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Fusion protein of Exendin-4 and mutational human serum albumin, and preparation method of fusion protein

A human serum albumin and fusion protein technology, applied in the field of long-acting recombinant fusion protein drugs, can solve the problems of small molecular weight, large changes in serum concentration, and inconvenience to patients, and achieve good application prospects and prolong the effect of action time

Inactive Publication Date: 2012-12-19
SHANGHAI ALLIST PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to its small molecular weight and rapid elimination by the kidneys, patients still need to inject twice a day to obtain a satisfactory therapeutic effect, and the serum concentration varies greatly. so much inconvenience

Method used

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  • Fusion protein of Exendin-4 and mutational human serum albumin, and preparation method of fusion protein
  • Fusion protein of Exendin-4 and mutational human serum albumin, and preparation method of fusion protein
  • Fusion protein of Exendin-4 and mutational human serum albumin, and preparation method of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: pTG19-(Exendin-4) 2 - Construction of HSA recombinant plasmid

[0046] Human serum albumin gene was obtained by RT-PCR method. Firstly, human hepatoma cells were obtained, and total RNA was prepared from them. cDNA was then obtained by the reverse transcription method. The human serum albumin gene was amplified by PCR molecular cloning technology. The specific instructions are as follows:

[0047] 1. Extraction of total RNA from human liver cancer cells

[0048] Human liver cancer cell SMMC7721, total RNA was extracted with BioFlux simply P total RNA extraction kit. Specifically, about 5×10 human liver cancer cell SMMC7721 6 After freezing and crushing, centrifuge at 5000rpm×1min, discard the supernatant, add 100μL of Solution R1 (Simply P Total RNA Extraction Kit), shake and mix for 30s, let stand at room temperature for 1min, and proceed to the next step. Add 600 μL of Solution R2 (Simply P Total RNA Extraction Kit) to the processed sample, mix thoro...

Embodiment 2

[0087] Example 2: pTG19-(Exendin-4) 2 - Construction of HSA(R410A)-OPT1 recombinant plasmid

[0088]The 410th position of human serum albumin is mutated from R to A, the coding gene is expressed as HSA(R410A)-OPT1, and the nucleotide sequence is the sequence SEQ ID NO:5. The gene encoding the fusion protein is (Exendin-4) 2 -HSA(R410A)-OPT1, the nucleotide sequence is the sequence of SEQ ID NO:7. The fusion protein recombinant plasmid is expressed as pTG19-(Exendin-4)2-HSA(R410A)-OPT1, and its construction process is as follows:

[0089] The expression vector plasmid pPicZαA was cut with XhoI / NotI. The specific conditions are as follows, expression vector plasmid pPicZαA, 25 μL; 10×H buffer 5.0 μL, XhoI 1.0 μL, NotI 1.0 μL, double distilled water 18 μL. The reaction was carried out in a constant temperature water bath at 37°C for 6 hours, and the linearized pPicZαA plasmid DNA with a molecular weight of 3600bp was recovered by agarose gel electrophoresis. pTG19-(Exendin-4...

Embodiment 3

[0103] Example 3: pTG19-(Exendin-4) 2 - Construction of HSA(R410A)-OPT2 recombinant plasmid

[0104] The 410th position of human serum albumin is mutated from R to A, and the codon is optimized. The coding gene is expressed as HSA(R410A)-OPT2, and the nucleotide sequence is the sequence SEQ ID NO:6. The gene encoding the fusion protein is (Exendin-4) 2 -HSA(R410A)-OPT2, the nucleotide sequence is the sequence of SEQ ID NO:8. The fusion protein recombinant plasmid is expressed as pTG19-(Exendin-4) 2 -HSA(R410A)-OPT2, the construction process is as follows:

[0105] The HSA(R410A)-OPT2 gene fragment was obtained by using the human serum albumin gene obtained in Example 1 for genetic modification through multiple rounds of PCR. The specific instructions are as follows:

[0106] The first round of PCR H1-10 fragment amplification

[0107] The pPicZα-HSA plasmid was used as a template, HSAoptU (1-10U), HSAopt (1-9D, HSAdoIIID) as primers for PCR. The specific primers are as ...

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Abstract

The invention provides a fusion protein of Exendin-4 and mutational human serum albumin and a preparation method of the fusion protein, belonging to the technical field of long-acting recombined fusion protein drugs. The invention relates to the fusion protein of Exendin-4 and human serum albumin (HSA) in which the 410-site is mutated to A (Ala alanine) from R (Arg arginine). The fusion protein comprises two polypeptide zones, wherein zone I is composed of two Exendin-4 repetitive sequences, and zone II is site-mutation human serum albumin (HSA); the zone I is directly connected with the N-terminal of the zone II by the C-terminal of the zone I without adding any connecting peptides therebetween; and the structure of the fusion protein is: (Exendin-4)2-HSA (R410A). Compared with Exendin-4 and (Exendin-4)2-HAS, (Exendin-4)2-HSA (R410A) fusion protein provided by the invention has the characteristic of longer-lasting effect in vivo, and can be used for preparing long-lasting preparation of non-insulin-dependent diabetes.

Description

technical field [0001] A fusion protein of insulin secretion-stimulating peptide and mutated human serum albumin and a preparation method thereof belong to the technical field of long-acting recombinant fusion protein drugs. Background technique [0002] Insulin-stimulating peptide (Exendin-4) is a peptide hormone that stimulates insulin secretion. It contains 39 amino acids. It was discovered in the early years from the saliva of the Gila monster that grew in the southwestern United States and the deserts of Mexico. 53% of its amino acid sequence is homologous to the human blood glucose regulating hormone, glucagon-like peptide-1 (GLP-1). The substance has the effect of directly lowering blood sugar and stimulating the secretion of insulin by the pancreas, and is a potential agonist of the GLP-1 receptor (GLP-1R); it can also slow down the absorption of food by slowing down the peristalsis of the gastrointestinal tract, thereby reducing The peak of glucose in the blood. A...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/19A61K38/38A61K38/22A61K47/48A61P5/50A61P3/10C12R1/84
Inventor 吴海洋梁岩郭建辉
Owner SHANGHAI ALLIST PHARM CO LTD
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