CN109266734A - Autoimmune disease diagnostic kit and application - Google Patents

Abstract

The invention provides a diagnostic kit and application for autoimmune diseases. The present invention firstly uses tRNA as an entry point to study the tRNA expression profile of patients with autoimmune diseases, and screens differentially down-regulated tRNA in patients with autoimmune diseases, which can be used as an important means for diagnosis and prognosis of autoimmune diseases. In order to make the diagnosis of the disease more convenient and accurate, so as to lay a relevant foundation for the treatment of future diseases.

Description

Autoimmune disease diagnostic kit and application

Technical field

The present invention relates to field of biotechnology, in particular to a kind of diagnosing autoimmune diseases kit and application.

Background technique

A kind of important non-coding RNA (non-coding of transfer ribonucleic acid (transfer RNA, tRNA) RNA, ncRNA), it is the key joints molecule of protein synthesis, and being responsible for will be close on mRNA (message RNA, mRNA) Numeral information is converted into the amino acid sequence information on nascent protein.In addition to this, tRNA is also maintained in the stability of body The normal life process of cell, and the unstable rule of tRNA may result in cell and denaturation, proliferation, some lifes such as death occur Reason process.Meanwhile it is existing studies have shown that tRNA has the function of a part of house-keeping gene, its overexpression can enhance cell Metabolic function and proliferative capacity, the overexpression of the tRNA found in the certain cancers such as epithelial tumor, lung cancer, liver cancer at present The viewpoint has all been proved to a certain extent.

But at present about whether there is between tRNA and autoimmune disease similar relationship yet there are no disclosure.Currently In the genetics research of autoimmune disease, mainly using DNA, mRNA as Study of Support.Therefore, it is necessary to be to cut with tRNA Access point furthers elucidate the pathogenesis of autoimmune disease, and then provides new opportunity for the diagnoses and treatment etc. of disease.

Summary of the invention

The object of the present invention is to provide a kind of autoimmune disease diagnostic kit and applications.

To achieve the above object, the technical solution adopted by the present invention is that:

A kind of kit of diagnosis and/or prognosis for autoimmune disease, kit include the table of quantitative tRNA Up to the reagent of amount, the sequence of tRNA is at least one of SEQ ID No.1-3.

Preferably, autoimmune disease is systemic loupus erythematosus, rheumatoid arthritis, at least one in dermatomyositis Kind.

It preferably, further include the extraction reagent of tRNA.

The reagent of the expression quantity of quantitative tRNA answering in the reagent of diagnosis and/or the prognosis of preparation autoimmune disease With the sequence of tRNA is at least one of SEQ ID No.1-3.

Preferably, autoimmune disease is systemic loupus erythematosus, rheumatoid arthritis, at least one in dermatomyositis Kind.

The beneficial effects of the present invention are:

The present invention has studied the tRNA express spectra of autoimmune disease patient, filters out for the first time using tRNA as point of penetration The otherness of patient lowers tRNA, can be used as one of the important means of diagnosis, prognosis of autoimmune disease, to energy It is enough it is more convenient, accurately the disease is diagnosed, so that relevant basis is laid in the treatment for disease in future.

Detailed description of the invention

Fig. 1 is the scatter plot of differential expression tRNA of the invention.

Specific embodiment

The technical effect of design and generation of the invention is clearly and completely described below with reference to experiment, to fill Ground is divided to understand the purpose of the present invention, feature and effect.

Systemic loupus erythematosus (systemic lupus erythematosus, SLE) be a kind of chronic systematicness from Body immunological disease.Patient's self immune system generates antibodies attack own cells and tissue, leads to generation and the histologic lesion of inflammation. The pathogenesis of SLE is still not clear, it is now recognized that it is multifactor multifaceted influence that it, which causes a disease, including immune, heredity, environment, Sex hormone etc., all multiple cytokines are participated.The present invention is by with systemic loupus erythematosus, this is common, typically itself exempts from Epidemic disease disease is illustrated as representative and embodiment.

Case is from the SLE patient to go to a doctor in Shenzhen people's hospital, according to " systemic lupus erythematosus diagnosis in 2010 And treatment guidelines ", the SLE classification standard recommended using American Society of Rheumatism 1997, in 11 of the classification standard, symbol 4 or 4 or more persons are closed, are infected except.Tumour and other connective tissues after being ill, the SLE patient made a definite diagnosis, then calculate SLEDAI Scoring (SLEDAI scoring>=8 is the Lupus activity phase, and<8 be the stable disease phase), chooses the patient in the Lupus activity phase and enters reality Test group.SLE group includes SLE patient 20, and the control group of normal health includes 20, and two groups of experimental subjects Sex, Ages etc. are basic Condition is mutually matched, and random controls is taken to design.The inclusion criteria of patient are as follows: sign informed consent form and have complete clinic With various other data；Exclusion criteria is not obtain informed consent or clinical imperfect with other data.

