Cloning And Prokaryotic Expression Of Moschus Sifanicus Prion Protein Gene

Objective This research is to obtain prion protein(PrP) gene DNA cloning and prokaryotic expression proteins of one musk deer. Then the character of gene structure and the properity of celluar isoform of musk deer PrP can be realized; the result will enrich the research of the PrP gene about ruminant in the world, and bring experimental materials for the research of protein structure and the mechanism of interspecies barrier for PrP infection. .Methods Adopting the molecular cloning and the recombinant DNA technology, using the total DNAs which were extracted from peripheral whole-blood of musk deer, the PrP genes were amplified by polymerase chain reaction (PCR) with a pair of primers, and then cloned into pMD 18-T Vector, the entire clone of PrP gene was constructed. After sequencing, the nucleotides were analyzed & the amino acid sequences and their homology were deduced by DNAstar software; taking the entire recombinant PrP gene plasmid, the needed gene for expression was amplified by PCR,and the recombinant expression plasmid pGEX-MuPrP(85-233) was constructed, then it was transformed into E.coli BL21(DE3), after optimizing the expression condition and inducing the expression of recombinant bacterium, the result was identified by SDS-PAGE and Western blotting, the prokaryotic expression bacterium of the recombinant must deer PrP(85-233) was constructed with success, which was named GST-MuPrP(85-233). Results The sequencing showed all the entire PrP coding sequences have the complete ORFs contained within a single exon , and the total length is 771 bp. The sequences of the musk deer PrP gene cloned in this experiment contained five copies of a short, G-C-rich element, which encoded the octapeptide Pro-His-Gly-Gly-Gly-Trp-Gly-Gln or the nonapeptide Pro-Gln/His-Gly-Ala/Gly-Gly-Gly-Trp-Gly-Gln. The amino acid sequences of the gene have a 24 amino acids of N-terminal signal peptide and a 23 amino acids of C-terminal signal peptide. The comparison of the genes with white-tailed deer and red deer PrP gene revealed that the nucleotide and its putative amino acid identities were 97.4%-98.2% and 97.7%-98.8%, respectively, it show the PrP genes of ruminant among the same family or different families are all conservative. The comparison of the genes with white-tailed deer and red deer PrP gene revealed there are five base substitutions produced amino acid mutation, e.g. G57A, S100N, N173S, T177N and M208I; the comparison of the genes with sheep PrP gene revealed there are 3 base substitutions produced amino acid mutation, e.g. G57A, S98T and S100N ; the comparison of the genes with bovine PrP gene revealed there are 6 base substitutions produced amino acid mutation, e.g. G57A, G100N, S146N, H158Y, E189Q and M208I, and they lack a octapeptide vs bovine PrP. The base substitution G57A lies in the fourth of the first nonapeptide, this mutation have not been published, it is owned by musk deer PrP gene. When the constructed recombinant expression plasmid pGEX-MuPrP(85-233) of musk deer PrP gene had been transformed into E.coli BL21(DE3), the recombinant prokaryotic expression bacterium E.coli GST-MuPrP(85-233) was obtained. The expectant molecular size about 42.4 ku of fusion proteins is produced by the rencombinant bacterium. Under the optimized expression condition, the recombinant expressed product is account for over 30% of overall bacterium protein; it could be responsed with the anti-bovine PrP monoclonal antibody 4C11 by Western blotting, it show all mutations do not effect epitode recognized by the monoclonal antibody 4C11.