阅读说明：本技术 一种草鱼细菌性肠炎生物标记物及治疗靶点的应用 (Grass carp bacterial enteritis biomarker and application of grass carp bacterial enteritis biomarker to treatment target ) 是由 瞿符发 刘臻 谢宜芳 邓张仁 唐建洲 房佳美 周勇华 彭亮 于 2019-11-29 设计创作，主要内容包括：本发明公开了一种草鱼细菌性肠炎生物标记物及治疗靶点的应用。本发明首次从草鱼肠道中克隆得到完整的p38α基因、p38β基因,它们的cDNA序列分别如SEQ ID NO.1和SEQ ID NO.2所示。本发明发现,p38α基因、p38β基因在患细菌性肠炎的草鱼肠道中表达水平显著上调,且干预p38活性后草鱼肠道炎症因子表达水平恢复至正常水平,因此,p38α基因、p38β基因可作为草鱼细菌性肠炎生物标记物和治疗靶点,实现对草鱼细菌性肠炎的诊断和治疗。(The invention discloses a biomarker for grass carp bacterial enteritis and application of a therapeutic target spot. The invention clones the complete p38 alpha gene and p38 beta gene from grass carp intestinal tract for the first time, and the cDNA sequences of the genes are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. The invention discovers that the expression levels of the p38 alpha gene and the p38 beta gene in grass carp intestinal tracts suffering from bacterial enteritis are obviously up-regulated, and the expression levels of inflammatory factors in the grass carp intestinal tracts are restored to normal levels after the intervention of the activity of p38, so that the p38 alpha gene and the p38 beta gene can be used as biomarkers and treatment targets of the grass carp bacterial enteritis, and the diagnosis and treatment of the grass carp bacterial enteritis are realized.)

The application of the p38 gene as a biomarker for bacterial enteritis of grass carps.

2. The use of claim 1, wherein the p38 gene is p38 a gene or p38 β gene; the cDNA sequence of the p38 alpha gene is shown as SEQ ID NO.1, and the cDNA sequence of the p38 beta gene is shown as SEQ ID NO. 2.

3. The application according to claim 2, wherein the application comprises: detecting the expression level of the p38 alpha gene or the p38 beta gene in grass carp intestinal tissues, and evaluating grass carp bacterial enteritis by taking the expression level of the p38 alpha gene or the p38 beta gene as an evaluation index.

4. The use of claim 3, wherein the expression level of p38 alpha gene or p38 beta gene in grass carp intestinal tissues is detected by using a detection kit; the detection kit comprises specific primers p38 alpha-F2 and p38 alpha-R2 designed according to a cDNA sequence of a p38 alpha gene, and specific primers p38 beta-F2 and p38 beta-R2 designed according to a cDNA sequence of a p38 beta gene; the sequences of the p38 alpha-F2 and the p38 alpha-R2 are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8; the sequences of the p38 beta-F2 and the p38 beta-R2 are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10.

The application of the p38 gene as a target for treating the bacterial enteritis of the grass carps.

6. The use of claim 5, wherein the p38 gene is the p38 a gene or the p38 β gene; the cDNA sequence of the p38 alpha gene is shown as SEQ ID NO.1, and the cDNA sequence of the p38 beta gene is shown as SEQ ID NO. 2.

7. The application according to claim 6, wherein the application comprises: preparing a medicament for treating bacterial enteritis of grass carps by taking the p38 gene as a target spot; the medicine contains substances for interfering the expression of p38 gene or inhibiting the activity of p 38.

8. The use according to claim 7, wherein the substance for inhibiting the activity of p38 is a p38 specific inhibitor; the p38 specific inhibitor is SB 203580.

Technical Field

The invention particularly relates to a biomarker for bacterial enteritis of grass carps and application of the biomarker to treatment of targets.

Background

Grass carp (Ctenophagodon idella) is the most important economic grass carp cultivated in China, is also the freshwater grass carp with highest cultivation yield in the world nowadays, and has a cultivation history of more than 1700 years. Currently, high-density intensive culture has become the main culture mode of grass carp. In the culture environment, the grass carps have poor disease resistance and are easily infected by pathogenic microorganisms. It has been demonstrated that infection of fish bodies mostly occurs in various mucosal tissues, especially intestinal mucosa, and necrosis of intestinal tissues of grass carp is caused by extensive contact with a large amount of viruses, bacteria, parasites and algal toxins in intestinal cavity, and severe inflammatory reaction of intestinal tract is accompanied. Bacterial enteritis is an infectious disease which frequently occurs and has serious harm in the process of grass carp large-scale culture, and is called four diseases of grass carp together with gill rot, red skin disease and viral hemorrhagic disease. Researches show that pathogenic bacteria such as Aeromonas hydrophila (Aeromonas hydrophila), Aeromonas sobria (Aeromonas sobria), Aeromonas veronii (Aeromonas veronii) and the like can infect intestinal tracts of grass carps to induce severe enteritis, so that large-scale death is caused, huge loss is caused to the production and breeding industry, and meanwhile, the water environment is polluted by a large amount of diseased and dead fishes to cause great harm.

