Cloning And Expression Of Homo Sapiens Neuropeptide Y Y5 Receptor And It's Bioinformatics Analysis

Obesity is a serious danger for human's life and health,and it can cause many diseases,for example type 2 diabetes,heart disease,high blood pressure,apoplexy,osteoarthritis,cancer and so on. 300 millions of one billion overweight people are obesity sufferer,the number in our country is 60 million and 200 million respectively(data from WHO).So,how to treat obesity and overweight have become a very important problem.Recently,neuropeptide Y(NPY)is implicated in energy homeostasis and contributing obesity.Neuropeptide Y Y5 receptor(NPY5R)is involved in mediating NPY-induced stimulation of food intake and body weight gain.Many NPY5R agonists can increase animal's food intake;NPY5R coding gene antisense Oligodeoxynucleotides can reduce animal's food intake by blocking the translation of NPY5R gene;the NPY5R inhibitors can also against obesity.All these results suggest that NPY5R is a potential targets in discovering the drugs for treating obesity and overweight,and the inhibitors of NPY5R will be effective drugs in obesity's therapy.So,Cloning,expressing and purifying hNPY5R,and then screening the inhibitors of hNPY5R from the high-throughput screen(HTS)based on hNPY5R should be useful for the discovery of the drugs against obesity.This article will design a pair of special primers base on the human NPY5R(hNPY5R)coding gene(gene ID:4889);amplify hNPY5R coding sequence from Homo Sapiens genome by PCR; digest the PCR products and plasmid pESP2 with endonucleases SphI and BamHI;insert the PCR products into the plasmid pESP2;transport the recombinant plasmid pESP2-hNPY5R into E.coli JM109 by CaCl₂ method;identify the recombinant plasmid by clony PCR,digesting and sequencing;electroporation the correct recombinant plasmid into S.pombe SP-Q01;identify positive clone by clony PCR;induce the positive clone for expressing the recombinant protein; electrophoresis detect the expression of recombinant protein using 12%SDS-PAGE after broken cells by ultrasonator;there is no exactly recombinant protein blot is found on the PAGE.The reason probably is that the recombinant protein is poisonous for the host bacteria or the protein expression level is very low.We also design another pair of special primers,amplify hNPY5R coding sequence by PCR; construct the recombinant plasmid pET28a(+)-hNPY5R;transport the correct recombinant plasmid into E.coli BL21(DE3);induce it for expressing the recombinant protein; electrophoresis detect the recombinant protein;there is no exactly recombinant protein blot being found on the PAGE.The recombinant protein maybe has toxicity for the host bacteria.Analyse NPY and NPY5R's structure,activity sites,signal peptide,transmembrane structure and homology by bioinformatics methods.The methods can be partitioned two groups, one group using the analysis soft VectorNTI 9.0,Mega,ClustalX1.83 and so on;another groups using on-line analysis soft,for example PSIPRED Server,SignalP and TMPRED.It is found that the recombinant protein's structure has no remarkable change compared with NPY5R.We also find several highly conserved motifs that may be involved in signal transduction on 3rd intracellular loop of hNPY5R.