] SEQ ID NO:5 cpmApple

[0198] VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA

[0199] SEQ ID NO:6

[0200] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI。

[0201] SEQ ID NO:7

[0202] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0203] SEQ ID NO:8

[0204] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNGKSVINKKIAQELGITIPESVLKEAGQVI

[0205] SEQ ID NO:9

[0206] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNKSVINKKIAQELGITIPESVLKEAGQVI

[0207] SEQ ID NO:10

[0208] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDFPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0209] SEQ ID NO:11

[0210] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKFPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0211] SEQ ID NO:12

[0212] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGWPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0213] SEQ ID NO:13

[0214] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKFPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0215] SEQ ID NO:14

[0216] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKWPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0217] SEQ ID NO:15

[0218] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIFPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0219] SEQ ID NO:16

[0220] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIWPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0221] SEQ ID NO:17

[0222] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAFPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0223] SEQ ID NO:18

[0224] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAWPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0225] SEQ ID NO:19

[0226] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADFPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0227] SEQ ID NO:20

[0228] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADWPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITIPESVLKEAGQVI

[0229] SEQ ID NO:21

[0230] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITFPESVLKEAGQVI

[0231] SEQ ID NO:22

[0232] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNSTGKSVINKKIAQELGITWPESVLKEAGQVI

[0233] SEQ ID NO:23

[0234] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDFPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0235] SEQ ID NO:24

[0236] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKFPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0237] SEQ ID NO:25

[0238] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKWPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0239] SEQ ID NO:26

[0240] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGWPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0241] SEQ ID NO:27

[0242] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKFPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0243] SEQ ID NO:28

[0244] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKWPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0245] SEQ ID NO:29

[0246] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIFPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0247] SEQ ID NO:30

[0248] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAWPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0249] SEQ ID NO:31

[0250] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADFPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0251] SEQ ID NO:32

[0252] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADWPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITIPESVLKEAGQVI

[0253] SEQ ID NO:33

[0254] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNTGKSVINKKIAQELGITFPESVLKEAGQVI

[0255] SEQ ID NO:34

[0256] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDFPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNGKSVINKKIAQELGITIPESVLKEAGQVI

[0257] SEQ ID NO:35

[0258] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKFPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNGKSVINKKIAQELGITIPESVLKEAGQVI

[0259] SEQ ID NO:36

[0260] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKWPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNGKSVINKKIAQELGITIPESVLKEAGQVI

[0261] SEQ ID NO:37

[0262] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGWPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNGKSVINKKIAQELGITIPESVLKEAGQVI

[0263] SEQ ID NO:38

[0264] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIFPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADTPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNGKSVINKKIAQELGITIPESVLKEAGQVI

[0265] SEQ ID NO:39

[0266] SNAKIGVLQFVSHPSLDLIYKGIQDGLAEEGYKDDQVKIDFMNSEGDQSKVATMSKQLVANGNDLVVGIATPAAQGLASATKDLPVIMAAITDPIGANLVKDLKKPGGNVTGVSDHNPAQQQVELIKALTPNVKTIGALYSSSEDNSKTQVEEFKAYAEKAGLTVETFAVPSTNEIASTVTVMTSKVDAIWVPIDNTIASGFPTVVSSNQSSKKPIYPSATAMVEVGGLASVVIDQHDLGVATGKMIVQVLKGAKPADFPVNVYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNGKSVINKKIAQELGITIPESVLKEAGQVI

[0267] SEQ ID NO:40

[0268]

Claims

1. Use of a reagent for detecting tryptophan derived from a blood sample or a reagent for detecting tryptophan derived from a blood sample and a tryptophan standard in the preparation of a kit for one or more of the following: diagnosing systemic lupus erythematosus, identifying the disease activity of patients with systemic lupus erythematosus, identifying the degree of renal involvement in patients with systemic lupus erythematosus, and judging the prognosis of lupus nephritis; Among them, The disease activity of the patients with systemic lupus erythematosus includes active systemic lupus erythematosus and inactive systemic lupus erythematosus, and the degree of renal involvement includes systemic lupus erythematosus with renal involvement and systemic lupus erythematosus without renal involvement; Preferably, the sample is a blood sample, such as whole blood, serum and / or plasma.

2. The use according to claim 1, characterized in that, The reagent for detecting tryptophan includes the reagents required for detecting tryptophan using one or more methods selected from the following: visible spectrophotometry, acidic ninhydrin method, chromatography, surface enhanced Raman spectroscopy and optical probe method.

3. The use according to claim 1 or 2, characterized in that, The reagent for detecting tryptophan includes reagents for transforming, enriching, separating or identifying tryptophan, Preferably, the reagent for detecting tryptophan includes one or more selected from the following: tryptophan-specific antibody, tryptophanase, hydroxylamine, sulfuric acid, TCEP, formic acid, hydrochloric acid, ninhydrin, ethanol, formic acid, acetonitrile, isopropanol, DEAM, sodium nitrite, tryptophan-binding protein or functional variants thereof.

