Design,Preparation And Activity Of Brain-targeting Cholera Toxin-like Chimeric Protein

Protein drugs have the characteristics of low toxicity,targeting and good efficacy.Research and development of protein drugs for treating brain diseases have always been a research hotspot.Although the blood-brain barrier（BBB）restricts the entry of potentially neurotoxic substances into the brain,it is also a major obstacle to the delivery of therapeutic drugs into the central nervous system to treat diseases.The blood-brain barrier exhibits low endocytosis and has a tight junction（TJ）,which forms a seal and high transendothelial resistance between opposing endothelial membranes.This high resistance results in extremely low permeability,which greatly limits the diffusion of drugs from the blood to the paracellular cells in the brain.One of the difficulties in the development of protein drugs in CNS biotherapeutics is to overcome the low permeability of the blood-brain barrier.On the one hand,fusion expression with trans-activated transcription activator（TAT）is one of the common methods to improve brain delivery.On the other hand,intranasal administration is a comfortable and convenient method for non-invasively bypassing the BBB from the nasal cavity and delivering it directly to the brain.The nasal mucosa in the nasal cavity is rich in GM1 ganglioside receptors,and the cholera toxin B subunit pentamer（CTB）₅ can specifically bind to GM1 ganglioside receptors and enhance endocytosis.Therefore,in this study,enhanced fluorescent protein（EGFP）or recombinant epidermal growth factor was fused to the N-terminus of CTA₂（cholera toxin A2 subunit）through genetic engineering method,and then renatured with（CTB）₅ in vitro.Designed and prepared a cholera toxin-like chimeric protein,studied its biological activity in vivo and in vitro,brain-targeting effect and brain entry efficiency.Methods:1.Obtaining the cholera toxin-like chimeric protein EGFP-CTA₂-TAT/（CTB）₅ containing enhanced green fluorescent protein:using Oligo 7software to design primer sequences,construct plasmid vectors and transform into recombinant E.coli.The fusion proteins EGFP-CTA₂-TAT and CTB were respectively expressed from supernatant and inclusion body.After the primary purification,EGFP-CTA₂-TAT and CTB were respectively purified by dialysis renaturation and column chromatography purification to obtain higher purity protein,and EGFP-CTA₂-TAT/（CTB）₅was obtained in vitro by citric acid-Tris alkali denaturation method.2.Study of brain-targeting effect of cholera toxin-like chimeric protein EGFP-CTA₂-TAT/（CTB）₅containing enhanced green fluorescent protein:after intranasal administration of CTB and EGFP-CTA₂-TAT/（CTB）₅,mice were respectively anesthetized and executed at 0,15,30 and60 minutes.The whole brain was taken for brain homogenization,and the fluorescence value of the brain homogenate supernatant was detected by a fluorescence spectrophotometer.3.Study of the time and location of the chimeric protein into the brain:after intranasal administration of EGFP-CTA₂-TAT/（CTB）₅,mice were respectively anesthetized and executed at 0,5 and 30 minutes.The olfactory bulb,cerebellum,hippocampus,hypothalamus and cerebral cortex were taken for brain homogenization,and the fluorescence value of the brain homogenate supernatant was detected by a fluorescence spectrophotometer.4.Obtaining the cholera toxin-like chimeric protein EGF-CTA₂-TAT/（CTB）₅ containing recombinant epidermal growth factor:using Oligo 7 software to design primer sequences,construct plasmid vector and transform into recombinant E.coli.The fusion protein EGF-CTA₂-TAT was expressed from inclusion body.After the primary purification,EGF-CTA₂-TAT was respectively purified by dialysis renaturation and on-column renaturation to obtain two EGF-CTA₂-TAT with different purity and different activities.Both were combined with CTB protein in vitro by using citric acid-Tris alkali denaturation method to obtain EGF-CTA₂-TAT/（CTB）₅.5.Mouse embryonic fibroblast proliferation experiment and GM1-ELISA experiment were respectively used to verify the activity of EGF and CTB of chimeric protein.6.The therapeutic effect of cholera toxin-like chimeric protein containing recombinant EGF on LPS-induced neuritis mouse model:after intranasal administration to mice by cholera toxin-like chimeric protein containing recombinant EGF,LPS was injected intraperitoneally to induce neuritis.Twenty-one days later,the effect of chimeric protein EGF-CTA₂-TAT/（CTB）₅ treatment on learning,memory and anxiety defects in model mice was tested by Morris water maze and elevated maze experiments.7.Using the ELISA kits to detect the level of TNF-α,NF-κB and ROS in model mice whether intranasal administration EGF-CTA₂-TAT/（CTB）₅ or not.Results:1.Through prokaryotic expression and acid-base renaturation assembly in vitro,successfully prepared cholera toxin-like chimeric protein containing enhanced green fluorescent protein or epidermal growth factor respectively;2.After 5 minutes of nasal administration,the cholera toxin containing the enhanced green fluorescent protein can be directed to the olfactory bulb,cerebellum and hippocampus.After about 30 minutes,it spread to the hypothalamus,but did not reach the cerebral cortex;3.The cholera toxin-like chimeric protein containing epidermal growth factor has the same ability to stimulate the proliferation of mouse embryonic fibroblasts as the commercially available EGF,and it retained the ability of CTB subunit to bind to GM1.There was no significant change before and after chimerization;4.Successfully established a mouse model of LPS-induced neuritis,and confirmed that cholera toxin-like chimeric protein containing epidermal growth factor has the activity of treating LPS-induced learning,memory and anxiety defects in animal models.5.Mechanism studies had shown that cholera toxin containing epidermal growth factor can reduce the levels of TNF-α,NF-κB and ROS in model mice.Conclusions:In addition to obtaining a candidate macromolecular drug that can effectively enter the brain through the nose and treat brain neuritis,this study also shows that cholera toxin-like chimeric protein is an effective tool in delivering protein drugs to the brain.The delivery system introduced in this study provides a molecular design model by which a series of macromolecular protein drugs with brain-targeting activity can be constructed for drug development in brain diseases.