1. sequence library construction

The patient of SLE group and Normal human peripheral's venous blood (on an empty stomach) 5ml of control group are collected with EDTA anticoagulant tube, 4 is small When interior processing sample the venous blood of collection is isolated using lymphocyte separation medium according to Ficoll density-gradient centrifugation method Peripheral blood mononuclear cells (PBMC).The total serum IgE that PBMC is extracted using TrizolTM reagent is mentioned using Trizol method, using RNA Purification kit carried out column purification to total serum IgE, and carried out corresponding quality testing.Product is subjected to demethylation processing, is gone End-o f-pipe -control and the acid processing that deaminizes, to filter out the tRNA of high quality.By round pcr, the library tRNA is constructed.

2. sequencing and analysis

Using Assemnler-3.4.4.0, Alignment 2.0,3.0 software of coverage Analysis, it is based on The sequence alignment program of 2 algorithm of fame is assembled and is compared to data with reference to genome with h38chrM (NC-012920), Allow the mispairing of 10 bases.Original series screen out filtration rate by pre-filtering and ratioization and are lower than lower than 40%, mapping rate 80%, the overburden depth sample data inhomogenous lower than 100X or coverage, finally screens to obtain control group and SLE group significant figure According to, searching otherness up-regulated expression tRNA, and calculate up-regulated expression amount.

1.tRNA differential expression analysis

Fig. 1 is the scatter plot of differential expression tRNA of the invention.As shown in Figure 1, wherein abscissa represents control group The logarithm of the CPM average value of tRNA, ordinate represent the logarithm of the CPM average value of the tRNA of SLE group, inside two dotted lines Point represent the tRNA (totally 174) of non-differential expression, the tRNA of differential expression up-regulation is represented above upper dashed line (altogether 50), the tRNA (totally 51) of differential expression downward, Pearson correlation coefficient=0.984 are represented below lower dashed line. After annotating to the tRNA of significant difference differential expression, comprehensive many factors have filtered out 3 and have lowered most significant difference TRNA (the nucleotide sequence of tRNA-Ala-AGC-13-1, tRNA-Leu-CAA-3-1, tRNA-Tyr-ATA-1-1 point of expression Wei SEQ ID No.1, SEQ ID No.2, SEQ ID No.3), specifying information is as follows:

1. correlation tRNA information of table

Wherein, FC value (Fold Change) is differential expression multiple, and p value is the p value that negative binomial distribution is examined, and s_CPM is The logarithm of the CPM average value of SLE group tRNA, z_CPM are the logarithm of the CPM average value of control group tRNA, and CPM is every million Read the reading long number of specific tRNA in long (reads).In the present invention, the synthesis for experiment sample amount and experiment accuracy is examined Consider, by log₂The threshold value of FC is set as -0.585, i.e. the threshold value of FC is set as 0.67.

It can be seen from the results above that these three tRNA expressed in SLE patient and Normal group in have obvious downward. According to above-mentioned experimental result, detected by the expression of these three tRNA in human peripheral blood or some other sample, phase The researcher of pass can quickly, easily diagnose autoimmune disease, especially systemic loupus erythematosus, and having can A kind of effective fast diagnosis method can be provided for diagnosis, the prognosis of autoimmune disease from now on；Meanwhile by above-mentioned Result of study is further analyzed expectation and obtains the relevant crucial epigenetic site of autoimmune disease, can send out whereby The now important regulating and controlling factor relevant to the occurrence and development of autoimmune disease, further to disclose its physiological mechanisms, seeking The treatment of related disease and pharmaceutically-active completely new target spot provide theoretical foundation.

The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any Belong to those skilled in the art in the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of, all answers It is included within the scope of the present invention.Therefore, protection scope of the present invention should be subject to the protection scope in claims.

SEQUENCE LISTING

<110>Shenzhen people's hospital

<120>autoimmune disease diagnostic kit and application

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<170> PatentIn version 3.3

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<212> DNA

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ggggaattag ctcaagcggt agagcgcttg cttagcatgc aagaggtagt gggatcgatg 60

cccacattct ccacca 76

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<212> DNA

<213> Homo sapiens

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gtcaggatgg ccgagtggtc taaggcgcca gactcaagtt ctggtctccg catggaggcg 60

tgggttcgaa tcccacttct gacacca 87

<210> 3

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<212> DNA

<213> Homo sapiens

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ccttcaatag ttcagctggt agagcagagg actataggtc cttaggttgc tggttcgatt 60

ccagcttgaa ggacca 76

Claims (5)

A kit for diagnosis and/or prognosis of an autoimmune disease, characterized in that the kit comprises an agent for quantifying the expression level of tRNA, and the sequence of the tRNA is SEQ ID No. 1-3 At least one of them. The kit according to claim 1, wherein the autoimmune disease is at least one of systemic lupus erythematosus, rheumatoid arthritis, and dermatomyositis. The kit according to any one of claims 1 to 3, further comprising an extraction reagent for tRNA. 4. Use of an agent for quantifying the expression level of tRNA in the preparation of a reagent for diagnosis and/or prognosis of an autoimmune disease, characterized in that the sequence of the tRNA is at least one of SEQ ID No. 1-3. The use according to claim 4, wherein the autoimmune disease is at least one of systemic lupus erythematosus, rheumatoid arthritis, and dermatomyositis.