Allophycocyanin (APC) and Annexin V protein coupling method

Abstract

The invention discloses an allophycocyanin (APC) and Annexin V protein coupling method. The APC and Annexin V protein coupling method comprises the following steps: 1, an Annexin V protein is expressed and purified: specifically, 1.1, a gene sequence is optimized, specifically the gene sequence of the human Annexin V protein is looked up, the human Annexin V gene sequence is optimized, a termination codon sequence is removed, the human Annexin V gene sequence is connected into a prokaryotic expression vector, front and back His tag proteins are retained, and preparation is made for protein purification and subsequent coupled fluorescein purification; and 1.2, the Annexin V protein is subjected to inducible expression, specifically, a recombinant plasmid single colony is picked and inoculated into an LB liquid culture medium containing kanamycin, and culturing is conducted until the concentration OD600 of bacterial liquid is 0.4-0.6, and an inducer is added into the bacterial liquid forinduction culture. When the Annexin V gene sequence is designed, His tags at the two ends of the protein are reserved, the Annexin V protein and an Annexin V-APC conjugate are convenient to purify, only conventional nickel column purification is needed, special purification equipment is not needed, the purification steps are few, the purification time is short, the purification cost is lowered, the good biological activity of the protein is maintained, and the Annexin V protein can be used for cell apoptosis detection.

Description

technical field [0001] The invention relates to a coupling method of allophycocyanin (APC) and Annexin V protein. Background technique [0002] Annexin V is a human endogenous protein, which belongs to the calcium ion-dependent phospholipid-binding protein family, has anticoagulant effect, is widely distributed, and is expressed in various tissue cells (such as cardiomyocytes, liver cells, endothelial cells, etc.). AnnexinV is encoded by the AnnexinV gene located on the fourth human chromosome 4q26-q28 on the chromosome, with a total of 320 amino acids, including repeating units of 70-80 amino acids, and a molecular weight of about 36kD. In the process of cell apoptosis, the apoptotic cell membrane changes, and the migration of PS from the inner layer of the lipid bilayer to the outer layer of the cell membrane is the initial event of the apoptosis cascade reaction. AnnexinV protein has a high affinity with PS on the cell membrane. Therefore, the use of AnnexinV high-affini...

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Problems solved by technology

[0006] Molecular sieve high-pressure liquid chromatography column purification has high purification purity and high resolution efficiency, but because it can only be purified in a small amount and cannot be mass-produced, there are great limitations in production;

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