Sequence Analysis Of Chicken Prion-like Protein Shadoo, Doppel Gene And Chicken PrP Gene’s Expression In Pichia Pastoris

【Objective】 By cloning and analyzing the chicken Pr P-like protein Shadoo & Doppel gene(SPRN, PRND), find out their relationship with Pr PC. Furthermore, to provide a theoretical & molecular basis for the study of avian Sho & Dpl’s structure & function and the mechanism of the relationship between them; At the same time, to construct a chicken Pr P gene’s eukaryotic expression vector p PIC9K-Ch Pr P(25-248), and through Pichia pastoris expression system to get chicken prion protein(Ch Pr P), to provide material for the study of Pr P’s folding form in different physiological state and the research of Pr PC to Pr PSc conversion mechanism.【Methods】 According to the Gen Bank reported chicken SPRN(Ch SPRN) sequence to design specific primers, and amplify the SPRN complete ORF by using chicken brain tissue genomic DNA as template, furthermore, analysis it by bioinformatics methods and softwares; By comparing the aa sequence of multi-species Dpl, find Dpl’s conservative region, and design the degenerate primers and optimize primers with chicken codon preference to reduce the degeneracy gradually. With the specific primers to amplify chicken PRND gene by polymerase chain reaction(PCR) and clone it to PMD-18-T vector. Upload the sequence to the Gen Bank database after identified. Besides, respectively analyze the relationship between Sho/Dpl and Pr P binding related researches; Based on the codon preferences, G+C content and other characteristics of Pichia pastoris, to codon-optimize the target gene Pr P(25-248) and synthesize it. At the same time, amplify the uncodon-optimized target sequence. Afterwards, connect both sequences in p PIC9 K, then transform them into GS115 separately. After identification with PCR, MM, and MD, to express through induce and detect by SDS-PAGE.【Results】 Ch SPRN is 354 bp, and there are two G�'A base mutations at 23 bp and 56 bp, both are C�'Y mutation. Analysis the amino acid sequence and build the tertiary structures of Ch Sho and Ch Pr P by the homologous method, we saw that their N-end homology is 44.25%, and C-end structural similarity is 23.33%; Through the alignments of multi-species Dpl’s amino acid sequence, we found that the section 11-150 bits(140 amino acid residues) is the conserved region of Dpl’s amino acid sequence, through designing primers aim to this area and PCR, we obtained about 417 bp target band(KP140962 in Gen Bank). Through Comprehensively analysize the related researches of Dpl and Pr PC, we found that although Dpl and Pr PC have a similar post-translational modifications and spatial structure, they mostly show antagonism. Especially the 23-88 th residues of Pr PC’s N-terminal peptide, containing ORR, which are crucial for the protection of neural cells from Dpl induced neurodegeneration; Through identify, we constructed p PIC9K-Pr P(25-248), then got expression in GS115. Compared with uncodon-optimized, codon-optimized protein has higher expression.【Conclusion】(1) Ch Sho and Ch Pr P show striking similarities in sequence and structure;(2) The first time successful cloned chicken PRND gene;(3) Successfully got chicken Pr P by Pichia pastoris GS115 eukaryotic expression system, and get higer expression through codon-optimization than before.