However, the pathogenesis of bacterial enteritis in low-grade vertebrate grass carp is not clear, so the diagnosis method of enteritis mostly adopts observation of external form and intestinal section of fish body, but the diagnosis method is simple and effective, but results can be diagnosed only in the later period of enteritis occurrence, the timeliness is poor, and the application range is limited. Moreover, for a long time, farmers mostly adopt antibiotics or chemical drugs to prevent and treat bacterial enteritis of the grass carps, but the effect is not satisfactory; and long-term and excessive use of chemical drugs and antibiotics can destroy the intestinal mucosal immune system of the grass carp, further reduce the disease resistance and inevitably cause environmental pollution. Compared with the research on bacterial enteritis diseases of higher mammals, the research on pathogenesis and treatment targets of bacterial enteritis in grass carp of lower vertebrates is especially lacked. Therefore, developing pathogenesis of the grass carp bacterial enteritis and developing gene targets for diagnosing and treating the grass carp bacterial enteritis have important scientific significance and application value.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a grass carp bacterial enteritis biomarker and application of a therapeutic target. The p38 kinase is an important member of the Mitogen-activated protein kinase (MAPK) family, and its homologue exists widely in biology, including lower yeast, Drosophila and nematode species. It has now been found that p38 is not a single protein and includes four different isoforms: p38 alpha gene, p38 beta gene, p38 gamma and p38 delta, which are encoded by different genes, respectively. The different translocation of the individual members of p38 upon external stimuli enables them to exert different physiological functions in cells. The four isoforms of p38 all belong to the serine-threonine protein kinases, all have the same threonine-glycine-tyrosine (Thr-Gly-Tyr, TGY) activation domain, and when both Thr180 and Tyr182 are phosphorylated, p38MAPK can be activated. p38MAPK is one of the most important stress signal transduction pathways and can be widely activated by external environmental factors, such as pathogens, heat shock, osmotic pressure, ultraviolet light, oxidative stress, etc. Under the action of external stimulation, p38 phosphorylates and activates downstream substrates, mainly transcription factors including ATF1/2/6, Sap1, p53, C/EBP beta and the like, and finally participates in specifically regulating the transcription and expression of various genes such as TNF, C-myc, Fas/FasL and the like. A large number of studies show that p38 is involved in inflammatory reactions of higher animals and plays an important biological function. However, no experimental results show that the kinase is related to the bacterial enteritis of the grass carp, and even no related experimental results prove that the p38 can be used as a target point for diagnosing and treating the bacterial enteritis of the grass carp. The complete cDNA sequences of the p38 alpha gene and the p38 beta gene are obtained by first separation, the p38 alpha gene and the p38 beta gene are found to have correlation with the bacterial enteritis of the grass carp, and the p38 specific inhibitor can inhibit the expression levels of inflammatory factors TNF-alpha, IL-15 and antibacterial peptide beta-defensin in the intestinal tract of the grass carp, so that a novel biomarker and a treatment target point are provided for the bacterial enteritis of the grass carp.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

provides an application of a p38 gene as a grass carp bacterial enteritis biomarker.

In the above application, further, the p38 gene is p38 a gene or p38 β gene; the cDNA sequence of the p38 alpha gene is shown as SEQ ID NO.1, and the cDNA sequence of the p38 beta gene is shown as SEQ ID NO. 2.

In the foregoing application, further, the application includes: detecting the expression level of the p38 alpha gene or the p38 beta gene in grass carp intestinal tissues, and evaluating grass carp bacterial enteritis by taking the expression level of the p38 alpha gene or the p38 beta gene as an evaluation index.

In the application, a detection kit is further adopted to detect the expression level of the p38 alpha gene or the p38 beta gene in the grass carp intestinal tissue; the detection kit comprises specific primers p38 alpha-F2 and p38 alpha-R2 designed according to a cDNA sequence of a p38 alpha gene, and specific primers p38 beta-F2 and p38 beta-R2 designed according to a cDNA sequence of a p38 beta gene; the sequences of the p38 alpha-F2 and the p38 alpha-R2 are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8; the sequences of the p38 beta-F2 and the p38 beta-R2 are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10.

