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Purification method of protein group of 30 kD in silkworm pupa

A purification method and silkworm chrysalis technology, applied in the field of protein purification, can solve the problems of high cost, low purification efficiency, complicated and cumbersome purification steps for 30kD protein groups, etc., and achieve the effects of reducing damage and loss and saving manpower and material resources.

Active Publication Date: 2014-07-09
TIANJIN YAOYU BIOLOGICAL TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] In order to solve the defects of complex and cumbersome purification steps of 30kD protein group in the prior art, high cost and low purification efficiency, the present invention provides a new purification method of 30kD protein group in silkworm chrysalis, which is mainly based on the molecular weight of the protein, through simple The 30kD protein group obtained by ammonium sulfate fractionation precipitation, centrifugation and ultrafiltration membrane package ultrafiltration method

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  • Purification method of protein group of 30 kD in silkworm pupa
  • Purification method of protein group of 30 kD in silkworm pupa
  • Purification method of protein group of 30 kD in silkworm pupa

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Embodiment 1

[0023] PBS formulation: 137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , 2mM KH 2 PO 4 , pH7.4~7.6.

[0024] 1. Homogenization and centrifugation

[0025] Take 100g silkworm chrysalis and homogenize with 1000mL pre-cooled PBS; centrifuge the homogenate at 0~2℃, 12000rpm for 30min, and collect the supernatant.

[0026] 2. Fractional precipitation of ammonium sulfate

[0027] (1) Make the supernatant to volume, according to the ammonium sulfate saturation calculator, add a certain amount of solid (NH 4 ) 2 SO 4 , to solution (NH 4 ) 2 SO 4 The saturation is 25%, stirred on a magnetic stirrer at 4°C for 30 minutes, and then rested at 4°C for 90 minutes. Centrifuge at 12000 rpm for 30 min at 4°C and collect the supernatant.

[0028] (2) Make the supernatant to volume, according to the ammonium sulfate saturation calculator, weigh a certain amount of solid (NH 4 ) 2 SO 4 , to solution (NH 4 ) 2 SO 4 The saturation is 65%, stirred on a magnetic stirrer at 4°C for 30 minu...

Embodiment 2

[0033] The present embodiment sets three different ammonium sulfate salting-out saturations and carries out three groups of comparison experiments, and each group of experiments carries out the following operations respectively:

[0034] PBS formulation: 137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , 2mM KH 2 PO 4 , pH7.4~7.6.

[0035] 1. Homogenization and centrifugation

[0036] Take 100g of silkworm chrysalis freeze-dried powder (provided by Zhejiang Zhongqi Biomedical Co., Ltd.), and 1000mL pre-cooled PBS homogenate; the homogenate is centrifuged at 0~2°C, 12000rpm for 30min, and the supernatant is collected.

[0037]2. Fractional precipitation of ammonium sulfate

[0038] (1) Make the supernatant to volume, according to the ammonium sulfate saturation calculator, add a certain amount of solid (NH 4 ) 2 SO 4 , to solution (NH 4 ) 2 SO 4 The saturation is 25%, stirred on a magnetic stirrer at 4°C for 30 minutes, and then rested at 4°C for 90 minutes. Centrifuge at 1...

Embodiment 3

[0044] PBS formulation: 137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , 2mM KH 2 PO 4 , pH7.4~7.6.

[0045] 1. Homogenization and centrifugation

[0046] Take 100g silkworm chrysalis freeze-dried powder (provided by Zhejiang Zhongqi Bio-Pharmaceutical Co., Ltd.), 1000mL pre-cooled (refrigerated at 4°C) PBS homogenate three times, each time for 1min, and the middle homogenate cup was cooled on ice for 1min; Centrifuge at 8000 rpm for 30 min, filter the supernatant with 9 layers of gauze, repeat three times, and collect the supernatant.

[0047] 2. Fractional precipitation of ammonium sulfate

[0048] (1) Make the supernatant to volume, according to the ammonium sulfate saturation calculator, add a certain amount of solid (NH 4 ) 2 SO 4 , to solution (NH 4 ) 2 SO 4 The saturation is 25%, stirred on a magnetic stirrer at 4°C for 30 minutes, and then rested at 4°C for 90 minutes. 4°C, 8000rpm, centrifuge for 30min, filter through 9 layers of gauze, and collect the supernata...

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Abstract

Disclosed is a purification method of a protein group of 30 kD in silkworm pupa, belonging to the technical field of purification of protein. The method comprises the purification steps: carrying out cell disruption on silkworm pupa, taking supernatant to conduct ammonia sulfate fractional precipitation after centrifugation of cell disruption liquid, suspending the sediment again and utilizing an ultrafiltration membrane to filter substances having a molecular weight of 80kD to 100kD, utilizing the ultrafiltration membrane to filter flow liquid having a molecular weight of 30kD to 50kD, collecting retaining liquid, and finally obtaining the protein group of 30 kD. The purification method of the present invention reduces damage and loss on interest protein, and the simplest steps can be utilized to obtain large amount of purified interest protein. The purification method is simple in operation and low in cost.

Description

technical field [0001] The invention relates to the technical field of protein purification, in particular to a method for purifying 30kD protein group in silkworm chrysalis. Background technique [0002] Silkworm chrysalis protein is a high-quality protein resource, containing 18 kinds of amino acids, of which 8 kinds of essential amino acids for the human body exceed 40% of the total amino acids, and its protein is easily hydrolyzed and digested by the human body, so it is an ideal source of protein. Studies have shown that silkworm chrysalis proteins and amino acids have special physiological activities, and their polypeptides have the effects of lowering serum cholesterol and antioxidation. According to reports, silkworm chrysalis compound amino acids have various physiological activities, such as significantly promoting the weight growth of young male rats, the ability of blood to transport oxygen and nutrients, promoting the body's protein synthesis, accelerating metab...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/36C07K1/34C07K1/30
CPCC07K14/43586C07K1/36
Inventor 张耀洲郭志超王鉴杜瑶瑶陈剑清舒特俊
Owner TIANJIN YAOYU BIOLOGICAL TECH
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