Preparation method and application of a dual-target fusion protein

Abstract

The invention discloses a preparation method and application of a double-target fusion protein, belonging to the technical field of medicine. The present invention transforms the sequence of FGF19 and fuses it with GLP-1. Compared with wild-type FGF19 and GLP-1, the obtained novel fusion protein has a longer-lasting and more stable effect, and further reduces the risk of FGF19 carcinogenesis. , can better improve liver damage and correct metabolic disorders, obesity, overweight, metabolic syndrome, diabetes, dyslipidemia and other diseases, and there are no side effects of gastrointestinal discomfort such as diet decline caused by GLP‑1 treatment during the treatment , has broad application prospects.

Description

technical field

[0001] The invention relates to a preparation method and application of a double-target fusion protein, belonging to the technical field of medicine. Background technique

[0002] Fibroblast growth factor 19 (FGF19) is a newly discovered metabolic regulator, which stimulates intestinal secretion and expression after bile acid secretion into the intestinal tract. After being secreted by the intestinal tract, FGF19 can enter the liver with the circulation and combine with FGFR4 in the liver to act. It has a hormone-like effect and plays an important role in metabolic regulation, such as regulating bile acid metabolism, regulating gallbladder filling, improving energy metabolism and reducing blood pressure. body mass, blood sugar improvement, etc. Since many previous studies have shown that FGF19 has a mitogenic effect, FGFR4 can promote the proliferation of FGF19 in the liver and has a tumor-promoting effect. In 2014, a study found that the N-terminal domain ...

Examples

Embodiment 1

[0039] Example 1: Construction, expression and purification of recombinant proteins

[0040] (1) Construction of GLP-FGF19-1, GLP-FGF19-2 and GLP-FGF19-3 expression vectors

[0041] The FGF19 shown in the amino acid sequence as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 is respectively fused with the GLP-1 shown in SEQID NO.4 (optional use in FGF19 and GLP-1 Commonly used protein linker peptides) to obtain three novel fusion protein genes, namely GLP-FGF19-1 (shown in SEQ ID NO.6), GLP-FGF19-2 (shown in SEQ ID NO.7) and GLP-FGF19- 3 (shown in nuclear SEQ ID NO.8), the corresponding coding gene sequence was designed according to the codon preference of Escherichia coli. These three genes were sent to Shanghai Jierui Biological Co., Ltd. for synthesis, and NdeI and BamHI restriction sites were designed at both ends of each gene. The three synthetic vectors and pET30a(+) containing their respective target gene fragments were double-digested with NdeI and BamHI respectively. After th...

Embodiment 2

[0045] Example 2: Detection of half-life of recombinant protein in vivo

[0046] For five proteins FGF19 (amino acid sequence shown in SEQ ID NO.9), GLP-1 (amino acid sequence shown in SEQ ID NO.4), GLP-FGF19-1, GLP-FGF19-2 and GLP-FGF19-3 in vivo half-life assay.

[0047] Twenty-five rabbits with a body weight of about 2 kg were selected and randomly divided into 5 groups. Each group was subcutaneously injected with 5 kinds of proteins FGF19, GLP-1, GLP-FGF19-1, GLP-FGF19-2 and GLP-FGF19-3, the dose was 5mg / kg, and after administration, 0h, 1h, 3h, 5h, At 7h and 24h, about 800 μL of blood was collected from the ear vein. Centrifuge at 12000r / m for 10min, take the supernatant and store it at -20°C for later use. The in vivo half-life of five proteins was determined by ELISA indirect method: FGF19, GLP-1, GLP-FGF19-1, GLP-FGF19-2 and GLP-FGF19-3 proteins (2 μg / mL, 0.2 μg / mL) were diluted with different concentrations , 200ng / mL, 20ng / mL and 2ng / mL) to establish the standard...

Embodiment 3

[0050] Example 3: Effects of recombinant protein on body weight, diet, blood lipids and diabetes-related indicators

[0051] Three kinds of proteins, GLP-FGF19-1, GLP-FGF19-2 and GLP-FGF19-3 were prepared according to the method of Example 1.

