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Crystal structure and application of recombinant apolipoprotein J and analogues thereof

Apolipoprotein domain technology, applied in the field of preparation of recombinant apolipoprotein J, can solve the problem of limited source of apolipoprotein J, can not be used in pharmaceuticals, etc., achieve obvious immunological activity, good application value, not easy to immunize The effect of the primordial reaction

Inactive Publication Date: 2021-01-12
厦门德馨尚品医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although apolipoprotein J is widely present in the human body at present, the source of this natural apolipoprotein J is extremely limited; commercialized recombinant apolipoprotein J is without exception all tagged (TAG), and these tagged Proteins cannot be used in pharmaceuticals

Method used

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  • Crystal structure and application of recombinant apolipoprotein J and analogues thereof
  • Crystal structure and application of recombinant apolipoprotein J and analogues thereof
  • Crystal structure and application of recombinant apolipoprotein J and analogues thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Extraction of Recombinant Apolipoprotein J

[0038] 1.1 Bacterial fragmentation

[0039] (1) Sufficiently resuspend each 1g of bacteria (including recombinant SEQ ID.NO10 sequence) with 10mL suspension solution, centrifuge at 8000rpm, 4°C for 15min, and discard the supernatant; (2) Resuspend with 10mL / g suspension solution again Suspend the bacteria, place the bacteria suspension under the probe of the ultrasonic breaker, power: 400W, ultrasonic 3s, intermittent 6s, and the total time is 15min to break until the break is complete. The construction of the bacterium is a prior art, and will not be repeated here. Preferably, the suspension solution: 50mM Tris-HCl, pH 8.0.

[0040] 1.2 Inclusion body washing

[0041] (1) Centrifuge the crushed bacteria at 15000g at 4°C for 30nim, discard the supernatant, wash with washing solution, add 10mL of washing solution per gram of bacteria, resuspend fully after adding the washing solution, and shake at 4°C Incubate fo...

Embodiment 2

[0057] APO-J (concentration 0.3 mg / mL) corresponding to the partial sequence (SEQ ID. NO3, 4, 7, 11, 12, 15, 16, 18, 19, 22, 23, 26, 27) after SEC chromatography SEC-HPLC detection was carried out, and the results are shown in Table 1. The detection conditions are: TSKGel chromatographic column (300mm×7.8mm), packing particle size 5μm; the experimental instrument is Agilent 1200-DAD GR11010670, the mobile phase is 100mM PBS+200mM urea, pH 6.7; the injection volume is 80μl; the flow rate is 0.7ml / min .

[0058] Table 1 The purity of APO-J corresponding to different sequences by SEC chromatography

[0059] corresponding sequence Sample purity, % Impurities, % corresponding sequence Sample purity, % Impurities, % SEQ ID. NO3 94.13 5.86 SEQ ID. NO18 96.12 3.85 SEQ ID. NO4 94.67 5.31 SEQ ID. NO19 95.21 4.76 SEQ ID. NO7 95.11 4.87 SEQ ID. NO22 94.97 5.01 SEQ ID. NO11 93.91 6.05 SEQ ID. NO23 94.38 5.58 SEQ ID. NO12...

Embodiment 3

[0061] Example 3 Optimizing the renaturation process

[0062] 3.1 Effect of different renaturation processes on sample stability

[0063] Experimental procedure: Take 6ml of the sample after cation exchange chromatography in Example 1, add different refolding buffers at a volume ratio of 1:16, add to the sample, incubate at 4°C for 4h, concentrate directly or desalt to 20mM PB , concentrated to 3-5mg / ml after pH 7.4, incubated with 3 times the mass of SKL at room temperature for 16 hours and passed Superdex 200pg, the mobile phase was 10mM PB, 200mM Na-citrate, 1% SKL, pH7.4. Place the sample at 4°C, 37°C, and RT (room temperature), take samples on day 0, day 1, day 2, day 3, and day 4, inject 0.5ml, and measure the stability of the sample. The results are shown in Table 3, Table 4. At the same time, each group of samples was repeatedly frozen and thawed 1, 2, 3, and 4 times, and the purity of the samples was measured after each freezing and thawing; the repeated freezing an...

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Abstract

The invention provides a crystal structure and application of recombinant apolipoprotein J and analogues thereof. The primary sequence of amino acid is arbitrary one of SEQ ID NO. 1-28. The recombinant apolipoprotein J has a space structure without an artificially added structure, cannot easily induce an immunogenic reaction, has no other toxic or side effect, and has strong druggability; Meanwhile, the purified product has remarkable immunological activity, biological activity and pharmaceutical effect, can be used for effectively treating diseases of tested animals, and has a very good application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant apolipoprotein J, a preparation method and an application. Background technique [0002] Apolipoprotein was first isolated from the testicular rete fluid of rams. Because it can promote the aggregation of Sertoli cells and support the maturation and development of sperm, it is also named as an aggregation factor. It is widely expressed in tissues and is associated with various physiology and pathology. The process is closely related, including apolipoprotein A-I, B, E, J. So far, the functions of overloaded lipoproteins have been reported to include inhibition of apoptosis (Kowolik et al. 2006), inactivation of complement factors (Correa-Rotter et al., 1992), lipid recycling and transport (Gelissen et al., 1998), the protection of cell membranes, and the maintenance of connections between cells or between cells and the matrix. [0003] Apolipoprotein J (apolipoprotein...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/775C07K1/18C07K1/22C07K1/20C07K1/16C07K1/34C07K1/30C07K1/113C07K1/36C07K1/14A61K38/17A61P17/00A61P17/02A61P39/00A61P37/04
CPCA61K38/00A61P17/00A61P17/02A61P37/04A61P39/00C07K14/775
Inventor 宇文镐陈翔杜佩云
Owner 厦门德馨尚品医疗科技有限公司
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