阅读说明：本技术 尿液免疫球蛋白κ可变区3D-7及其多肽片段在烧伤中的应用 (Application of urine immunoglobulin kappa variable region 3D-7 and polypeptide fragment thereof in burn ) 是由 张曼 王佶图 于 2020-06-05 设计创作，主要内容包括：本发明提供一种尿液免疫球蛋白κ可变区3D-7(Immunoglobulin kappa variable 3D-7)及其多肽片段的应用,具体为尿液免疫球蛋白κ可变区3D-7及其多肽片段在制备用于烧伤诊断、鉴别诊断、烧伤面积及程度评价、治疗效果评价、监测、预后评估及机理研究等制剂的应用。烧伤是日常生活中常见的重要创伤,每年每100万人中约有5000～100000人烧伤,据世界卫生组织统计,每年全球死于烧伤患者超过30万人,严重烧伤救治存活率仍处于较低水平。本发明通过研究证实,与健康人(正常对照组)相比,尿液免疫球蛋白κ可变区3D-7及其多肽片段在烧伤患者中表达升高。可用于烧伤患者的各种目的应用检测。本发明发挥尿液标本获取无创、可大规模重复取样、保存方便的优势,利用尿液标本检测尿液免疫球蛋白κ可变区3D-7及其多肽片段。(The invention provides application of a urine Immunoglobulin kappa variable region 3D-7 (Immunoglobulin kappa variable 3D-7) and a polypeptide fragment thereof, in particular to application of the urine Immunoglobulin kappa variable region 3D-7 and the polypeptide fragment thereof in preparation of preparations for burn diagnosis, differential diagnosis, burn area and degree evaluation, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like. The burn is an important common wound in daily life, about 5000-100000 people burn in every 100 million people every year, according to the statistics of the world health organization, more than 30 million people die of burn patients all year around the world, and the survival rate of the serious burn treatment is still at a lower level. The research proves that the expression of the urine immunoglobulin kappa variable region 3D-7 and the polypeptide fragment thereof is increased in burn patients compared with healthy people (normal control group). Can be used for various purposes of detecting burn patients. The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient storage of the urine specimen, and utilizes the urine specimen to detect the urine immunoglobulin kappa variable region 3D-7 and the polypeptide fragment thereof.)

1. The urine immunoglobulin kappa variable region 3D-7 and the polypeptide fragment thereof are applied to the preparation of preparations for burn diagnosis, differential diagnosis, burn area and degree evaluation, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.

2. The use of claim 1, wherein the urine immunoglobulin kappa variable region 3D-7 has an amino acid sequence as set forth in SEQ ID No. 1; or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1 and has the same function with the amino acid sequence shown in SEQ ID NO. 1.

3. The use of claim 1, wherein the agent is a detection kit for immunoglobulin kappa variable region 3D-7 and polypeptide fragments thereof from urine of burn patients.

4. Use according to claim 3, wherein the kit comprises an immunization method for antigen-antibody reaction and kits thereof such as one or more of aptamer antibodies or antibody fragments capable of specifically binding to immunoglobulin kappa variable region 3D-7 and polypeptide fragments thereof.

5. The use of claim 3, wherein the detection method comprises mass spectrometry and related kits for direct detection of immunoglobulin kappa variable region 3D-7 and its polypeptide fragments.

6. The use of claim 3, wherein the detection method comprises detecting directly the immunoglobulin kappa variable region 3D-7 and its polypeptide fragments or its related nucleic acid and its related kit.

7. The use according to claim 3, wherein the kit further comprises a component selected from the group consisting of: the kit comprises a solid phase carrier, a diluent, a reference substance, a standard substance, a quality control substance, a detection antibody, a second antibody diluent, a luminescent reagent, a washing solution, a color development solution and a stop solution, wherein the solid phase carrier is any one or a combination of a plurality of the solid phase carrier, the diluent, the reference substance, the standard substance, the quality control substance, the detection antibody, the second antibody and the second antibody diluent.

8. The use of claim 7, wherein the standard comprises an immunoglobulin kappa variable region 3D-7 standard, a humanized tag antibody standard; preferably, the quality control product comprises: immunoglobulin kappa variable region 3D-7 control, humanized label antibody quality control; preferably, the solid support comprises: particles, microspheres, glass slides, test strips, plastic beads, liquid phase chips, micro-porous plates or affinity membranes and other carriers with the same functions.

9. The use of claim 8, wherein the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide, cellulose, and carriers with similar functions.

