Preparation Of Antiserum Of Chicken Prion Protein And Its Distribution In Nervous System

[Objective] Physiological functions of the mammalian prion protein and structure transition mechanism hasbeen in-depth study, while less research on amphibians and birds prion protein. The chicken was firstdiscovered containing the PRNP gene in birds. however, we have not fand research in the chicken prionprotein function and expression mechanisms. Therefore,this study intended by prokaryotic expression ofchicken recombinant prion protein, purified recombinant prion protein, then producting anti-serum of thechicken recombinant prion protein.we identify prion protein positioning and distribution byimmunohistochemical method in the neural tissue inside gallinaceous body.we provide the scientific basis forillustrating the physiological function of chicken prion protein.[Methods]According to the reported ChPRNP gene group sequence, we extract the genome of healthyRoman chicken as the material by molecular biology methods, such as primers design,PCRamplification,cloning,sequencing and others to obtain73bp-741bp,286bp-741bp,520bp-741bp genefragments of ChPRNP gene. after sequencing,we apply DNAstar of molecular biology software to analysisChPRNP sequence；cloned chicken prion protein gene as Substrate,we obtain ChPRNP gene73bp-741bp,286bp-741bp,526bp-741bp gene fragments by PCR method,to recombine expression vector which arepGEX-ChPrP (25-247), pGEX-ChPrP (96-247), pGEX-ChPrP (174-247) plasmids. We have explored the bestexpressed conditions, induction optimal IPTG concentration and expression time. We identify expressionproducts by SDS-PAGE. A large number of bacteria pGEX-ChPrP (25-247) have been cultured.we havelysised bacteria by ultrasonic wave, to obtain inclusion bodies,then denaturation and refolding by urea.Refolding product traverse GSTtrap affinity chromatography column to collect the GST-ChPrP (25-247)fusion protein. fusion protein GST-ChPrP (25-247) with Freund adjuvant immunized New Zealand whiterabbits and BABL/c mices, to obtain the anti-GST-ChPrP (25-247) polyclonal antiserum and determinationof their antibody price. We detect antiserum immunogenicity by western blotting method and ChPrPdistribution in various tissues of the chicken by immunohistochemical method.[Results](1)we have used whole blood DNA of healthy Roman chicken as a template and designed the PRNPgene-specific primers for PCR. Ultimately we have obtained ChPRNP fragments about685bp,472bp and232bp.The target gene and the pGEX-4t-1vector have connected together, then was transformed into E. coliBL21(DE3). for expression bacteria pGEX-ChPrP (25-247), pGEX-ChPrP (96-247), pGEX-ChPrP(174-247),we have repplied PCR and Eoc RI\Bam HI restriction endonuclease digestion, Sequence analysisshowed that the recombinant expression plasmid were inserted669bp,456bp,216bp chPRNP fragmentsand no frameshift.(2) expression products were found protein with molecular weight approximately50000,42000,32000by SDS-PAGE analysis. Molecular weight of expression target protein and forecast ChPrPwere consistent. vector pGEX-4T-1was transformed into E. coli BL21,to obtain target protein about26000tagged protein.(3)using recombinant pGEX-ChPrP(25-247),we optimized expression conditions, the resultsshowed that the GST-ChPrP(25-247) had the highest expression level accounted for30%to45%of the totalbacterial protein in1.0mmol/L IPTG and6~8h induced at37°C.(4)We extracted inclusion body frompGEX-ChPrP(25-247), then inclusion body denaturation and refolding.howere,we purified the target proteinpurity by affinity column chromatography.it can reach more than90%.(5)we have immunized rabbits andmice, ultimately, to obtain the titer about1:12800of the anti-serum (6)Western blotting experimentsconfirmed that the fusion protein GST-ChPrP (25-247) lanes have a protein of molecular weight about50,000specific bands showed that expression GST-ChPrP, fusion proteins have a better immune activity.(7)Astrocytes and pyramidal cells express the prion protein in chicken brain.Axons of neurons express the prion protein in spinal cord gray matter areas by immunohistochemistry method.[Conclusion](1) chicken prion protein has a strong immunogenicity and reactogenicity.(2) Immunohistochemical Identification of chicken prion protein chicken brain tissue expression of thechicken prion protein in the nervous system play an important role.