阅读说明：本技术 一种分泌型fndc5蛋白及其制备方法和应用 (Secretory FNDC5 protein and preparation method and application thereof ) 是由 唐东起 李世武 李慧 于 2019-09-04 设计创作，主要内容包括：本发明公开了一种分泌型FNDC5蛋白,不含跨膜结构域,可直接分泌并释放到血液中；所述分泌型FNDC5蛋白的氨基酸序列为SEQ ID NO：1；本发明的分泌型FNDC5蛋白结构简单,获得方便；本发明的分泌型FNDC5蛋白能用于治疗肥胖相关代谢紊乱；高脂喂养的肥胖小鼠模型腹腔注射分泌型FNDC5重组蛋白后,可有效改善葡萄糖和胰岛素耐量,表明此蛋白可明显改善肥胖患者糖代谢紊乱和胰岛素抵抗；可明显缓解脂肪肝发生,降低血浆胆固醇、游离脂肪酸及甘油三酯的水平,表明此蛋白可改善肥胖患者脂代谢紊乱。本发明的分泌型FNDC5蛋白,可以作为新的治疗代谢性疾病的药物靶点进行开发。(The invention discloses a secretory FNDC5 protein, which does not contain a transmembrane domain and can be directly secreted and released into blood; the amino acid sequence of the secretory FNDC5 protein is SEQ ID NO: 1; the secretory FNDC5 protein has simple structure and is convenient to obtain; the secreted FNDC5 protein can be used for treating obesity-related metabolic disorders; after secretory FNDC5 recombinant protein is injected into the abdominal cavity of an obese mouse model fed with high fat, the glucose and insulin tolerance can be effectively improved, which shows that the protein can obviously improve the glucose metabolism disorder and the insulin resistance of obese patients; can obviously relieve the occurrence of fatty liver and reduce the levels of plasma cholesterol, free fatty acid and triglyceride, which indicates that the protein can improve the lipid metabolism disorder of obese patients. The secretory FNDC5 protein can be developed as a new drug target for treating metabolic diseases.)

1. A secreted FNDC5 protein, wherein: the secretory FNDC5 protein does not contain a transmembrane structural domain, and can be directly secreted and released into blood; the amino acid sequence of the secretory FNDC5 protein is SEQ ID NO: 1.

2. the process for producing secreted FNDC5 protein as claimed in claim 1, wherein: the method comprises the following steps:

① designing primers from different sites of the fndc5 gene, carrying out PCR and running electrophoresis, and detecting other isomers besides the known fndc5 isomer;

wherein the primer is as follows:

upstream primer FNDC 5-F: TGAGGCCGAGAAGATGGCCTCTAA, respectively;

downstream primer FNDC 5-R: TAGTGACAATGGCTGCTCTCTGCC, respectively;

② transforming the cDNA sequence corresponding to the new isomer protein into DH5a competent cell, plating, selecting single clone, extracting plasmid, enzyme digestion confirming, transferring into x-33 yeast, YPD culture medium for enlarging culture, protein expression with BMMH solution containing 0.5% methanol, and purifying the protein expression supernatant to obtain secreted FNDC5 protein.

3. The method of claim 2, wherein the cDNA sequence corresponding to the new isoform protein detected in step ② is ligated to the PPIC _ ZaA plasmid and transformed into DH5a competent cells.

4. The use of secreted FNDC5 protein as claimed in claim 1, wherein: use in the treatment of obesity-related metabolic disorders.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a secretory FNDC5 protein and a preparation method and application thereof.

Background

Obesity is a common metabolic disease, threatens the health of people all over the world, and the obesity patients have higher probability of complicating cardiovascular and cerebrovascular diseases, type 2 diabetes, respiratory distress syndrome and musculoskeletal diseases. When the energy intake of the human body is higher than the energy consumption, the residual heat is stored in the body in the form of fat tissue, and when the content of the fat tissue exceeds the physiological requirement, the obesity is developed. Therefore, increasing energy expenditure has gradually become a powerful means for the potential prevention and treatment of obesity and its related diseases.

