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ae1d active peptide and its preparation method and application

A technology of active peptide, 1.ae1d, which is applied to the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve the problems of high technical threshold and low degree of product homogeneity

Active Publication Date: 2021-12-14
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of Pichia pastoris has the problem of low homogeneity of the product, and compared with the preparation of E. coli, there is a higher technical threshold
There is no report on the use of E. coli to produce Ae1d

Method used

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  • ae1d active peptide and its preparation method and application
  • ae1d active peptide and its preparation method and application
  • ae1d active peptide and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction and use of expression vector pWE-Ae1d

[0045] Using the commercialized pET-43a vector (purchased from Novagen) as a template, a PCR-based seamless cloning method was used to obtain the target plasmid. To put it simply, using the homologous sequence (Tm>60 degrees) between the fragment and the vector, using a commercially available high-fidelity DNA polymerase, the target gene is integrated into the relevant vector by PCR.

[0046] PCR system: 1× buffer, 10pmol primers, 0.5mM dNTP, 1mM MgSO 4 , 1U DNA polymerase and 1ng-60ng template DNA.

[0047] The PCR reaction conditions were: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 68°C (2 kb / min), a total of 30 cycles.

[0048] The original NusA sequence was replaced by the common PCR-based seamless cloning method in the art, and then the sequence encoding SUMO (SEQ ID NO.2) was inserted downstream of 6×His, and finally the sequence en...

Embodiment 2

[0065] Example 2 Induced Expression of Expression Plasmid pWE-Ae1d

[0066] Using the conventional calcium chloride transformation method, the expression plasmid was transformed into E. coli SHuffle TM , and then spread it on ampicillin-resistant (100 μg / mL ampicillin) LB solid medium, and place it upside down at 37°C for 16 hours. The next day, pick 1-2 single colonies, inoculate them in 5 mL of ampicillin-resistant 505 culture solution, and cultivate them at 37°C and 220 rpm for 8 hours (this step is the enrichment of the seed medium). Transfer 5 mL of seed culture medium into fresh 500 mL of 5051 auto-induction (100 μg / mL ampicillin) culture medium, and continue culturing at 30°C and 220 rpm. Choose SHuffle TM strain because it can provide intracellular oxidative conditions that promote the folding of disulfide bonds.

[0067] Wherein, the 5051 auto-induction culture medium can be configured with reference to the document Studier, F.W. "Protein Production by Auto-Inducti...

Embodiment 3Ae1

[0068] Example 3 Preliminary Separation and Purification of Ae1d Fusion Protein and Protein Kinase Digestion

[0069] The thalline that the induction in embodiment 2 is completed, 4500g centrifugal 10 minutes, obtain thalline. Then add an appropriate volume of PBS buffer to resuspend the thalline, followed by high-pressure homogenizer circulation and crushing. After the crushing is completed, centrifuge at 13000rpm for 40 minutes to obtain supernatant and precipitate. The supernatant was purified by nickel column affinity chromatography, and the PBS solution eluate of 200mM imidazole was collected, concentrated and desalted by a 10kDa pore size ultrafiltration tube, and the tube solution in the ultrafiltration tube was collected and an appropriate dose of ULP1 kinase was added (for releasing Ae1d), digest overnight at 4°C.

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Abstract

The invention belongs to the technical field of polypeptide preparation, and specifically discloses the Ae1d active peptide and its preparation method and application. The present invention provides an ideal recombinant expression scheme for Ae1d using Escherichia coli by selecting a suitable expression strain and a suitable carrier and adopting an automatic induction method. This method can stably obtain 0.55 mg / L highly pure recombinant Ae1d (rAe1d), the amino acid sequence of which is shown in SEQ ID NO.11. rAe1d can significantly inhibit voltage-sensitive potassium channel (VGPC) current on rat DRG cells at 0.5 μM level.

Description

technical field [0001] The invention belongs to the technical field of polypeptide preparation, in particular, it relates to Ae1d active peptide and its preparation method and application. Background technique [0002] Compared with small molecular drugs, peptide drugs have better selectivity and no accumulation in the body, and are more and more favored by major pharmaceutical companies. Ants, as a species as old as dinosaurs, record the use of ants for treating diseases in "Compendium of Materia Medica", and also have detailed descriptions in the dictionary "Erya". Ants have very high medicinal value, and are called micro-animal nutrition treasure house and natural medicine processing plant, which can not only keep fit but also cure diseases, and are very suitable for development and utilization as a new medicinal resource. But at present, the development of ants is only limited to the whole medicine, and the biological activity of its single component has not been studie...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C12N15/70C12N15/66C07K1/34C12N15/11
CPCC12N15/66C12N15/70C07K7/08
Inventor 袁维峰郭晓宇鑫婷贾红侯绍华朱鸿飞
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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