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Cloning and Expression of PTD-NPY Fusion Protein Gene and Its Identification

Author: ZhouFengQiu
Tutor: ZhouGui
School: Jilin Agricultural University
Course: Basic Veterinary Science
Keywords: PTD-NPY Fusion Protein Gene Cloning Expression Identification
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 45
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Abstract


Neropeptide Y（NPY） is a 36-amino acid peptide,it widely distributed in both the central nervous system（CNS） and the peripheral nervous System（PNS）,especially contain abundantly in telencephalon, preoptic area,optic tectal slices and hypothalamus.NPY has been implicated in various physiological processe.It is closely relately with important physiological regulations of food intake and in the control of blood pressure in vertebrates.Moreover,NPY has been linked to the regulation of circadian rhythms respiration,neuroendocrine,blood vessel contraction,action of smooth muscle and sexual behavior in mammals.This trial was conducted to study expression of PTD-NPY fusion protein gene on the Pichia pastoris and obtain high pureness fusion protein.The fusion protein gene PTD-NPY whose DNA fragement was about 170bp,was constructed by overlapping PCR with template of itself,specific primer was planned.A specific DNA fragement about 170bp was recovered.Ligation of the DNA with the vector PMD-18T simple was done and then transformed into competent host strain Escherichia coli DH5a.Plasmids were isolated from recombinant clones,and PCR identification of the recombinant plasmid.According to multiple cloning site and gene sequence of the Pichia yeast expression vector pPICZαA, two specific primers were designed,and E.coil＆XbaI were respectively belongded to upstream and downstream primers.A specific DNA fragement about 170bp was recovered.Ligation of the DNA with the vector PMD-18T simple vector was done and then transformed into competent host strain E.coil DH5α.Plasmids were isolated from recombinant clones and PCR identification of the recombinant plasmid. The fusion gene PTD-NPY DNA was cut from the recombinant PMD- PTD-NPY with EcoRI＆XbaI,was inserted into the yeast expression vector pPICZαA by the same digestion.Positive recombinant plasmid was sequenced,and results showed that hybrid gene PTD-NPY sequence was the exact fragment.The recombinant yeast expression plasmid was named as pPICZαA-PTD-NPY.The recombinant plasmids of correct sequencing were transformed into the Pichia yeast host cells GS115 by eletroporation.The positive clones were screened by ZeocinTM resistance,and their genomic DNA were isolated and then amplified by PCR,using vector primers5’AOX1 paired primers3’AOX1,and specific primers P1 paired primers P4,to determine the integration of the interest gene into the Pichia genome.Most clones with ZeocinTM resistance were confirmed to be positive Pichia integrants by PCR amplification.When the recombinant Pichia strain was induce by adding of methanol to a final concentration of 1%,mRNA transcripted from the interest gene was detected by PCR amplification and the band of interest protein was found in the condensed supernatant by SDS-PAGE and Western blot.

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