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The Preparation of the Recombinant VIP-TAT and its Effects on the Scopolamine-induced Amnesia in Mice

Author: ZengZhiXing
Tutor: YuZuoJie
School: Jinan University
Course:
Keywords: VIP VIP-TAT scopolamine amnesia cell-penetrating peptide
CLC: R96
Type: Master's thesis
Year: 2013
Downloads: 3
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Abstract


Objetive：Vasoactive intestinal Peptide （VIP）is a very important signal moleculeof neurotransmitter, which participates in information transfer and physiologicalregulation. So it was a potential therapeutic neuropeptide, but unstable instructure.The11-amino acid peptide TAT is a cell penetrating peptide，able to deliverprotein cargoes across the cell membrane. In this study, the preparation of arecombinant VIP-TAT composed of VIP and TAT was achieved using geneticengineering principle and technology. After its bioactivity of cell penetrating andtraversing biological barriers was detected, a mice model with amnesia induced byscopolamine was used to test the function of VIP-TAT and VIP.Methods: Based on the natural sequence of VIP, VIP-TAT gene was designed andsynthesized according to the expression bias of E.coli. VIP-TAT gene was acquired bytwo steps PCR using four primers and cloned into the expression vector pKYB.Engineering bacteria pKYB-VIP-TAT-ER2566was constructed, and high-efficientpreparation of VIP-TAT was achieved with the IMPACT (Intein Mediated Purificationwith an Affinity Chitin-binding Tag) protein expressing and purification system.Western blot was used to identify the immunological activity of recombinantVIP-TAT，and the fluorescence labeling technology was used to test its ability topenetrate into the cells and traverse the biological barriers into blood and brain. Theinhibitory effects of VIP-TAT medicated by atomization on the food intake were alsoassayed. At last a mice model with scopolamine-induced amnesia was used to assaythe effects of VIP-TAT. After one-time and long-term intervention trial with VIP-TATand VIP, passive avoidance test was used to test the learning and memory of mice, thelevels of MDA (malondialdehyde) and SOD (superoxide dismutase) in the brain weredetermined, and the antioxidant activities and oxidative stress indicators were assayedin the brain and blood. The mechanism about the VIP-TAT againstscopolamine-induced amnesia in mice was explored from the content of ACHE (acetylcholin esterase) in the brain.Results: The engineering strain pKYB-VIP-TAT-ER2566was constructed, and thefermentation conditions acquired for the preparation of VIP-TAT were optimized. Therecombinant VIP-TAT was obtained with IMPACT system, SDS-PAGE and westernblot were used to characterize the recombinant VIP-TAT. VIP-TAT labeled withfluorescence FITC was found to penetrate into the CHO cells more effectively thanthe VIP labeled with FITC under the fluorescence microscope. VIP-TAT administratedby atomization was shown to get into blood through the respiratory tract and thentraversed the blood brain barrier into brain. Compared VIP-TAT with VIP, the resultsshowed VIP-TAT inhibited the food intake more effectively than VIP and VIP-TAThad enhanced ability to traverse the biological barrier. During the treatment of thescopolamine-induced amnesia in mice, whether administrated in one-time orlong-term, in intraperitoneal injection or atomization, unlike VIP, VIP-TAT stablyprolonged the incubation period of passive avoidance and ameliorated the amnesia inmice. VIP-TAT also decreased the levels of MDA and AChE in brain more effectively.It was also found that VIP-TAT not only promoted the SOD levels in blood and inbrain, but also increased the red cells counts and hemoglobin (HGB) level in blood.These results indicated that VIP-TAT delivered by atomization had significantlyefficient function on improving the learning and memory dysfunctions mice.Conclusion: High-efficient preparation of recombination VIP-TAT with enhancedability to traverse biological barriers was accomplished with genetic engineeringprinciple and technology. In vivo experiment, VIP-TAT was proven more effectiveagainst mice with amnesia induced by scopolamine than VIP. The finding thatVIP-TAT has the ability to improve the mice with learning and memory dysfunctionswhile medicated by atomization will lay the foundation for its further application.

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