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The Construction of Mutation Libraries and Screening Method of α-N-acetylgalactosaminidase

Author: LinYiGang
Tutor: XiaGang
School: East China Normal University
Course: Genomics
Keywords: α-N- acetyl- galactosamine enzyme Site-directed mutagenesis High-throughput screening
CLC: Q785
Type: Master's thesis
Year: 2011
Downloads: 19
Quote: 0
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α-N- acetyl galactosamine enzymes are blood type conversion tool enzymes that can degrade type A red blood cell surface antigens , eliminating the antigen -antibody reaction , transfusion therapy, avoids the inconvenience of blood type matching . From our laboratory reported α-N- acetyl galactosamine best enzyme activity of the enzyme selected to construct α-N- acetylgalactosamine enzyme prokaryotic expression vector , expression and purification of α-N- acetyl galactosamine enzyme , detecting enzyme activity . Site-directed mutagenesis techniques , mutations at critical loci with high-throughput screening technologies mutation screening library , hoping to get a higher activity of α-N- acetyl galactosamine enzymes. The research topics include the following two parts : 1.α-N- acetyl galactosamine build in order to establish a new enzyme α-N- acetylgalactosamine enzyme screening , detection methods , we extract the meninges golden genomic DNA of Bacillus , was amplified by PCR as a template α-N- acetyl- galactosamine enzyme (A4). The A4 was cloned into pET-24a vector and transformed into the expression strain B121 protein . Purified by affinity chromatography using the His-A4 enzyme , a chromogenic substrate selected authentication activity. We improved the traditional ELISA method , the red cell membrane coated directly on ELISA test plates to red cell surface antigen as a direct substrate , enzyme activity was detected by ELISA . This study established a novel ELISA assay method , this method proved A4 enzyme activity of this enzyme that can effectively reduce the red blood cell surface antigen-antibody reaction, and a concentration and time dependent . 2.α-N- acetyl- galactosamine library screening mutant enzyme nature of the α-N- acetyl- galactosamine activity is low, not enough for mass production of the enzyme preparation of universal donor blood . In this experiment , the use of site-directed mutagenesis technology combined with high-throughput screening methods , expect higher activity glucosidase . We mutated enzyme six amino acid sites are 96,98,179,181,225,228 , screened about 2,400 recombinants from the experiment shows that the position mutated enzyme 179,181,225,228 activity decreased substantially , so that four loci on the enzyme activity is essential. Both point mutations 96, 98 , the activity of four mutations rather , they change in activity after mutation mechanism needs further study.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering) > Transformation and cloning
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