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Preparation and Functional Study of Recombinant Protein PTD-MAX with Transmembrane Activity

Author: ZengLe
Tutor: XuMingFang；YuZuoJie
School: Jinan University
Course: Microbiology
Keywords: Recombinant protein PTD-MAX Preparation Penetrating ability Biological activity Blood-brain barrier Corneal alkali burn
CLC: Q51
Type: Master's thesis
Year: 2010
Downloads: 36
Quote: 1
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Abstract


The PAC1 specific receptors of the pituitary adenylate cyclase activating peptide (Pituitary adenylate cyclase activating polypeptide, PACAP), mediated by a variety of biological functions. Maxadilan neural peptides extracted from the sandfly salivary glands, it is a the PAC1 receptor specific agonist, nerve repair, regulating energy metabolism and balance a variety of biological functions. PTD (Protein transduction domain) is a structural domain of the TAT (Trans-activator transcription) protein, has powerful penetrating function, can effectively guide the be connected thereto macromolecular proteins wear film role. In this paper, the principle of genetic engineering technology to achieve recombinant PTD-MAX's preparation and its wear film capacity, biological activity and drug application, its biological function in the development and in the brain, eyes and other nerve drug application in the intensive parts of the foundation. The PTD sequence, our laboratory has successfully constructed on the basis of the natural Maxadilan protein gene, synthesis the PTD-MAX gene by overlapping PCR technology, engineering bacteria pKYB-PTD-MAX-ER2566; explore through fermentation conditions, when the shake-flask conditions control: 2% glycerol carbon source, temperature 37 ° C train 2-3 hours of cultured cells to OD600 reached 0.5-1.0, IPTG (Isopropylβ-D-thiogalactoside) to a working concentration of 0.5 mmol / L, induced expression 4 hours, to obtain optimal expression of fusion protein; amplified by a 100L fermenter culture to obtain large amounts of engineering bacterial cells on this basis; using IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) protein purification system, by affinity Single Column chromatography protein purification obtained 12.1mg / L, up to 95% purity of PTD-MAX protein, the preparation of the recombinant protein PTD-MAX. Fluorescently labeled with FITC (Fluoresceine isothiocyanate) the PTD-MAX penetrating ability to conduct research, the results showed that the PTD-MAX effectively through the CHO cell membrane into the cell across the blood-brain barrier in mice and the mice and rabbit corneas. The PTD-MAX wearing blood-brain barrier efficiency was significantly higher than Maxadilan (P lt; 0.01), and through the mouse and rabbit corneas experiments, PTD-MAX's penetrating efficiency is Maxadilan of 4.7 times and 7.2 times, respectively. The The PTD-MAX transient elevated blood glucose, and the ability to inhibit food intake has more significant than the natural Maxadilan protein; Description recombinant PTD-MAX is able to activate the PAC1 receptor and good research on the biological activity of the PTD-MAX The retention of the biological activity of natural Maxadilan. PTD-MAX can be effective in promoting healing corneal ulcers, reduce the inflammatory response after alkali burns, and promote the recovery of corneal sensitivity after alkali burn the PTD-MAX rabbit eye alkali burns, corneal repair. Showed that the recombinant PTD-MAX has a therapeutic effect on corneal alkali burns.

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