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Recombinant Scolotoxin-Ssm2.1 gene and expression and purification method and application thereof

An expression purification and anti-insect technology, which is applied in the field of DNA recombination, can solve the problems of weak research on centipede anti-insect toxin and no recombinant expression of centipede anti-insect toxin gene, and achieve strong neurotoxicity and lethal effects

Active Publication Date: 2014-05-14
JIANGSU INSTITUTE OF EDUCATION
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  • Abstract
  • Description
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Problems solved by technology

However, compared with the in-depth research on nearly a hundred anti-insect toxins in spiders and scorpions, the research on centipede anti-insect toxins is quite weak (E.F.Schwartz, C.B.F.Mourao, K.G.Moreira, T.S.Camargos, M.R.Mortari, Pept Sci. 2012 . 98(4):385–405.), so far there is no case of recombinant expression of centipede anti-insect toxin gene

Method used

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  • Recombinant Scolotoxin-Ssm2.1 gene and expression and purification method and application thereof
  • Recombinant Scolotoxin-Ssm2.1 gene and expression and purification method and application thereof
  • Recombinant Scolotoxin-Ssm2.1 gene and expression and purification method and application thereof

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Embodiment 1

[0039] Embodiment 1: Extraction of total RNA of centipede venom gland and cloning and sequence determination of natural toxin gene Scolotoxin-Ssm2.1 of centipede

[0040] Centipede venom glands were isolated by vivisection and immediately placed in liquid nitrogen. After crushing the venom glands in liquid nitrogen, total RNA was extracted with the Trizol reagent (Invitrogen Company) kit, followed by SMART TM The cDNA Library Construction Kit manual operates to synthesize the first strand of cDNA, synthesize the second strand by LD-PCR amplification, digest with proteinase K, digest with SfiI and recover the cDNA from the column, connect with the vector λTriplEx2, and package. The vector with the insert is poured by electroporation into E. coli DH10B competent cells, SOC medium 37 ℃ 1h100rpm recovery, spread on the agar medium with inducer IPTG and X-Gal chromogenic reagent and cultivate overnight. Pick the white spot, and quickly identify the size of the inserted fragmen...

Embodiment 2-1

[0042] Example 2-1 Construction of recombinant expression vector pSUMO-M-Scolotoxin-Ssm2.1

[0043] (1) Optimization and synthesis of the recombinant anti-insect toxin gene Scolotoxin-Ssm2.1

[0044] According to the codon preference of Escherichia coli, the cDNA sequence of the natural toxin gene Scolotoxin-Ssm2.1 of the spiny centipede was optimized, and a restriction endonuclease Xho I restriction site was added to the end of the gene to synthesize a full-length For the comparison of the nucleotide sequence of the recombinant anti-insect toxin gene Scolotoxin-Ssm2.1 of the spiny centipede and the natural toxin, please refer to the appendix figure 2 .

[0045] (2) Construction of recombinant expression vector pSUMO-M-Scolotoxin-Ssm2.1:

[0046] The synthesized Scolotoxin-Ssm2.1 DNA was digested with the restriction enzyme XhoI and ligated into the expression vector pSUMO-Mutant, and the ligated product was transformed into Escherichia coli DH5α competent cells for sequenc...

Embodiment 2-2

[0047] Example 2-2 Efficient prokaryotic expression of recombinant anti-insect toxin rScolotoxin-Ssm2a

[0048] (1) Expression of recombinant anti-insect toxin fusion protein

[0049] Prepare Escherichia coli BL21 (DE3) competent cells, transform the recombinant expression vector pSUMO-M-Scolotoxin-Ssm2.1 with correct sequencing into Escherichia coli BL21 (DE3) by heat shock method (42°C heat shock for 45 seconds) Engineering strains containing recombinant plasmids, their construction basis and construction process reference Figure 4 and Figure 5 , build result detection see Figure 6 .

[0050] Pick a single colony of the engineered strain and inoculate it in Kan + LB liquid rich medium, and cultivate overnight at 37°C with shaking. The next day, the overnight bacteria were inoculated in fresh Kan + LB rich medium at a volume ratio of 1:100, and amplified with vigorous shaking at 37°C until OD 600 About 0.6, add IPTG to a final concentration of 0.5mmol / L, and induce ex...

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Abstract

The invention belongs to DAN (deoxyribonucleic acid) recombinant technologies, in particular relates to a gene for encoding animal protein, and more specifically relates to a recombinant Scolotoxin-Ssm2.1 gene and an expression and purification method and an application thereof. According to the sequence of Scolotoxin-Ssm2a, a DNA sequence suitable for expression in escherichia coli is designed and is subjected to recombinant expression in the escherichia coli, so that Scolotoxin-Ssm2.1 having obvious anti-insect activity is obtained. The obtained recombinant Scolotoxin-Ssm2a (rScolotoxin-ssm2a) has strong neurotoxicity and lethal effect on insects. The recombinant Scolotoxin-Ssm2.1 gene can be used for preparing neurobiological research reagents and biological insecticides, and can also be used for researching centipede neurotoxin space structures and pharmacological activity.

Description

technical field [0001] The invention belongs to DNA recombination technology, in particular to a gene encoding animal protein, more specifically to a recombined centipede anti-insect toxin gene, its expression and purification method and access. Background technique [0002] Centipede (centipede) is the general term for Chilopoda animals belonging to Arthropoda phylum Arthropoda, and there are about 3300 species in the world (Edgecombe, G.D., Giribet, G., Ann. Rev. Entomol. 2007. 52, 151-170.). The centipede family Scolopendridae centipede (hereinafter referred to as centipede all belong to this category) is one of the most ferocious predators, mainly feeding on insects and small invertebrates; large centipedes can also prey on vertebrae such as toads, snakes, bats and rats animal. Centipedes prey by injecting venom into their prey, either directly killing or paralyzing it. Like spiders, scorpions and other predatory arthropods, the centipede venom system has developed a s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/70C07K14/435A01N47/44A01P7/04
Inventor 华卫建
Owner JIANGSU INSTITUTE OF EDUCATION
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