Method for largely and quick extracting tachyplesin peptide

Abstract

The invention relates to a method for largely and quickly extracting tachyplesin peptide. The method comprises the following steps: (1) collecting horseshoe crab blood cells for reservation, suspending the collecting horseshoe crab blood cells in Tris-HCl and NaCl buffer solution homogenate, performing centrifugation for removing supernatant liquor, and washing sedimentation with the same buffer solution twice; (2) depositing, adding acid liquor homogenate according to the ratio of solid to liquid of 1: (6.5-8), performing centrifugation for collecting supernatant liquor, and re-extracting the sedimentation for three times by using acid liquor to obtain acid extraction liquid; (3) adjusting the pH of the acid extraction liquid to 6.5 by using NaOH, performing centrifugation to remove sedimentation, and performing filtration by use a 0.1-0.22 [Mu]m film to obtain supernatant liquor; (4) performing boiling water bath processing on the supernatant liquor, performing centrifugation to remove sedimentation, collecting supernatant liquor, performing filtration by use the 0.1.22 [Mu]m film to obtain supernatant liquor; (5) performing processing on ultrafiltration membrane with the molecular weight cut off of 10 KD; (6) performing processing on ultrafiltration membrane with molecular weight cut off of 3 KD; (7) performing desalination processing; and (8) performing freeze drying on the obtained product to be made into dry powder. The tachyplesin peptide obtained by using the method is high in yield and purity; the process is simplified; the time is greatly shortened; the cost is greatly reduced; and the throughout is increased significantly.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a limulus peptide, in particular to a method for rapidly extracting a large amount of limulus peptide. Background technique [0002] Limulus, also known as horseshoe crab, belongs to the marine arthropods of Merostomata (Merostomata) and Xiphosura (Xiphosura). There are 4 species in total, which have high medicinal value. Limulus blood cell extract "Limulus reagent" can accurately and quickly detect whether the internal tissues of the human body are infected with bacteria; in the pharmaceutical and food industries, it can be used to monitor toxin pollution. The meat and eggs of horseshoe crab are edible, and its shell, tail, eggs, meat and blood can be used as medicine. [0003] Limulus peptide is derived from Limulus blood cell extract and is a polypeptide containing 17 amino acid residues. Amino acid sequence: NH 2 -K-W-C-F-R-V-C-Y-R-G-I-C-Y-R-R-C-R-CONH 2 . Contains two pairs of...

AI Technical Summary

Problems solved by technology

Among the three methods, the genetic engineering method is the most advanced, and it does not need to take blood from the horseshoe crab once and for all. However, this method requires technical difficulties, cumbersome steps, expensive reagents and equipment, and the constructed clones are easily destroyed during the expression process. The host is additionally modified, and the activity of the target protein is not ideal

The loading amount of gel chromatography is 1-5% of the volume of the column bed. According to the calculation, the loading volume of this method is 1.59ml-7.85ml, and it takes 1100 minutes (18.3h) to collect the elution peak, and it cannot be operated continuously. , without the second step of ion exchange chromatography, the disadvantages of long time consumption and small processing volume are already obvious

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