P-5m-Fc fusion protein and expression gene thereof, and preparation method and applications of P-5m-Fc fusion protein

A technology for fusion protein and gene expression, applied in the field of medicine and biology, can solve the problems of short half-life and poor stability, and achieve the effects of prolonging half-life, improving drugability and improving drug efficacy

Active Publication Date: 2016-10-26
BEIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, after the administration of P-5m octapeptide, it has the defects of poor stability and short half-life, and it is still difficult to apply in clinical practice.

Method used

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  • P-5m-Fc fusion protein and expression gene thereof, and preparation method and applications of P-5m-Fc fusion protein
  • P-5m-Fc fusion protein and expression gene thereof, and preparation method and applications of P-5m-Fc fusion protein
  • P-5m-Fc fusion protein and expression gene thereof, and preparation method and applications of P-5m-Fc fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Construction of expression vector of P-5m-Fc fusion protein

[0058] 1. Method.

[0059] 1. Extraction and purification of bacterial plasmids

[0060] After analysis, the amino acid sequence of P-5m is GHGKHKNK. After screening through a large number of experiments, the inventor synthesized the positive and negative chains of the P-5m peptide gene by using the codons preferred by Escherichia coli, adding ATG to the 5' end and 6 to the 3' end. Glycine codons (GGT GGT GGT GGT GGT GGT part below), and in order to increase the titer, the P-5m peptide gene and glycine codons were repeated for two cycles, after annealing, the 5' end and the 3' end were respectively Generate Nde I recognition site (CAT ATG) and BamH I recognition site (GGA TCC): positive strand 5'-C CAT ATG GGC GAT GGC AAA GAT AAG AAC AAG GGT GGT GGT GGTGGT GGT GGC GAT GGC AAA GAT AAG AAC AAG GGA TCC GCG-3' (the nucleotide sequence shown in SEQ ID NO.2); 5'-CGC GGA TCC CTT GTT CTT ATC TTT GCC ATC GCC CAT ACC...

Embodiment 2

[0085] Expression and purification of recombinant protein in host bacteria

[0086] 1. Method.

[0087] 1. Conversion

[0088] The positive recombinant plasmid p was transformed into E. coli competent BL21 (DE3) by heat shock method, and cultured on LB resistance (containing 50 μg / ml ampicillin) agar plate at 37°C for 12-16h. Pick the recombinant plasmid transformed bacteria of a single strain, inoculate 10ml of LBG medium containing 50μg / ml ampicillin, and culture at 37°C until OD 600 When the concentration reaches 0.4-0.6, add IPTG to a final concentration of 0.3mmol / L, and continue culturing at 200rpm for 3.5h.

[0089] 2. Induced expression

[0090] Take 1ml of the above-mentioned induced product, centrifuge at 5000rpm to induce bacterial culture, discard the supernatant, add 100μL PBS to the precipitate and mix well, add 100μL of 2×SDS loading buffer and mix well, boil for 5min, centrifuge at 8000rpm for 5min at 4°C, take 20μL of supernatant Perform SDS-PAGE electroph...

Embodiment 3

[0123] Activity detection of P-5m–Fc fusion protein

[0124] 1. Western-blotting test

[0125] The protein bands (Fc and P-5m-Fc fusion protein) analyzed by SDS-PAGE were transferred to PVDF membrane, blocked with 1% BSA overnight at 4°C, washed 3 times with 0.05% PBST, 10 min each , join 2000 × Diluted positive serum, acted at 37°C for 2 hours, washed 3 times with 0.05% PBST, 10 minutes each time, added 25000 × Diluted rabbit anti-human IgG enzyme-labeled antibody, washed 3 times with 0.05% PBST, 10 minutes each time, added PIERCE luminescent substrate to react at room temperature for 3 minutes, exposed in the dark room, fixed after developing, the result is as follows Image 6 shown.

[0126] The above results indicated that Fc could not bind to P-5m polyclonal antibody, but P-5m-Fc could specifically bind to P-5m polyclonal antibody.

[0127] 2. Indirect ELISA test

[0128] 1. Preparation of polyclonal antibody against P-5m octapeptide

[0129] (1) Antigen preparation...

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Abstract

The invention relates to a P-5m-Fc fusion protein and an expression gene thereof, and a preparation method and applications of the P-5m-Fc fusion protein, and belongs to the technical field of medicine and biology. The P-5m-Fc fusion protein is formed by linking a functional unit and the Fc portion of human immunoglobulin IgG1, wherein the functional unit is formed by linking an amino acid sequence represented by SEQ ID NO.1 and a connexon, the connexon is a polypeptide formed by six sequentially-linked glycines, the P-5m-Fc fusion protein has at least one functional unit, and when the P-5m-Fc fusion protein has a plurality of the functional units, the plurality of the functional units are sequentially linked, and then are linked to the Fc portion of immunoglobulin. According t the present invention, with the P-5m-Fc fusion protein, P-5m and the Fc portion of the human IgG1 are subjected to fusion expression to prepare the peptide, such that the efficacy of the P-5m octapeptide can be increased, the half-life of the drug can be significantly prolonged, the good patent medicine property is obtained, and the experiment results prove that the significant anti-tumor-metastasis function is provided.

Description

technical field [0001] The invention relates to the technical field of medicine and biology, in particular to a P-5m-Fc fusion protein and its expression gene, preparation method and application. Background technique [0002] Current research shows that tumor metastasis is the root cause of cancer patients' death. P-5m octapeptide is a polypeptide with eight amino acids derived from the fifth domain (D5) of macromolecular kininogen, which is the shortest polypeptide derived from D5 to effectively anti-tumor metastasis found so far. [0003] Studies have shown that P-5m octapeptide can inhibit the adhesion and invasion of mouse melanoma cells at the cellular level, and can inhibit the lung metastasis of mouse melanoma, and has the effect of inhibiting tumor cell metastasis in human HCCLM3 liver cancer cells . [0004] However, P-5m octapeptide has the disadvantages of poor stability and short half-life after administration, and it is still difficult to be applied clinically...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/57A61K47/48A61P35/04
CPCA61K38/00C07K14/8139C07K2319/30C12N15/62C12N15/70C12N2800/101
Inventor 韩笑杜培革安丽萍徐广宇孙静波苑广信郭笑赵南晰王曼力李洪宇盛瑜
Owner BEIHUA UNIV
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