4. The use according to claim 3, characterized in that, The functional variant of the tryptophan-binding protein includes a tryptophan optical probe, Preferably, The tryptophan optical probe comprises a tryptophan-binding protein and an optically active polypeptide, More preferably, the optical probe has one or more of the following characteristics: The tryptophan-binding protein has the sequence shown in SEQ ID NO:1 or a functional fragment thereof, or a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity therewith; The optically active polypeptide is located at one or more of the following sites of the tryptophan-binding protein: 114 / 115, 114 / 116, 114 / 117, 114 / 118, 115 / 116, 115 / 117, 115 / 118, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 233 / 235, 234 / 235, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 264 / 268, 265 / 266, 265 / 267, 265 / 268, 266 / 267, 266 / 268 and / or 267 / 268, The tryptophan-sensitive polypeptide contains mutations at the following sites: H13, T71, L84, D93, K105, N117, T130, V170, V192, F202, K214, Y217, K255, T259, I283, and the amino acid mutations include modifications, substitutions or deletions of amino acids; Preferably, the mutation is selected from one or more of H13F, H13W, T71F, T71W, L84F, L84W, D93F, D93W, K105F, K105W, N117F, N117W, T130F, T130W, V170F, V170W, V192F, V192W, F202W, K214F, K214W, Y217F, Y217W, K255F, K255W, T259F, T259W, I283F and / or I283W.

5. A method for diagnosing systemic lupus erythematosus, identifying the disease activity of patients with systemic lupus erythematosus, identifying the degree of kidney involvement in patients with systemic lupus erythematosus, and judging the prognosis of lupus nephritis, comprising: (1) Detect tryptophan in a sample derived from blood, (2) compare with a control level, wherein, the tryptophan level in patients with active systemic lupus erythematosus is lower than the control level; preferably, the control level is the tryptophan level of a healthy subject or a patient with non-active systemic lupus erythematosus, the tryptophan level in patients with systemic lupus erythematosus with kidney involvement is lower than the control level; preferably, the control level is the tryptophan level in a corresponding sample of a healthy subject or a patient with systemic lupus erythematosus without kidney involvement, the tryptophan level in patients with active lupus nephritis is lower than the control level; preferably, the control level is the tryptophan level in a corresponding sample of a healthy subject, a patient with active systemic lupus erythematosus without kidney involvement or a patient with non-active systemic lupus erythematosus, the tryptophan level in the non-complete remission group of lupus nephritis is lower than the control level, and the tryptophan level in the complete remission group is lower than that of healthy subjects; preferably, the control level is the tryptophan level in a corresponding sample of a healthy subject and the complete remission group.

6. A kit for detecting tryptophan, the kit comprising reagents for detecting tryptophan and instructions, and the instructions record methods for diagnosing systemic lupus erythematosus by detecting tryptophan, identifying the disease activity of patients with systemic lupus erythematosus, identifying the degree of kidney involvement in systemic lupus erythematosus or judging the prognosis of lupus nephritis, Preferably, the reagents for detecting tryptophan include reagents required for detecting tryptophan using one or more methods selected from the following: visible light photometry, acidic ninhydrin method, chromatography, surface enhanced Raman spectroscopy and optical probe method, More preferably, the reagents for detecting tryptophan include reagents for converting, enriching, separating or recognizing tryptophan, More preferably, the method is as described in claim 5.

7. The kit according to claim 6, characterized in that, The reagents for detecting tryptophan include one or more of the following: tryptophan-specific antibodies, tryptophanase, hydroxylamine, sulfuric acid, TCEP, formic acid, hydrochloric acid, ninhydrin, ethanol, formic acid, acetonitrile, isopropanol, DEAM, sodium nitrite, tryptophan-binding protein or functional variants thereof, The serum / plasma processing reagents include: 3NPH_HCl, EDC, buffer; preferably, the buffer is a phosphate-based buffer or Tris.

8. The kit according to claim 6, characterized in that, The kit contains a tryptophan optical probe and a buffer, and the tryptophan optical probe contains a tryptophan-binding protein and an optically active polypeptide, Preferably, the optically active polypeptide is located within the sequence, at the N-terminus or C-terminus of the tryptophan-binding protein, Preferably, the buffer is a phosphate-based buffer or Tris, such as HEPES, PBS, etc.

9. The kit according to claim 8, characterized in that, The optical probe has one or more of the following characteristics: The tryptophan-binding protein has the sequence shown in SEQ ID NO:1 or a functional fragment thereof, The optically active polypeptide is located at one or more of the following sites of the tryptophan-binding protein: 114 / 115, 114 / 116, 114 / 117, 114 / 118, 115 / 116, 115 / 117, 115 / 118, 116 / 117, 116 / 118, 117 / 118, 233 / 234, 233 / 235, 234 / 235, 263 / 264, 263 / 265, 263 / 266, 263 / 267, 263 / 268, 264 / 265, 264 / 266, 264 / 267, 264 / 268, 265 / 266, 265 / 267, 265 / 268, 266 / 267, 266 / 268 and / or 267 / 268, The tryptophan-sensitive polypeptide contains mutations at one, two or three or more of the following sites: H13, T71, L84, D93, K105, N117, T130, V170, V192, F202, K214, Y217, K255, T259, I283, and the amino acid mutations include modifications, substitutions or deletions of amino acids; Preferably, the mutations are selected from one or more of H13F, H13W, T71F, T71W, L84F, L84W, D93F, D93W, K105F, K105W, N117F, N117W, T130F, T130W, V170F, V170W, V192F, V192W, F202W, K214F, K214W, Y217F, Y217W, K255F, K255W, T259F, T259W, I283F and / or I283W.

10. The kit according to any one of claims 6-9, characterized in that, The kit further includes: reagents for detecting systemic lupus erythematosus and / or lupus nephritis.

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