Safe preparation method of botulinum neurotoxin
A technology of botulinum toxin and a production method, applied in the field of safe production, can solve the problems of low yield purification process, difficulty in forming disulfide bonds, no economy, etc., and achieves the advantages of overcoming high complexity, improving extensibility and shortening production time. Effect
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[0093] Regarding the preparation / production of botulinum toxin, conventional production of botulinum toxin is carried out in the following manner: After culturing Clostridium botulinum, botulinum toxin is purified by separation and ion exchange chromatography. However, this method has the problems of low efficiency and low protein yield. In addition, botulinum (C.botulinum) is a spore-forming bacterium, which requires special culture equipment that is not required when cultivating bacteria such as Escherichia coli (Escherichia coli), and since botulinum toxin is a very deadly toxin, it is therefore A corresponding security level is required. Therefore, although there are known attempts to produce recombinant botulinum toxin in commercial strains such as Escherichia coli (E. coli), the situation that safety equipment is required remains unchanged because neurotoxins are produced. The method of production in recombinant E. coli has the following problems:
[0094] 1) It is dif...
Embodiment 1
[0101] Example 1. Preparation of plasmids for the production of botulinum toxin split fragments
[0102] 1-1. Plasmids for production of binary fragments
[0103] In order to prepare H for the production of LC (light chain, light chain) and HC (heavy chain, heavy chain) N (Translocation domain, translocation domain) [LC-H N ] with H of HC C (Receptor binding domain or RBD, receptor binding domain) [H C ] of the botulinum toxin split protein formed by 2 fragments ( Figure 2a4 and 3), using the vector and T4 deoxyribonucleic acid (DNA) polymerase (polymerase) to clone pET 28b, duet and pCola duet. Specifically, using the pET 28b vector and LC-H N -Cfa N A polymerase chain reaction (PCR) was performed with primers designed so that the mutual complementarity (complementary) between both ends of the insert (insert) was 15 bp. In the pETduet, pCola duet vectors, use the LC designed at the RBS 1 site, the H at the RBS 2 site N -Cfa N The primers with mutual complementarity ...
Embodiment 2
[0123] Example 2. Confirmation of soluble expression of botulinum toxin fragments
[0124] The plasmid prepared in Example 1 above was used to produce botulinum toxin split protein. Specifically, Escherichia coli BL21(DE3) strain transformed into competent cells was stabilized in ice after being subjected to heat shock for 45 seconds at a temperature of 42°C and transduced with the plasmid prepared above. The transduced Escherichia coli was smeared on LB ampicillin solid selection medium and cultured at 37°C for one day. A colony grown in the selective medium was put into 10 mL of liquid LB medium and 10 μL of kanamycin was added, and then pre-cultured at 37° C. for 12 hours. The pre-cultured recombinant strain was re-inoculated in 50 mL of LB medium supplemented with kanamycin at a concentration of 1%, and cultured at a temperature of 37° C. until the O.D. (wavelength: 600 nm) value reached 0.5. Then 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and cultured ...
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