Technical Field

The invention relates to a new application of a urine immunoglobulin kappa variable region 3D-7 and a polypeptide fragment thereof, in particular to an application of the urine immunoglobulin kappa variable region 3D-7 and the polypeptide fragment thereof in burn diagnosis, differential diagnosis, burn area and degree evaluation, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.

Background

Burn refers to the injury of skin, tissue and even deep viscera of human body caused by chemical substances such as flame, high-temperature gas, scorching solid or liquid, radioactive rays, electric energy, strong acid and strong alkali, etc., and is a systemic comprehensive disease. After burn, a large amount of reactions such as necrosis, infection, shock, blood coagulation dysfunction and the like of wound tissues can cause a series of pathophysiological changes of organisms. Burns of different degrees have different influences on human bodies, serious burns can damage the environment in the human bodies, the burn patients have pathophysiological changes of complexity of various systems, and relevant detection indexes can correspondingly change along with the difference of the severity of the burns. Timely detection of changes in these indices can provide valuable reference for clinicians in many areas, such as disease diagnosis, disease judgment, treatment selection, and patient prognosis assessment.

However, patients with severe burns have poor skin integrity, and clinical use of hematological tests as invasive tests on the skin in such patients has presented difficulties, and repeated blood draw tests can also exacerbate patient pain. Urine as ultrafiltrate of blood contains abundant biological information, and the collection process has the advantages of non-invasive and convenient operation, and the like, which is particularly obvious in the detection of burn patients. The biomarker which is helpful for burn diagnosis and reflects disease change is searched in urine, so that the life quality and compliance of burn patients can be improved, the pain of blood collection for many times is relieved, and a reference basis which is favorable for disease diagnosis and treatment is better provided for clinicians.

Immunoglobulin kappa variable region 3D-7 (Alpha-1-antichymotrypsin, IGKV 3D-7) is involved in antigen recognition. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. Plays an important role in humoral immunity, in the recognition phase, membrane-bound immunoglobulins act as receptors, triggering clonal expansion of B lymphocytes upon specific antigen binding and differentiation into immunoglobulin-secreting plasma cells. Secreted immunoglobulins mediate an effector phase in humoral immunity that results in elimination of bound antigen. The antigen binding site is formed by the variable domain of one heavy chain and the variable domain of its associated light chain. Thus, each immunoglobulin has two antigen binding sites with significant affinity for a particular antigen. The variable domains are amenable to somatic hypermutation by V- (D) -J rearrangement and, upon exposure to antigen and selection, affinity maturation to the particular antigen. In the research, the expression of the immunoglobulin kappa variable region 3D-7 in the urine of the burn patient is up-regulated compared with that of a healthy human group, and the protein content in the urine of the burn patient is increased.

Compared with the common clinical blood sample, the urine can be collected in a non-invasive, continuous and large amount; without homeostatic regulation, more various and larger changes can be accumulated, and many pathophysiological changes of the body can be reflected in urine. Some protein polypeptides with relatively small molecular weight, such as hormones and cytokines, are excreted into urine quickly after entering blood, and the probability that the proteins and polypeptides are detected in urine is much higher than that in blood; before urine is collected, a possible protein degradation process in urine is completed, so that urine protein can be kept stable for a longer time. In order to relieve the pain of multiple blood sampling of burn patients, the experiment is expected to realize the diagnosis and disease monitoring of the burn patients by painless, convenient, quick and easily repeated urine detection through the research of urine protein or polypeptide on the basis of the methodology exploration of the early stage, and also lays a foundation for the further research of the urine polypeptide detection kit.

Disclosure of Invention

The invention aims to provide application of urine immunoglobulin kappa variable region 3D-7 and polypeptide fragments thereof in preparation of preparations for burn diagnosis, differential diagnosis, burn area and degree evaluation, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.

Preferably, the amino acid sequence of the urine immunoglobulin kappa variable region 3D-7 is shown in SEQ ID NO.1 (MEPWKPQHSF FFLLLLWLPD TTGEIVMTQS PATLSLSPGE RATLSCRASQ SVSSSYLSWY QQKPGQAPRL LIYGASTRAT GIPARFSGSG SGTDFTLTIS SLQPEDFAVY YCQQDYNLP); or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1 and has the same function with the amino acid sequence shown in SEQ ID NO. 1.

Preferably, the preparation is a detection kit for the immunoglobulin kappa variable region 3D-7 and polypeptide fragments thereof in urine of burn patients.

Preferably, the kits include methods of immunization for antigen-antibody reactions and kits thereof such as one or more of aptamer antibodies or antibody fragments capable of specifically binding immunoglobulin kappa variable region 3D-7 and polypeptide fragments thereof.

