Method for extracting high-density lipoprotein and separating and purifying apolipoprotein apoA-I from human plasma

Abstract

The invention provides a method for extracting high-density lipoprotein (HDL) and separating and purifying high-purity apolipoprotein apoA-I from normal human plasma. The method comprises the following steps: (1) regulating plasma to be of different densities by utilizing neutral salt, and eliminating impure protein by utilizing a sequential gradient density supercentrifugation method, so as to obtain a high-purity HDL component; (2) carrying out degreasing on the obtained high-purity HDL component in a flowingly adding manner by utilizing a certain proportion of alcohol ether solvents under a low-temperature freezing state, so as to obtain degreased apoHDL precipitates; (3) redissolving the apoHDL precipitates by utilizing a TBS buffer liquid system containing 1-8M urea, filtering, carrying out separation and purification by molecular sieve chromatography of filtrate and anion exchange chromatography in sequence to obtain a high-purity apoA-I solution. According to the method, albumin components which are very difficult to remove can be separated from the HDL; the apoA-I prepared by utilizing the method has very high purity and activity, the purity can reach above 98%, and the apoA-I can generate clear precipitin reaction with antiserum after being diluted by 64 times; the method is safe and convenient to operate, is short in extraction period and is suitable for industrial production.

Description

technical field [0001] The invention relates to the field of biological technology, and specifically relates to a method for extracting high-density lipoprotein (HDL) from normal human plasma and separating and purifying high-purity apoA-I. Background technique [0002] Epidemiological investigations have shown that plasma high-density lipoprotein (HDL) levels are negatively correlated with the incidence of atherosclerosis (AS), and have an anti-AS effect. [0003] HDL is mainly composed of apolipoprotein and lipid, which is the most dense but extremely heterogeneous type of lipoprotein in plasma. The protein in HDL is mainly composed of apoA-I and apoA-II, and also contains a small amount of apoA-IV, CI, CII, CIII and E, among which apoA-I has the most content and is the main functional protein of HDL, so it is used The content of apoA-I can reflect the content of HDL, and indirectly diagnose the onset of AS. apoA-I also has anti-inflammatory and anti-endotoxin functions,...

AI Technical Summary

Problems solved by technology

[0004] At present, the common methods to obtain HDL are ultracentrifugation, polymer and salt ion sequence precipitation, etc., but these methods have some unavoidable shortcomings: 1) The processing method is cumbersome, because HDL is the highest density in plasma but its composition is extremely heterogeneous A class of lipoproteins with complex components and conditions, HDL cannot be completely separated from other components by precipitation alone, especially albumin, which is the most abundant in plasma, and once albumin is introduced in the HDL extraction stage, it is difficult It will be removed in the subsequent chromatographic purification; 2) The whole process of precipitation method requires the HDL components to undergo multiple liquid phase / solid phase conversion processes. Loss of protein activity, unable to be used later

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