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Cloning, Expression and Function of Partial Fragment of Bovine Prion Protein and Studying of Prion Protein Detection

Author: ZhangDaChuan
Tutor: LiuZhiGuo
School: Wuhan Polytechnic University
Course: Microbial and Biochemical Pharmacy
Keywords: Partial fragment of bovine prion E.coli BL21(DE3) clone prokaryotic expression FGP
CLC: S852.659.7
Type: Master's thesis
Year: 2011
Downloads: 14
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Abstract


Partial fragment of bovine prion Protein(PrP) was cloned and expressed,it was used to study the structure,properties and detection of prion. According to the gene sequence of the local fragment of bovine prion(90~231 Amino acids),12 oligonucleotide fragments were designed with E.coli-preferred codon. Then the gene of PrP was synthesized by overlap extension PCR. After sequencing was finished, it was cloned into plasmid pET-28a(+).The recombinant plasmid was transformed into E.coli BL21(DE3) by calcium chloride transformation method. Recombinant protein was induced by IPTG and expression conditions were optimized . With the tag of 6×His in the plasmid pET-28a(+), the recombinant protein was purified by a single step affinity chromatography with Ni+-NTA.After purification was finished,the recombinant protein was useed to study the structure, properties and detection of prion.The result showed that the gene of PrP was synthesized successfully.The recombinant plasmid pET-28a(+)-PrP was constructed and the recombinant protein was expressed in E.coli BL21(DE3). When the IPTG concentration was 0.8 mmol/L and induction time was 6 hours,PrP was expressed highly.After PrP was purified,its purity reached 95% and its molecular weight was the expected 19.15 kilo Da. Recombinant PrP can be completely hydrolyzed by proteinase K, This fragment of PrP has a significant specific binding Affinity for FGP (FGP has a specific binding Affinity for PrPsc),the Binding constant is about 105.It was confirmed that the recombinant PrP can replace PrPsc to study the structure and properties of the nomal PrPsc, and it is safe for studying.FGP was used to concentrate PrP in the Sample to be detected, and then detect the PrP in the Concentrate by Western blot, the Detection limit is 0.5pmol/L. Campared with traditional methods( detection limit is 3~5pmol/L) , this way of detection is more sensitive.In this study , the recombinant plasmid pET-28a(+)-PrP was constructed and high expression of PrP provided convenience for the study of PrP.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology > Prion
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