Irisin (irisin) is a 112 amino acid residue protein found in 2012 and is the product of cleavage of the fibronectin type III domain containing protein 5(FNDC5) by an unknown protease. The three FNDC5 subtypes found so far all contain a transmembrane domain (exon5) to anchor protein on cell membranes, and the irisin is the C-terminal cleavage product of FNDC5, the sequence of the product is highly conserved in mammals, and the amino acid sequences of the irisin of mice, rabbits and humans are completely identical. The irisin can be produced by various histiocytes of skeletal muscle, cardiac muscle, liver, kidney, nerve, fat, pancreas and the like, and researches prove that the irisin has good application prospect in the aspects of improving various metabolic diseases such as obesity and the like. It is presumed that irisin is cleaved from FNDC5 by an unknown protease and released into the blood, but the protease and the exact cleavage site have not been found so far, and therefore the irisin release pathway is still under question.

The FNDC5 subtype which does not contain transmembrane domain and can be directly secreted and the application thereof in lipid metabolism have not been reported yet.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a secretory FNDC5 protein and a preparation method and application thereof.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a secreted FNDC5 protein, which does not contain a transmembrane domain, and which can be secreted directly and released into the blood; the amino acid sequence of the secretory FNDC5 protein is SEQ ID NO: 1.

a preparation method of secreted FNDC5 protein comprises the following steps:

① designing primers from different sites of the fndc5 gene, carrying out PCR and running electrophoresis, and detecting other isomers besides the known fndc5 isomer;

wherein the primer is as follows:

upstream primer FNDC 5-F: TGAGGCCGAGAAGATGGCCTCTAA, respectively;

downstream primer FNDC 5-R: TAGTGACAATGGCTGCTCTCTGCC, respectively;

② transforming the cDNA sequence corresponding to the new isomer protein into DH5a competent cell, plating, selecting single clone, extracting plasmid, enzyme digestion confirming, transferring into x-33 yeast, YPD culture medium for enlarging culture, protein expression with BMMH solution containing 0.5% methanol, and purifying the protein expression supernatant to obtain secreted FNDC5 protein.

The preferred method of preparation, step ②, involves ligating the cDNA sequence corresponding to the new isoform protein detected to the PPIC _ ZaA plasmid and transforming into DH5a competent cells.

The invention also comprises the application of the secreted FNDC5 protein in treating obesity-related metabolic disorders.

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

the secretory FNDC5 protein is a novel protein which does not contain a transmembrane structural domain and can be directly secreted, and has simple structure and convenient acquisition; the secreted FNDC5 protein can be used for treating obesity-related metabolic disorders; after secretory FNDC5 recombinant protein is injected into the abdominal cavity of an obese mouse model fed with high fat, the glucose and insulin tolerance can be effectively improved, which shows that the protein can obviously improve the glucose metabolism disorder and the insulin resistance of obese patients; can obviously relieve the occurrence of fatty liver and reduce the levels of plasma cholesterol, free fatty acid and triglyceride, which indicates that the protein can improve the lipid metabolism disorder of obese patients. The secretory FNDC5 protein can be developed as a new drug target for treating metabolic diseases.

Drawings

FIG. 1 is a graph showing the results of a glucose tolerance test;

FIG. 2 is a graph showing the results of an insulin resistance experiment;

FIG. 3 is a graph showing the serum cholesterol levels in the sera of three groups of mice;

FIG. 4 is a graph showing the content of triglycerides in serum in the sera of three groups of mice;

FIG. 5 is a graph showing the levels of free fatty acids in serum in the sera of three groups of mice;

FIG. 6 is a schematic of oil red staining of liver of three groups of mice.

Detailed Description

The PPIC _ ZaA plasmid was purchased from Invitrogen (USA).