Bombyx natural immune protein Hemolin and purifying method thereof

A technology of natural immunity and purification method, which is applied in chemical instruments and methods, peptide preparation methods, organic chemistry, etc. It can solve the problems of large mobile phase consumption, limited purification amount, and large initial investment, so as to prevent inactivation and denaturation Effect

Inactive Publication Date: 2014-09-24
TUKE TIANJIN PHARMA TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although people generally use preparative HPLC for the final step of purification of genetic engineering products, because HPLC has high requirements for hardware and software, large initial investment, limited purification amount, high price, and difficulty in analyzing biological macromolecules and inorganic ions, mobile phase Disadvantages such as large consumption and toxicity

Method used

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  • Bombyx natural immune protein Hemolin and purifying method thereof
  • Bombyx natural immune protein Hemolin and purifying method thereof
  • Bombyx natural immune protein Hemolin and purifying method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1: Preparation of silkworm immune protein mother solution

[0041] Step 1: Silkworm chrysalis stored at -20°C (purchased from Zhejiang Zhongqi Bio-Pharmaceutical Co., Ltd.) and sterilized 4°C pre-cooled ddH 2 O was mixed at a weight percentage of 1:3 and homogenized on ice for 2 min, and the homogenate was placed on ice for 5 min, and repeated 9 times;

[0042] Step 2: Centrifuge at 15,000 rpm at 4°C for 1 hour, discard the supernatant, resuspend the pellet in water, then centrifuge at 15,000 rpm at 4°C for 1 hour, repeat this 5 times, and filter with 4 layers of gauze for the last time;

[0043] Step 3: Take 200mL of the filtered supernatant and mix it with water at a volume of 1:1, filter with a 0.22μm membrane bag, repeat 10 times, and the supernatant solution returns to the volume of 200mL;

[0044] Step 4: After the obtained supernatant was centrifuged at 50,000 rpm at 10°C for 40 minutes, the black mass that appeared was resuspended in ultrafiltrate fo...

Embodiment 2

[0046] Embodiment 2: Separation and purification of natural immune protein of silkworm

[0047] (1) set the activity higher than 2 6 (The protein content of the sample was detected by the BCA method, activity=2n×4 / sample concentration (mg / mL), n=the number of wells where erythrocyte agglutination occurred) took 200mL and the desugared component at a volume ratio of 1:10 and passed the macro The method of aspiration is to remove sugar by passing through a 300K membrane bag. The final volume was restored to 200mL;

[0048] (2) Mix the desugared mother liquor and protein extract at a volume ratio of 1:2.5, and slowly stir overnight in a refrigerator at 4°C;

[0049] (3) Preliminary separation through a weak anion exchange column (DEAE, purchased from GE Healthcare): mix the protein sample obtained in the above step (2) with DEAE at a volume ratio of 2:1, and store in a refrigerator at 4°C After stirring slowly for 2 hours, pour the DEAE filler into the column; balance the colu...

Embodiment 3

[0052] Embodiment 3: the mass spectrometric identification of silkworm natural immune protein Hemolin

[0053] 1. Sample preparation: After tapping the second strip of target protein No. 47 in Figure 4A, cut it into 1mm 3 Left and right small pieces were put into centrifuge tubes. Send it to a mass spectrometry company. Note: Ensure the cleanliness of the sample and all items in contact with it to prevent contamination by impurities and other pollutants.

[0054] 2. The result of mass spectrometry is: the above-mentioned sample is identified as the natural immune protein Hemolin of silkworm, such as Figure 7 As shown, it is a first-order mass spectrum, such as Figure 8 As shown, it is a secondary mass spectrogram; the peptide fingerprint of the protein and the protein number gi69146821 in the protein library of Southwest Agricultural University have a coverage rate of 49%, and its amino acid sequence is obtained as Figure 9 And as shown in SEQ ID NO.1, it is proved that...

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Abstract

The invention relates to a bombyx natural immune protein Hemolin and a purifying method of the bombyx natural immune protein Hemolin and belongs to the field of protein purifying. Silkworms are taken as raw material, a component containing immune proteins is obtained with different centrifugation methods and then through ultrafiltration, supernatant liquid containing the target immune protein is subjected to separation and purification through a simple chromotography technique in an AKTA explorer 10 protein purification system, and the target immune protein Hemolin is obtained. The purifying method of the bombyx natural immune protein Hemolin mainly use Superdex 200 and Superdex 75 gel chromatography technique, provides a novel solution method for natural immune protein purification, and is of significant meaning.

Description

technical field [0001] The invention relates to a silkworm natural immune protein Hemolin and a purification method thereof, belonging to the technical field of proteins. Background technique [0002] As a kind of defense protein unique to insects, Hemolin is of great significance in the study of the immune mechanism of insects. At present, the research on its structure and function has achieved certain results, but there are few researches on its function in vitro. Therefore, this experiment obtained relatively pure Hemolin through simple purification, laying the foundation for further research on the structure and function of the protein. It is also possible to explore its structure more deeply on this basis. [0003] Because the components of genetic engineering products are complex, the purity requirements of the products are high, and the protein tends to aggregate and denature during the purification process, its preparation and purification are much more difficult th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/36C07K1/34C07K1/18C07K1/16
Inventor 张耀洲杜瑶瑶刘玲玲陈剑清舒特俊
Owner TUKE TIANJIN PHARMA TECH CO LTD
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