New application of Edwardsiella fish-sourced protein EseO or coding gene thereof

Abstract

The invention relates to an application of Edwardsiella fish-sourced protein EseO or a coding gene thereof in preparation of medicines for treating and / or preventing obesity. The edwardsiella fish source protein EseO screened by the invention can inhibit lipid metabolism of cells HeLa and mice C57 / BL6J, which proves that the edwardsiella fish source protein EseO has an obvious fat reducing effect and can be applied to feed additives, medical products and the like in the aquaculture industry related to weight loss.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a new use of a protein EseO derived from Edwardsiella piscicida or a gene encoding the protein EseO. Background Art

[0002] Edwardsiella piscicida is a major aquatic pathogen. It is a short, non-capsulated, Gram-negative bacterium with peritrichous flagella. It is widespread in the ocean and can infect over 20 major commercially cultivated fish species, including mullet, catfish, eel, and turbot. Edwardsiella piscicida can cause diseases such as hemorrhagic septicemia and gastroenteritis in fish, leading to mass mortality and significant economic losses to the aquaculture industry. During infection, Edwardsiella piscicida utilizes multiple proteins of its own to interfere with lipid metabolism in turbot and other fish.

[0003] Currently, in the treatment of obesity, many weight loss drugs are restricted from the market or withdrawn from clinical use. For example, some of these drugs fail to achieve the expected effects or cause adverse reactions in patients.

[0004] Therefore, there is an urgent need to develop an effective method or drug for treating obesity and related diseases with few side effects. Summary of the Invention

[0005] The main purpose of the present invention is to address the above problems and provide a new use of the protein EseO or its encoding gene derived from Edwardsiella piscicidalis.

[0006] To achieve the above objectives, the first aspect of the present invention provides the use of a protein derived from Edwardsiella piscicida or its encoding gene in the preparation of a drug for treating and / or preventing obesity. The present invention has found that the protein EseO has weight loss and fat reduction effects.

[0007] Preferably, the amino acid sequence of the protein EseO is as shown in SEQ ID NO: 1, and SEQ ID NO: 1 is specifically: MKTPPINGLYRYKLSGKALRQPPSAPHGWVIFLACLAIAGLTLLLIHRL.

[0008] Preferably, the nucleotide sequence of the gene encoding the protein EseO is shown in SEQ ID NO: 2, and SEQ ID NO: 2 is specifically:

[0009] ttacagccggtggatcagcagcagcgtcagcccggcgatggccaggcaggccaggaaaatgacccagccgtggggagcgctcggt ggttggcgcagtgctttgccgctgagtttgtagcgatacagtccattgatcgggggcgttttcat.

[0010] Preferably, the protein EseO or its encoding gene is used for fat reduction.

[0011] The second aspect of the present invention provides a recombinant expression vector, the main feature of which is that the recombinant expression vector comprises a gene encoding the EseO protein derived from Edwardsiella piscicida to express the EseO protein.

[0012] Preferably, the recombinant expression vector is a recombinant expression AAV vector, and a nucleic acid fragment encoding the EseO protein is inserted into the recombinant expression vector to stably express the EseO protein.

[0013] The protein EseO derived from Edwardsiella piscicida screened by the present invention can inhibit lipid metabolism in HeLa cells and C57 / BL6J mice, and has been shown to have a significant fat-reducing effect. It can be applied to aquaculture feed additives, pharmaceutical products, and the like related to weight loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 These are confocal images of lipid droplet numbers in wild-type Edwardsiella piscicidal strain, type III secretion system-deficient strain, EseO-deficient strain, EseO-complemented strain, and uninfected HeLa cells.

[0015] Figure 2 This is an immunofluorescence staining image of EseO expression in mice, where blue is the nuclear stain DAPI, green is the lipid dye BODIPY, and red is the Flag-tagged EseO protein.

[0016] Figure 3 Figure 2 is the relative curve of body weight change of C57 / BL6J male mice, where ND is the normal diet group, HFD is the high-fat diet group, and EseO+HFD is the high-fat diet group injected with an AAV vector containing the EseO fragment.

[0017] Figure 4 Four blood lipid indicators were tested in C57 / BL6J male mice eight weeks after the AAV vector was injected. Among them, TG is triglyceride, T-CHO is cholesterol, LDL-C is low-density lipoprotein, HDL-C is high-density lipoprotein, ND is the normal diet group, HFD is the high-fat diet group, and EseO+HFD is the high-fat diet group injected with the AAV vector containing the EseO fragment.

[0018] Figure 5 The results of oil red staining of the liver of C57 / BL6J male mice eight weeks after the injection of AAV vectors. ND is the normal diet group, HFD is the high-fat diet group, and EseO+HFD is the high-fat diet group injected with AAV vectors containing EseO fragments.

[0019] Figure 6 The following are the results of HE staining of the liver of C57 / BL6J male mice eight weeks after the injection of AAV vectors. ND is the normal diet group, HFD is the high-fat diet group, and EseO+HFD is the high-fat diet group injected with AAV vectors containing EseO fragments. DETAILED DESCRIPTION

[0020] In order to more clearly understand the technical content of the present invention, the following embodiments are given in detail. However, it should be noted that these descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the claims of the invention.

[0021] Unless otherwise specified, the reagents and methods involved in the examples are commonly used in the art.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] The present invention has confirmed through research that the EseO protein derived from Edwardsiella piscicida can inhibit the formation of lipid droplets in HeLa cells; and after being expressed in C57 / BL6J male mice via an AAV vector, the body weight, oil red staining and blood lipid levels were tested to prove that it has a good fat-reducing effect, and HE staining proved that it has no harm to mice.

