Method for separating III type precollagen amino terminal peptides

A kind of original amino, procollagen technology, applied in the field of separation of type III procollagen amino terminal peptide

Inactive Publication Date: 2011-06-15
BEIJING NORTH INST OF BIOLOGICAL TECH
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Problems solved by technology

[0007] The present invention is to solve the problems of extraction method, e

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  • Method for separating III type precollagen amino terminal peptides

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Embodiment Construction

[0008] In order to obtain antibodies with good immune activity, PIIINP was extracted from human cancerous ascites and immunized rabbits. Take 2 liters of ascites from cancerous patients and centrifuge at 10,000 RPM to remove tissue blocks and cancerous cells; this step must wear protective gloves and glasses, because most liver cancer patients carry hepatitis virus;

[0009] Collect the precipitates whose concentration of ammonium sulfate reaches 40-60% (varies according to different patients) in stages, add sulfur Stir overnight after acid ammonia, centrifuge at 10,000 RPM, collect the precipitate, and discard the supernatant;

[0010] As the content of ascites miscellaneous protein reaches 80 mg / ml, but the effective content is 60-600 ng / ml, which is only one part per million, so it is impossible to separate trace pIIINP directly by column chromatography. According to sequence analysis, the isoelectric point of PIIINP is ~5.71. Therefore, dialyze the above precipitate wi...

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Abstract

The invention relates to a method for separating III type precollagen amino terminal peptides, which solves the problems of an extraction method, the efficiency, the purity and the activity of the III type precollagen amino terminal peptides. An ammonia sulfate fractionation method is adopted, so that the quantity of impurities is reduced by 5.5 percent, and the yield is increased by 11.7 percent in a first step; Qsepharose FF (Qsepharose Fast Flow) is adopted, so that the loading amount is large, the flow rate is high, and further separation is achieved; a buffer solution with the pH value of 4.5 and a buffer solution with the pH value of 8.5 are not used for eluting alternatively, but hydrophobic chromatography which plays a role in protecting proteins is adopted, so that purity and activity are greatly enhanced; Sephacryl 200 is replaced by high-resolution Superdex 200, so that the purity is further enhanced; and by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) analysis, the purity is up to 98 percent, and the activity is 10 times that of the prior art.

Description

technical field [0001] The present invention relates to a method for isolating amino terminal peptide of type III procollagen. Background technique [0002] The existing purification methods all use DEAE-Sephacel ion exchange columns and Sephacryl molecular sieves. It has been verified that the purification efficiency of this method is not high, and the impurity protein is inseparable from the active peak, and the activity of the purified pIIIINP is not high, and the activity drops extremely fast. The purity cannot meet the requirements of high-precision experiments. The most important thing is that the yield is too low. According to calculation (RIA-Gnost Prokollagen-III-Peptid), the effective content of PIIINP needs to be more than 100mg to purify 95% of PIIINP1mg by this method. [0003] The first step of adding (NH4)2SO4 without fractionation will bring most of the impurities to the subsequent stage, and this step can only remove 10% of the impurities. [0004] [0...

Claims

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Application Information

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IPC IPC(8): C07K1/36C07K1/30C07K1/20C07K1/18C07K1/16
Inventor 赵玉军
Owner BEIJING NORTH INST OF BIOLOGICAL TECH
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