As a general inventive concept, the invention also provides an application of the p38 gene as a target for treating the bacterial enteritis of the grass carps.

In the above application, further, the p38 gene is p38 a gene or p38 β gene; the cDNA sequence of the p38 alpha gene is shown as SEQ ID NO.1, and the cDNA sequence of the p38 beta gene is shown as SEQ ID NO. 2.

In the foregoing application, further, the application includes: preparing a medicament for treating bacterial enteritis of grass carps by taking a p38 alpha gene and a p38 beta gene as targets; the medicine contains substances for interfering the expression of p38 alpha gene and p38 beta gene or inhibiting the activity of p 38.

In the above application, further, the substance for inhibiting the activity of p38 is a p38 specific inhibitor; the p38 specific inhibitor is SB 203580.

Compared with the prior art, the invention has the advantages that:

(1) the complete cDNA sequences of the p38 alpha gene and the p38 beta gene are firstly cloned from grass carp intestinal tracts, so that a support is provided for developing a detection kit for detecting the expression levels of the p38 alpha gene and the p38 beta gene by subsequently designing quantitative primers, and a foundation is laid for further carrying out RNAi (ribonucleic acid interference) and gene knockout experiments on the basis of the sequences.

(2) The expression levels of the p38 alpha gene and the p38 beta gene in grass carp intestinal tracts are obviously increased after the bacterial MDP is injected for 6 hours, and the good correlation between the p38 alpha gene and the p38 beta gene and grass carp bacterial enteritis is disclosed, so that the p38 alpha gene and the p38 beta gene can be used as biomarkers of the grass carp bacterial enteritis, the expression levels of the p38 alpha gene and the p38 beta gene can be used for effectively diagnosing the grass carp bacterial enteritis, and the sensitivity and the specificity of the diagnosis of the grass carp bacterial enteritis can be greatly improved.

(3) In the application of the p38 gene as a biomarker for bacterial enteritis of grass carps, the expression level of the p38 alpha or p38 beta gene in intestinal tissues of the grass carps is detected by using a detection kit, and the bacterial enteritis of the grass carps is evaluated by using the expression level of the p38 alpha or p38 beta gene as an evaluation index. The detection kit comprises a specific primer designed according to a cDNA sequence of a p38 alpha or p38 beta gene, and the detection kit is used for carrying out fluorescence quantitative PCR (polymerase chain reaction) technical detection, so that the expression levels of the p38 alpha gene and the p38 beta gene can be accurately and quickly detected, the diagnosis of the grass carp bacterial enteritis is realized, the method is simple and reliable, the timeliness is good, and the defects of the traditional grass carp enteritis diagnosis method can be overcome.

(4) The invention discovers that the expression level of grass carp intestinal inflammatory factors can be restored to the level without being infected by bacterial MDP 6 hours after the specific inhibitor is injected by taking the p38 alpha gene and the p38 beta gene as targets, and discloses that the p38 alpha gene and the p38 beta gene have good effects when being taken as the target for treating grass carp bacterial enteritis.

(5) In the application of the invention, the p38 gene is used as a target for treating the bacterial enteritis of the grass carp, the p38 alpha gene and the p38 beta gene are used as targets for preparing the medicine for treating the bacterial enteritis of the grass carp, the medicine contains substances for interfering the expression of the p38 alpha gene and the p38 beta gene or inhibiting the activity of p38, and the medicine treats the bacterial enteritis of the grass carp by interfering the expression of the p38 alpha gene and the p38 beta gene or inhibiting the activity of the p 38. The p38 specific inhibitor is used as a therapeutic drug, can obviously inhibit the expression levels of the fish intestinal inflammatory factors TNF-alpha, IL-15 and the antibacterial peptide beta-defensin, and has good therapeutic effect on grass carp enteritis. Compared with the existing antibiotics or chemical drugs, the grass carp bacterial enteritis treatment drug has better specificity, can greatly reduce the dosage, reduce the risks of damage to the intestinal mucosa immune system and reduction of disease resistance of grass carp caused by excessive use of disease-resistant drugs, and reduce pollution to the water environment.

Drawings

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.

FIG. 1 shows the expression level of p 38. alpha. gene under stimulation by bacterial MDP in example 3 of the present invention.

FIG. 2 shows the expression level of p 38. beta. gene under stimulation by bacterial MDP in example 4 of the present invention.

FIG. 3 is a graph showing the expression levels of inflammatory factors in the intestine after intervention in p38 activity in example 5 of the present invention.

Detailed Description

The invention is further described below with reference to the drawings and specific preferred embodiments of the description, without thereby limiting the scope of protection of the invention.

In the following examples, all the raw materials and instruments used are commercially available unless otherwise specified.