Method for largely and quick extracting tachyplesin peptide
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A limulus peptide and rapid technology, applied in the field of bioengineering, can solve the problems of unsatisfactory target protein activity, inability to operate continuously, technical difficulty, etc., and achieve obvious bacteriostatic effect, increase processing capacity, and simplify the effect of process.
Active Publication Date: 2013-09-04
BEIHAI SINLON BIOTECH CO LTD
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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
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Embodiment 1
[0066] The lysate of Limulus blood cells is prepared by using a tangential flow ultrafiltration system to prepare Limulusin peptides. The process steps are as follows:
[0067] (1) Raw material processing: extract about 1000mL of fresh Limulus blood with sterile non-pyrogenic equipment, centrifuge at 1500 rpm for 5 minutes, collect Limulus blood cells, weigh 100g Limulus blood cells (wet weight), suspend Limulus blood cells in 200mL, 20mmol / L Tris-HCl, PH8.8 containing 50mmol / L NaCl buffer for homogenization, then centrifuged at 8000r / min for 20min, discard the supernatant, and wash the collected precipitate twice with the same buffer;
[0068] (2) Acid hydrolysis: After washing, the precipitate was suspended in 700mL, 20mmol / L HCl and homogenized, the homogenate was centrifuged at 8000r / min, 4°C for 20min, and the supernatant was collected. Supernatant, and finally discard the precipitate, about 1200ml of the supernatant collected three times is the required limulus blood cel...
Embodiment 2
[0087] The process steps of preparing limulus peptide by using the ultrafiltration tube to prepare the remaining waste from the production of Limulus reagent are as follows:
[0088] (1) Raw material processing: collect the cell lysate mass produced in the production process of the Limulus reagent and weigh 10g (wet weight), suspend the Limulus blood cells in 50mL of 20mmol / L Tris-HCl, pH8.8 containing 50mmol / L NaCl buffer for homogenization Then centrifuge at 8000r / min for 20min, discard the supernatant, and wash the collected precipitate twice with the same buffer;
[0089] (2) Acid hydrolysis: After washing, the precipitate was suspended in 70mL, 20mmol / L HCl and homogenized, the homogenate was centrifuged at 8000r / min, 4°C for 20min, and the supernatant was collected. Supernatant, and finally discard the precipitate, about 120ml of the supernatant collected three times is the required limulus blood cell acid extract;
[0090] (3) Adjust the pH value to remove precipitatio...
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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...
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