Expression Of Neusopeptide Y(NPY) Fusion Protein Of Spinibarbus Sinensis Bleeker, In E. Coli

Neuropeptide Y is an important neurotransmitter in living organisms, It is closely related to individual growth and life behavior.s. sinensis bleeker (Spinibarbus sinensis Bleeker), commonly is one of freshwater fishes in our country. This study was designed to obtain NPY with biologicalactivity of s.sinensis bleeker by prokaryotic expression, which can lay the foundation for studying the structure and biological function in vitro of NPY, By linking s. sinensis bleeker NPY gene with novagen pET-32a, then translate it into strain Transetta, it could bioactive NPY.Experiment 1 Clone full length NPY gene of s.sinensis bleekerAfter analysied the reported s.sinensis bleeker sequence, we designed primers to clone full-length of S.sinensis bleeker. First, we extracted total RNA from cerebellum of S.sinensis bleeker, then amplificated NPY gene fragments by PCR. After purified PCR products, cloned, blue-white screening, white colonies on plates were picked for verification with PCR, PCR product of full-length S.sinensis bleeker NPY fragment was about 750bp, the sequence between the two primers base consist of 722bp, with 291bp Of open reading frame(orf), encoding 96 amino acids, before and after orf is 53bp and 373bp, respectively. Coded amino acids, include 31 hydrophobic amino acids,32.3 percent of total amino acids.Experiment 2 Construction of Spinibarbus sinensis Bleeker NPY prokaryot ic expression recombinantAccording to the coined NPY full-length sequence in experiment(1), we invesigated the 291bp open reading frame,and designed for primers colning the NPY open reading frame, and added restriction senzyme sites Nco I and Xho I on 5'ends of upstream and downstream primer, respectively. First of all, open reading frame sequence of Spinibarbus NPY 316bp (ie NPY316) was cloned into E.coli DH5a, while the expression vector pET-32a was also transformed into E.coli DH5a. Two groups of bacteria were cultured to expand, large-scale preparation of plasmid, and double digests, purified the products. connecting fragments and pET-32a vector, constructing recombinant pET-32a/NPY316, transformed into the prokaryotic strain Transetta. Finally, colonies on the culture plate were picked to expand, verification by PCR,double digests. The results of the PCR, restriction enzyme digestion, and sequencing show that the target fragment NPY316 was successfully connected with the pET-32a, and transformed the Transetta. Sequence analysis showed that the Spinibarbus NPY316 gene does not mutate.Experiment 3 Expression of precursor protein of Spinibarbus sinensis Bleeker NPY by Prokaryotic InductionAfter Inducing Transetta Recombinant strain with IPTG, We collected induced cell by Centrifugation, SDS-PAGE electrophoresis was used to detect the induced protein bands. Finally, Western Blot was carry out detect the expression of the target protein. The results of SDS-PAGE showed there was one new protein band at about 29KDa position by comparing it with control groups. Western Blot test proved that the protein is the precursor fusion protein induced by IPTG in recombinant Transetta bacteria in vitro.