Dissertation > Excellent graduate degree dissertation topics show

Expression of Neusopeptide Y(NPY) Fusion Protein of Spinibarbus Sinensis Bleeker, in E. Coli

Author: HuChunYan
Tutor: LiYingWen
School: Chongqing Normal University
Course: Biochemistry and Molecular Biology
Keywords: China Spinibarbus Neuropeptide Y (NPY) Clone pET-32a vector The prokaryotic expression Transetta strains
CLC: S917.4
Type: Master's thesis
Year: 2011
Downloads: 23
Quote: 0
Read: Download Dissertation

Abstract

Neuropeptide Y (Neuropeptide Y, NPY) is an important neural regulatory factors in vivo, and is closely related to the growth and development of the individual as well as the behavior of living. China barb gills (Spinibarbus sinensis Bleeker) is one of the China's unique freshwater fish. This study was designed by in vitro expression of precursor the bioactive Spinibarbus gills NPY, to lay the foundation for the structure and biological functions of the in vitro studies of NPY hope the NPY gene fragment of Chinese Spinibarbus by prokaryotic expression vector pET-32a connection transferred Transetta bacteria, expression of NPY with biological activity. Experiment 1 Chinese Spinibarbus of NPY full-length gene the clone the careful analysis known the China Spinibarbus sequence, design cloned the full-length primer the Chinese Spinibarbus N-PY. Extracting total RNA from China denticulatus cerebellum, the gene fragment of the PCR amplified double stranded NPY, The resulting PCR product was purified, cloned, and the blue-white screening, the plates white colonies were picked for PCR verification, and sequenced, and for cloning the right pMD18-T/NPY recombinant basis. NPY of PCR amplification Spinibarbus gills length fragment about 750bp position, the sequence between the two primers by 722bp nucleotide open reading frame has 291bp bases, before and after the 58bp and 373 bp, respectively. 291bp open reading frame encoding 96 amino acids, including 31 hydrophobic amino acids, accounting for 32.3% of the total number of amino acids. To Experiment 2 Chinese Spinibarbus of NPY prokaryotic expression recombinants cloned in our laboratory NPY full-length sequence, design the the cloned NPY open reading frame sequence primer and add restriction sites downstream primer 5 'end, respectively, in the previous point Nco Ⅰ and Xho Ⅰ. Chinese barbs gill of NPY open reading frame of 316 bp fragment (i.e. NPY316) is first cloned into E.coli DH5a bacteria, while the expression vector pET-32a transformed into E.coli DH5a bacteria. Respectively expanding culture of the two groups of bacteria, plasmid was extracted and digested. The double target fragment was digested vector pET-32a, building pET-32a/NPY316 recombinants, and then transferred to the prokaryotic expression the strains Transetta in. Plate colonies were picked for PCR validation, the the double digestion verify and sequenced. The validation results show that the target sequence after NPY316 pET-32a successful transformed Transetta bacteria. Sequence analysis showed that the the Chinese Spinibarbus NPY316 gene fragment had no mutation. Experiment 3 induced expression China denticulatus NPY precursor protein with the IPTG induction Transetta recombinant bacteria were collected by centrifugation induced bacterial cells, the processing performed after SDS-PAGE electrophoresis induced by detecting whether the target protein band. Finally, through the Western Blot test to determine whether the expression of the target protein. SDS-PAGE electrophoresis results showed that, compared with the control group, the experimental group about 29KDa position significantly increase a protein bands by Western Blot test proved that the protein is the to restructuring Transetta Upon induction with IPTG expression of NPY precursor fusion protein.

Related Dissertations

CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquatic basic science > Aquatic Biology > Aquatic Zoology
© 2012 www.DissertationTopic.Net Mobile