[0052] Take 50 SPF 8-week-old male db / db mice, weigh them after pre-feeding for 1 week, fast for 6 hours the next day, take blood from the tail vein to measure the fasting blood glucose of the mice, remove abnormal weight, and screen blood glucose and body weight 36 model mice whose values ​​were relatively close to the mean value were randomly divided into saline injection group (Saline), FGF19 group, GLP-1 group, GLP-FGF19-1 group, GLP-FGF19-2 group and GLP-FGF19-3 group Group, 6 in each group. At about 8:30 every morning, the corresponding test substance was given to the experimental group once, intraperitoneally, at a dose of 2 mg / kg, and the normal saline group was injected with the same volume of normal saline, and the administra...

technical field

[0001] The invention relates to a preparation method and application of a double-target fusion protein, belonging to the technical field of medicine. Background technique

[0002] Fibroblast growth factor 19 (FGF19) is a newly discovered metabolic regulator, which stimulates intestinal secretion and expression after bile acid secretion into the intestinal tract. After being secreted by the intestinal tract, FGF19 can enter the liver with the circulation and combine with FGFR4 in the liver to act. It has a hormone-like effect and plays an important role in metabolic regulation, such as regulating bile acid metabolism, regulating gallbladder filling, improving energy metabolism and reducing blood pressure. body mass, blood sugar improvement, etc. Since many previous studies have shown that FGF19 has a mitogenic effect, FGFR4 can promote the proliferation of FGF19 in the liver and has a tumor-promoting effect. In 2014, a study found that the N-terminal domain ...

Examples

Embodiment 1

[0039] Example 1: Construction, expression and purification of recombinant proteins

[0040] (1) Construction of GLP-FGF19-1, GLP-FGF19-2 and GLP-FGF19-3 expression vectors

[0041] The FGF19 shown in the amino acid sequence as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 is respectively fused with the GLP-1 shown in SEQID NO.4 (optional use in FGF19 and GLP-1 Commonly used protein linker peptides) to obtain three novel fusion protein genes, namely GLP-FGF19-1 (shown in SEQ ID NO.6), GLP-FGF19-2 (shown in SEQ ID NO.7) and GLP-FGF19- 3 (shown in nuclear SEQ ID NO.8), the corresponding coding gene sequence was designed according to the codon preference of Escherichia coli. These three genes were sent to Shanghai Jierui Biological Co., Ltd. for synthesis, and NdeI and BamHI restriction sites were designed at both ends of each gene. The three synthetic vectors and pET30a(+) containing their respective target gene fragments were double-digested with NdeI and BamHI respectively. After th...

Embodiment 2

[0045] Example 2: Detection of half-life of recombinant protein in vivo

[0046] For five proteins FGF19 (amino acid sequence shown in SEQ ID NO.9), GLP-1 (amino acid sequence shown in SEQ ID NO.4), GLP-FGF19-1, GLP-FGF19-2 and GLP-FGF19-3 in vivo half-life assay.

[0047] Twenty-five rabbits with a body weight of about 2 kg were selected and randomly divided into 5 groups. Each group was subcutaneously injected with 5 kinds of proteins FGF19, GLP-1, GLP-FGF19-1, GLP-FGF19-2 and GLP-FGF19-3, the dose was 5mg / kg, and after administration, 0h, 1h, 3h, 5h, At 7h and 24h, about 800 μL of blood was collected from the ear vein. Centrifuge at 12000r / m for 10min, take the supernatant and store it at -20°C for later use. The in vivo half-life of five proteins was determined by ELISA indirect method: FGF19, GLP-1, GLP-FGF19-1, GLP-FGF19-2 and GLP-FGF19-3 proteins (2 μg / mL, 0.2 μg / mL) were diluted with different concentrations , 200ng / mL, 20ng / mL and 2ng / mL) to establish the standard...

Embodiment 3

[0050] Example 3: Effects of recombinant protein on body weight, diet, blood lipids and diabetes-related indicators

[0051] Three kinds of proteins, GLP-FGF19-1, GLP-FGF19-2 and GLP-FGF19-3 were prepared according to the method of Example 1.

[0052] Take 50 SPF 8-week-old male db / db mice, weigh them after pre-feeding for 1 week, fast for 6 hours the next day, take blood from the tail vein to measure the fasting blood glucose of the mice, remove abnormal weight, and screen blood glucose and body weight 36 model mice whose values ​​were relatively close to the mean value were randomly divided into saline injection group (Saline), FGF19 group, GLP-1 group, GLP-FGF19-1 group, GLP-FGF19-2 group and GLP-FGF19-3 group Group, 6 in each group. At about 8:30 every morning, the corresponding test substance was given to the experimental group once, intraperitoneally, at a dose of 2 mg / kg, and the normal saline group was injected with the same volume of normal saline, and the administra...

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