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Bioactive peptide kss1b and preparation method and application thereof

A kss1b, biologically active peptide technology, applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., can solve the problems of high technical threshold, inability to talk about activity, and low degree of product homogeneity.

Active Publication Date: 2018-11-20
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 E. coli preparation, there is a higher technical threshold
At present, there is no report of using E. coli to prepare kss1b, let alone its activity

Method used

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  • Bioactive peptide kss1b and preparation method and application thereof
  • Bioactive peptide kss1b and preparation method and application thereof
  • Bioactive peptide kss1b and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Construction and use of embodiment 1 expression vector pWE-kss1b

[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, 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-kss1b

[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. On 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 solution, 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] Among them, the 5051 auto-induction culture medium can be configured with reference to the document Studier, F.W. "Protein Production by...

Embodiment 3

[0068] Example 3 Preliminary separation and purification of kss1b fusion protein and protein kinase digestion

[0069] The thallus that has been induced in Example 2 was centrifuged at 4500 g for 10 minutes to obtain the thallus. Then add an appropriate volume of PBS buffer to resuspend the bacteria, followed by a high-pressure homogenizer for cyclic disruption. 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 kss1b), digested overnight at 4°C.

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Abstract

The invention belongs to the technical field of polypeptide preparation, and specifically discloses a bioactive peptide kss1b and a preparation method and application thereof. The invention provides an ideal recombinant expression scheme for kss1b using Escherichia coli by selecting an appropriate expression strain and an appropriate vector and adopting automatic induction. 1 mg / L high-purity recombinant kss1b (rkss1b) can be stably obtained by the method, and the amino acid sequence thereof is SEQ ID NO. 11. The rkss1b can significantly inhibit TTX-S type voltage gated sodium channel (VGSC) current on rat DRG cells at a 1 muM level.

Description

technical field [0001] The invention belongs to the technical field of polypeptide preparation, in particular, it relates to bioactive peptide kss1b and its preparation method and application. Background technique [0002] The traditional Chinese medicine centipede is to use the whole body of the giant centipede with few spines of the Centipede family, and the main active component of it is centipede toxin. The centipede toxin has high medicinal value, and has the functions of attacking poison and dispelling stagnation, quenching wind and relieving pain, and anti-cancer. Its medicinal value is highly valued by domestic and foreign medical experts. Centipede toxin contains quite a lot of small peptide molecules, which have different physiological activities and are potential medicinal molecules. However, compared with scorpion toxin, there are fewer researches on the activity of single active peptide molecules derived from centipede in my country. [0003] kss1b is a toxin o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/70C12N15/62C07K1/20C12N15/11
CPCC12N15/70C07K14/00C07K2319/00
Inventor 袁维峰侯绍华贾红郭晓宇鑫婷朱鸿飞
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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