阅读说明：本技术 尿液α1-抗糜蛋白酶及其多肽片段在烧伤中的应用 (Application of urine alpha 1-antichymotrypsin 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α1-抗糜蛋白酶(Alpha-1-antichymotrypsin)及其多肽片段的应用,具体为尿液α1-抗糜蛋白酶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α1-抗糜蛋白酶及其多肽片段在烧伤患者中表达升高,升高程度随烧伤程度加重而增高。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α1-抗糜蛋白酶及其多肽片段。(The invention provides urine Alpha 1-antichymotrypsin (Alpha-1-antichymotrypsin) and application of polypeptide fragments thereof, in particular to application of urine Alpha 1-antichymotrypsin 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. 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 compared with healthy people (normal control group), the expression of urine alpha 1-antichymotrypsin and polypeptide fragments thereof in burn patients is increased, and the increase degree is increased along with the increase of the burn degree. Can be used for various purposes of detecting burn patients. The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient preservation of the urine sample, and utilizes the urine sample to detect the urine alpha 1-chymotrypsin and the polypeptide fragment thereof.)

1. The urine alpha 1-antichymotrypsin and its polypeptide fragment are used in preparing preparation for burn diagnosis, differential diagnosis, burn area and degree evaluation, treating effect evaluation, monitoring, prognosis evaluation, mechanism research, etc.

2. The use according to claim 1, wherein the urinary α 1-antichymotrypsin has the amino acid sequence shown 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 kit for detecting alpha 1-antichymotrypsin and its polypeptide fragments in urine of burn patients.

4. Use according to claim 3, wherein the kit comprises one or more of an immunological method of antigen-antibody reaction and kits thereof such as aptamer antibodies or antibody fragments capable of specifically binding to α 1-antichymotrypsin and polypeptide fragments thereof.

5. The use of claim 3, wherein the detection method comprises mass spectrometry and related kits for directly detecting alpha 1-antichymotrypsin and its polypeptide fragments.

6. The use of claim 3, wherein the detection method comprises a method for directly detecting alpha 1-antichymotrypsin and related nucleic acid detection of polypeptide fragments thereof and related kits.

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 alpha 1-antichymotrypsin standard, a humanized tag antibody standard; preferably, the quality control product comprises: alpha 1-chymotrypsin-resistant 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.

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 new application of urine alpha 1-antichymotrypsin and polypeptide fragments thereof, in particular to application of urine alpha 1-antichymotrypsin and polypeptide fragments 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.

Alpha 1-Antichymotrypsin (ACT) is one of the members of the serine protease inhibitor superfamily, specifically inactivating serine proteolytic enzymes, the target enzymes of which are cathepsin H, mast cell chymase and chymotrypsin. Several proteolytic enzymes, which play a key role in tissue remodeling and development, play important regulatory roles in the homeostasis of proteolytic enzymes. Genetic defects in alpha 1-antichymotrypsin have been found to be associated with a number of diseases (asthma, chronic obstructive pulmonary disease, chronic liver disease, alzheimer's disease). In the research, alpha 1-antichymotrypsin in urine of burn patients is up-regulated compared with that of healthy people, and has a certain correlation with the burn degree, and the more serious the burn degree is, the higher the protein content is.

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 alpha 1-antichymotrypsin 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 alpha 1-antichymotrypsin is shown in SEQ ID NO.1 (MERMLPLLAL GLLAAGFCPA VLCHPNSPLD EENLTQENQD RGTHVDLGLA SANVDFAFSL YKQLVLKAPD KNVIFSPLSI STALAFLSLG AHNTTLTEIL KGLKFNLTET SEAEIHQSFQ HLLRTLNQSS DELQLSMGNA MFVKEQLSLL DRFTEDAKRL YGSEAFATDF QDSAAAKKLI NDYVKNGTRG KITDLIKDLD SQTMMVLVNY IFFKAKWEMP FDPQDTHQSR FYLSKKKWVM VPMMSLHHLT IPYFRDEELS CTVVELKYTG NASALFILPD QDKMEEVEAM LLPETLKRWR DSLEFREIGE LYLPKFSISR DYNLNDILLQ LGIEEAFTSK ADLSGITGAR NLAVSQVVHK AVLDVFEEGT EASAATAVKI TLLSALVETR TIVRFNRPFL MIIVPTDTQN IFFMSKVTNP KQA); 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 alpha 1-antichymotrypsin and polypeptide fragments thereof in urine of burn patients.

Preferably, the kit includes one or more of an immunological method of antigen-antibody reaction and kits thereof such as aptamer antibodies or antibody fragments capable of specifically binding to alpha 1-antichymotrypsin and polypeptide fragments thereof.

Preferably, the detection method comprises methods such as mass spectrometry for directly detecting alpha 1-antichymotrypsin and polypeptide fragments thereof and related kits thereof.

Preferably, the detection method comprises related nucleic acid detection methods for directly detecting alpha 1-antichymotrypsin 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 alpha 1-antichymotrypsin standard, a humanized tag antibody standard; preferably, the quality control product comprises: alpha 1-chymotrypsin resistant 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 patients with different burn degrees, centrifugates for 5min at 4000r/min, absorbs supernatant, determines 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 inventor identifies the differential polypeptide with statistical significance, and utilizes a database to search to obtain the differential protein alpha 1-anti-chymotrypsin.

Compared with healthy people, the alpha 1-antichymotrypsin and the polypeptide fragment thereof are proved to be highly expressed in urine of burn patients and are increased along with the aggravation of the burn degree through research, and the alpha 1-antichymotrypsin and the polypeptide fragment thereof have better consistency with clinical diagnosis. Therefore, the detection of urine alpha 1-antichymotrypsin and polypeptide fragments 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 preservation of the urine sample, and utilizes the urine sample to detect the urine alpha 1-chymotrypsin 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 showing the content of urine alpha 1-antichymotrypsin and its polypeptide fragments in burn and healthy control groups.

FIG. 2 is a schematic representation of the involvement of alpha 1-antichymotrypsin 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 alpha 1-antichymotrypsin is highly expressed in urine of burn patients as shown in figure 1, the main biological processes involved in the alpha 1-antichymotrypsin are shown in figure 2, and the expression of the alpha 1-antichymotrypsin in urine of a normal control group and a burn group is remarkably different and is increased along with the increase of the burn degree.

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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