[0024] Example 1

[0025] EseO inhibits lipid droplet accumulation in HeLa cells after infection

[0026] The EseO gene deletion strain, overexpression strain and type III secretion system gene deletion strain were constructed by homologous recombination. Edwardsiella piscicida EIB202 (CCTCC M208068) and EIB202 strain EseO gene deletion strain, EseO gene overexpression strain and type III secretion system gene deletion strain were added at an MOI of 10:1. 7Each well was inoculated with 1 ml of Opti-MEM medium. The cells were inoculated in an incubator with 5% CO2 at 35°C. After 2 hours of infection, the cells were rinsed twice with PBS. Then, 1 ml of Opti-MEM medium containing 1000 μg / ml gentamicin was added. After 20 minutes, the cells were rinsed twice with PBS and then 1 ml of Opti-MEM medium containing 10 μg / ml gentamicin was added for 4 hours. After 4 hours, the cells were rinsed twice with PBS and stained for lipid droplets and nuclei with Bodipy 493 / 503 (green) and DAPI (blue), respectively. The cells were then observed using a confocal microscope.

[0027] The experimental results are as follows Figure 1 As shown, infection of HeLa cells with the EseO-deficient strain stimulates the formation of lipid droplets in the cells. After infection of the cells with the EseO-overexpressing strain, the size and number of lipid droplets in the cells decreased significantly.

[0028] Example 2

[0029] EseO significantly reduced the body weight, blood lipids and other related indicators of mice

[0030] The present invention synthesizes an AAV vector containing EseO by Guangdong Paizhen Biotechnology, such as Figure 2 As shown, EseO is expressed in mice, where blue is the nuclear stain DAPI (from Shanghai Beyotime Biotechnology), green is the lipid dye BODIPY (from Thermo Fisher Scientific (China)), and red is the Flag-tagged EseO protein (Flag antibody from Shanghai Beyotime Biotechnology).

[0031] To verify whether EseO also has a fat-reducing effect in vivo, 36 5-week-old C57 / BL6J male mice were fed for 7 days and then divided into groups according to body weight, including a normal feed-fed group, a high-fat feed-fed group, and a high-fat feed group injected with the above-mentioned AAV vector containing EseO, and recorded as week 0. The normal feed-fed group was fed with breeding irradiated mouse feed provided by JaseGen, and the high-fat feed group was fed with D12492 feed provided by Research Diet. These 36 mice were weighed weekly. The results are shown in Figure 2. Figure 3 As shown, the weight gain of mice in the high-fat diet group was the fastest, while the weight of the high-fat diet group injected with the AAV vector containing EseO was comparable to that of the normal diet group.

[0032] The weight loss ratio was calculated based on the body weight of each group. The results are shown in Table 1 below. HFD refers to the high-fat diet group, and EseO+HFD refers to the high-fat diet group injected with an AAV vector containing the EseO fragment. The weight loss ratio for the high-fat diet group injected with the EseO AAV vector relative to the high-fat diet group was 9.10%.

[0033] Table 1

[0034] HFD Eseo+HFD Initial average weight 19.53 20.38 Final average weight 28.60 27.17 Weight loss ratio 0 9.10％

[0035] The mice were raised to the eighth week and tested for triglycerides, cholesterol, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) using a Nanjing Jiancheng Biological Kit. 2.5 μL of mouse orbital blood was added, followed by 250 μL of the working solution. The plate was shaken to mix thoroughly, and the absorbance of each well was measured using a microplate reader after incubation at 37°C. The results were as follows: Figure 4 The triglycerides, cholesterol, low-density lipoprotein and high-density lipoprotein of mice in the high-fat diet group were the highest, while the body weight of the high-fat fed group injected with the AAV vector containing EseO was comparable to that of the normal chow fed group.

[0036] At the same time, the livers of mice raised to the 8th week were sent to Worden Pathology Company for oil red staining. The tissues were first fixed with 10% neutral formaldehyde and prepared into frozen sections, and then stained with filtered diluted oil red storage solution for 10 minutes. Figure 5 As shown, the fat content of the oil red-stained sections of the high-fat diet group mice was the highest, while the fat content of the high-fat diet group injected with the AAV vector containing EseO was relatively low and there was a tendency for fat accumulation.

[0037] The above experimental results show that EseO has a good fat-reducing effect in the body and can affect lipid metabolism.

[0038] Example 3

[0039] AAV vectors containing EseO are not harmful to mice

[0040] HE staining was performed on the liver of the mice raised to the 8th week in the above embodiment. Figure 6 As shown, no lesions were produced in the HE-stained sections of the normal feed-fed group, the high-fat feed-fed group, and the high-fat feed-fed group injected with the AAV vector containing EseO, indicating that the AAV vector containing EseO is not harmful to mice.

[0041] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the description is to be regarded as illustrative rather than restrictive.

Claims

1. Use of the protein EseO derived from Edwardsiella piscicida or its encoding gene in the preparation of a drug for treating and / or preventing obesity.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein EseO is shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that The nucleotide sequence of the gene encoding the protein EseO is shown in SEQ ID NO:

2.

4. The use according to claim 1, characterized in that The protein EseO or its encoding gene is used for fat reduction.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises a gene encoding the EseO protein derived from Edwardsiella piscicida to express the EseO protein.

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