Construction,Expression And In Vitrio Activity Studies Of Cell-penetrating Carrier Similar To Toxin

The existing drugs for the treatment of central nervous system diseases need to pass through the BBB(Blood Brain Barrier).However, for most drugs, only litle or no part of them can pass through the BBB. Currently, protein delivery systems have attracted much attention for its low toxicity, accuracy and high efficiency. Therefore, this research aimed to build a kind of brain-targeting drug delivery carrier which can overcome the BBB and deliver the drug to the brain with an efficient and noninvasive manner.The natural cholera toxin (CT) is consisting of cholera toxin subunit B (CTB) and cholera toxin subunit A (CTA). CTB is a pentamer with a hole in the center, which can bind to the GMl-ganglioside. CTA is located in the hole in the center of the pentamer. CTB can bind to the GM1 on the mucosa to allow the entire CT molecular to anchor on the mucosa. CTA enter the cell by the guidance of the hole. After entering the cell, CTA will be rapidly hydrolyzed into part A1 and A2. Al can interact with an enzyme, which may cause the output of ions in the cell, make the cell in a dehydration state even fataldehydration, and thus threaten the life. A2 is a non-toxicsubunit which is a key part connected to CTB. By simulating the structure of CT, this study is to find a way to obtain a kind of drug deliverycarrier similar to the CT structure. Non-toxicreport proteinof enhancedgreen-fluorescent protein (EGFP)was used to replace A1 in the form of fusion protein. Thus, the originalCTA1-CTA2 structure was replaced by EGFP-CTA2. As the receptor of GMl-ganglioside which is rich on the nasal mucosa, (CTB)5 can guide the EGFP-CTA2 enter the body in a noninvasive manner. However, EGFP-CTA2 needs to go through many membranes and the BBB after entering the body. In order to enhance the transmembrane ability of the EGFP-CTA2 and deliver EGFP into the brain, a short-peptide TAT (the trans-activated factor) was integrated with it to design a kind of new drug deliverycarrier protein "EGFP-CTA2-TAT/(CTB)5".In order to improve the method to get the carrier EGFP-CTA2-TAT/(CTB)5, the method of respectively express and in-vitro reassembly was found after exploration. Firstly, we cloned CTB to pETduet-CTB multiple cloning site 1. After inducible expression, the condition was optimized as 160xg.37 degree. Sixteen hours later, the maximum CTB protein could be obtained, and after washing, purification and renaturation to get the (CTB)5. In addition EGFP-CTA2-TAT was cloned into the vector pET22b, the maximum expression quantity of EGFP-CT2-TAT in supernatant could be obtained 16 hours later with the optimization of 120r/min,25 degree. The EGFP-CTA2-TAT could be obtained after the NI-NTA purification. Ultrafiltration and desalination were conducted to (CTB)5 and EGFP-CTA2-TAT, and the two proteins were mixed at the proportion of 5:1. Through acid denaturation and alkali renaturation, the hexamer protein EGFP-CTA2-TAT/(CTB)5 was identified. The GM1-ganglioside ELISA experiment and fluorescence experiment showed that the hexamer protein had good bioactivity. The hexamer protein kept efficient transmembrane ability even in the in vitro transmembrane experiment.