| Objective: Vasoactive intestinal peptide(VIP) is a brain- intestinal peptide with high homology with neuropeptide PACAP(Pituitary adenylate cyclase activating polypeptide), which plays a potential role in the treat ment of neurodegenerative disesase, such as Alzheimer’s diseases(AD). VIP traverses the blood-brain barrier effect(BBB) in non-specific traversing membrane manner. The short half- life in vivo of VIP and the low traversing bio-barriers efficiency prevent its application in the central nervous system. TAT composed of 11 amino acid residues is a kind of cell penetrating peptide. In this research, we used the principles and techniques of genetic engineering to construct a novel fusion peptide VIP-TAT(M/L) that crossed the BBB efficiently, and utilized the intein- mediated protein purification technique combined with Sephadex G-50 chromatograph to achieve its preparation, study its anti-apoptotic activity in the apoptosis model off neurocells, and try to invesit igate the mechanism of its biological activities.Method: A new gene encoding VIP-TAT(M/L) was synthesized and cloned into the expression vector p KYB to construct the recombinant vector p KYB-VIP-TAT(M/L), which was transformed into Escherichia coli ER2566. The target protein VIP-TAT(M/L) was purified using IMPACT(Intein Mediated Purification with an Affinity Chitin-binding Tag) system combined with Sephadex G-50 chromatograph technique. In addition, we tried to prepare the recombinant polypeptide VIP-TAT(M/L) through combining the inclusion body(IBs) denaturation-renaturation with intein- mediated purification. Then the biological activity of VIP-TAT(M/L) was detected by its proliferation ability on the C hinese hamster ovary(C HO) cells expressing receptors PAC1, VPAC1 and VPAC2 respectivly; A apoptosis model was constructed utilizing scopolamine(Scop) in mice neuroblastoma(Neuro-2a) cells. After the incubation of the apoptotic cells with different concentrations of VIP-TAT(M/L) or VIP, the cell viabilities remained were determined, the level of intracellular anti-apoptotic protein Bcl2 and the activity of apoptosis protein caspase 3 were assayed for the detection of VIP-TAT(M/L) against Scop induced apoptosis in Neuro-2a cells. And the anti-apoptotic ability of recombinant polypeptide VIP-TAT(M/L) was also checked intuitively by TUNEL assay. To explore the mechanism of its anti-apoptotic function, we detected the levels of intracellular c AMPinduced by recombinant polypeptide VIP-TAT(M/L)/VIP with or without receptor PAC1 specific antagonist PACAP(6-38) in PAC1-CHO cells with high expression of PAC1 and Neuro-2a cells. The secondary structures of TAT and pituitary adenylate cyclase activating polypeptide PACAP(28-38) were also analyzed and compared.Results: In this study, the recombinant polypeptide VIP-TAT(M/L) was successfully prepared with purity of 84 % through the IMPACT purification system combined with Sephadex G-50 gel chromatography purification technology; Further more, the efficiency of preparing VIP-TAT(M/L) using IBs denaturation-renaturation and intein- mediated purification technology was low, which needs further improvement. The experiments on the effects of recombinant protein VIP-TAT(M/L) against Scop induced apoptosis in Neuro-2a cells showed that VIP-TAT(M/L) not only inhibited the apoptosis induced by Scop in Neuro-2a cells more significantly than VIP with decreasing intracellular caspase 3 levels and promoting intracellular Bcl2 concentration, but also promoted the proliferation of PAC1-CHO cells, while VIP did have proliferative acitity. The further mechanism study showed that VIP-TAT(M/L) induced the increase of intracellular c AMP level inhibited by PAC1 antagonist PACAP(6-38). And TAT had similar a-helix structure with PACAP(28-38).Conclusion: The recombinant polypeptides VIP-TAT(M/L) was successfully prepared in this experiment, while IBs denaturation-renaturation combined with intein- meiated purification provided a new way for preparation of VIP-TAT(M/L). The datas on the enhanced effects of recombinant peptide VIP-TAT(M/L) against Scop induced apoptosis in Neuro-2a cells indicated that the preferring activation of VIP-TAT(M/L) on PAC1 mediated its significant anti-apoptotic activities. This study laid the foundation for further potential application of VIP-TAT(M/L) targeting PAC1 in neurodegenerative disease. |
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