Preferably, the detection method comprises methods such as mass spectrometry for directly detecting the immunoglobulin kappa variable region 3D-7 and polypeptide fragments thereof and related kits.

Preferably, the detection method comprises related nucleic acid detection methods for directly detecting the immunoglobulin kappa variable region 3D-7 and polypeptide fragments thereof and related kits.

Preferably, the kit further comprises a component selected from the group consisting of: the kit comprises a solid phase carrier, a diluent, a reference substance, a standard substance, a quality control substance, a detection antibody, a second antibody diluent, a luminescent reagent, a washing solution, a color development solution and a stop solution, wherein the solid phase carrier is any one or a combination of a plurality of the solid phase carrier, the diluent, the reference substance, the standard substance, the quality control substance, the detection antibody, the second antibody and the second antibody diluent.

Preferably, the standard comprises an immunoglobulin kappa variable region 3D-7 standard, a humanized tag antibody standard; preferably, the quality control product comprises: immunoglobulin kappa variable region 3D-7 quality control substances and humanized label antibody quality control substances; preferably, the solid support comprises: particles, microspheres, glass slides, test strips, plastic beads, liquid phase chips, micro-porous plates or affinity membranes and other carriers with the same functions.

Preferably, the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose, and has similar functions.

The inventor firstly collects urine samples of healthy people and burn patients, centrifugates for 5min at 4000r/min, absorbs supernatant, measures the concentration of extracted protein by a Bradford method, and carries out SDS-PAGE enzymolysis. The Label-free mass spectrometry of the urine samples was performed by the OrbitrapFasion type mass spectrometer. And performing quantitative calculation on data obtained in the mass spectrum of the burn group and the normal control group. The differential polypeptide is screened by using the difference of protein expression amount more than 1.5 times and P <0.05 as a reference standard through statistical test. Then, the inventors identified the statistically significant differential polypeptides and searched the database to obtain the immunoglobulin kappa variable region 3D-7 of the differential protein.

Compared with healthy people, the immunoglobulin kappa variable region 3D-7 and the polypeptide fragment thereof are highly expressed in urine of burn patients and have better consistency with clinical diagnosis. Therefore, the detection of the urine immunoglobulin kappa variable region 3D-7 and the polypeptide fragment thereof can be used for auxiliary diagnosis or disease monitoring of burns.

The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient storage of the urine specimen, and utilizes the urine specimen to detect the urine immunoglobulin kappa variable region 3D-7 and the polypeptide fragment thereof.

In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.

Drawings

FIG. 1 is a graph of the content of urine immunoglobulin kappa variable region 3D-7 and its polypeptide fragments in burn and healthy control groups.

FIG. 2 is a schematic representation of the involvement of the immunoglobulin kappa variable region 3D-7 in major biological processes.

Detailed Description

Example 1Collection and processing of urine specimens

Burn patients were selected as the burn group, and contemporary physical examiners were selected as the normal control group. 30ml samples of fresh morning urine were collected from each group of subjects after admission, and those who failed to urinate normally collected their morning urine from their catheters and placed in dry, clean containers. Centrifuging the collected urine specimen at 4000r/min for 5min, sucking supernatant, subpackaging 2ml per tube, and storing in a refrigerator at-80 ℃.

Example 2Mass spectrometry and screening of urine polypeptides

Extracting protein from urine sample, and determining the concentration of extracted protein. Mass spectrometry of urine samples was performed by orbitrapfuision type mass spectrometry. And performing quantitative calculation on data obtained in the mass spectrum of the experimental group and the normal control group. The comparison among groups adopts t-test to carry out differential analysis, and differential expression proteins are screened by using the difference of protein expression quantity more than 1.5 times and taking the statistical test that P <0.05 as a reference standard.

Example 3Identification and analysis of differential Polypeptides

The used database is a Unit _ Homo database, the generated mass spectrum original file is processed by MaxQuant software, and the retrieval parameter setting is shown in Table 1.

Compared with healthy people, the immunoglobulin kappa variable region 3D-7 is highly expressed in urine of burn patients as shown in figure 1, the main biological processes involved in the human immunoglobulin kappa variable region 3D-7 are shown in figure 2, and the expression of the immunoglobulin kappa variable region 3D-7 in urine of normal control groups and burn groups is remarkably different.

Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

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Met Glu Pro Trp Lys Pro Gln His Ser Phe Phe Phe Leu Leu Leu Leu

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Trp Leu Pro Asp Thr Thr Gly Glu Ile Val Met Thr Gln Ser Pro Ala

20 25 30

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

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Ser Gln Ser Val Ser Ser Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Pro

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Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr

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Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

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Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys

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Gln Gln Asp Tyr Asn Leu Pro

115