Expression and purification of BDNF-TAT fusion protein in E.coli and its distribution in brain
10.3760/cma.j.issn.1671-8925.2011.06.010
- VernacularTitle:BDNF-TAT融合蛋白在大肠杆菌中的表达纯化及脑中分布
- Author:
Dao-Fei LUO
1
;
Wen-Shi LI
;
Shan CHEN
;
Ai-Min JI
Author Information
1. 南方医科大学珠江医院
- Keywords:
BDNF;
Fusion protein;
Purification;
Targeting
- From:
Chinese Journal of Neuromedicine
2011;10(6):578-581
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the expression and purification of BDNF-TAT fusion protein in E.coli, and study its brain-targeting and distribution in brain. Methods Plasmid PET-30(a) -BDNF-TAT was transfected into the E.coli BL21 (DE3)pLysS;the expression of PET-30(a)-BDNF-TAT was induced by isopropyl-beta-d-thiogalactopyranoside (IPTG);this BDNF-TAT fusion protein was purified by SP-Sepharose cation exchange column and then renatured by 0.4 mol/L arginine. According to the random number table method, KM mice were divided into 3 groups: BDNF-TAT treatment group (giving 4 μg BDNF-TAT through intravenous injection, n=3), negative control group (giving same volume of saline through intravenous injection, n=3) and blank control group (without giving any treatment, n=3). The whole brain protein was collected 3 h after the injection;Western blotting was used to identify the expression of BDNF-TAT fusion protein;the distributions of BDNF-TAT in the brain tissues were examined by SABC immunohistochemistry. Results BDNF-TAT fusion protein was purified with the purity about 90%. The relative expression of BDNF-TAT fusion protein was 1.897±0.286 in the BDNF-TAT treatment group, 0.615±0.234 in the negative control group, 0.335±0.154 in the blank control group;significant differences between each 2 groups were noted (F=39.019, P=0.000).Western blotting indicated that the content of BDNF-TAT in the mice brain in BDNF-TAT treatment group was obviously higher than that in the negative control group and blank control group (P<0.05);BDNF-TAT fusion protein were widely distributed in CA1,CA3 and DG areas of the hippocampus of brain tissues in the BDNF-TAT treatment group, while no obvious positive staining was noted in the negative control group and blank control group. Conclusion BDNF-TAT fusion protein abundantly expresses in E.coli in the form of inclusion bodies, and can target the brain tissue via a systemic route.
