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Cloning, Prokaryotic Expression of E.coli cysE, cysM and Preparation of Their Antibodies

Author: Fang
Tutor: ChenYuLin
School: Northwest University of Science and Technology
Course: Animal Genetic Breeding and Reproduction
Keywords: E. coli (Escherichia coli) Serine acetyltransferase gene ( cysE ) O-acetyl- homoserine sulfhydrylase gene ( cysM ) Gene The prokaryotic expression Antibody
CLC: S827
Type: Master's thesis
Year: 2011
Downloads: 74
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Abstract


The main components of the cashmere keratin and keratin is the combination of many amino acids, one of the highest concentrations of glutamate and cystine, cystine is a sulfur-containing amino acids. Sheep and other mammals can not direct the synthesis of cysteine, methionine can only rely on trans-vulcanization indirect synthesis of cysteine. Using transgenic technology cysteine ??synthase transferred to Cashmere goats, the sheep body itself to efficient synthesis of cysteine, greatly contribute to improve the quality of cashmere role. The purpose of this study is to be amplified by PCR using E. coli (Escherichia coli) homoserine acetyl transferase gene (cysE) the hydrogen sulfide and O-acetyl homoserine-lyase gene (cysM), to construct plasmid vectors for prokaryotic expression, and the corresponding protein was prepared polyclonal antibody, for the production of cysteine ??synthase transgenic cashmere goat lay method and technical basis. (1) cysE gene was amplified from E. coli genomic DNA, a length of 822 bp, encoding 274 amino acid residues, cysM gene of full-length 912 bp, encoding 304 amino acid residues, each of a complete open reading frame. The sequence obtained after the sequencing, published in Genbank cysE (8177668) and cysM (8176794), compared to the sequence homology reaches 100%. Successfully constructed by gene recombination prokaryotic expression vector the pET32a-cysE and the the pET32a-cysM, and can be induced in E. coli BL21 (DE3) to achieve efficient expression. (2) the original expression vector the pET32a-cysE and pET32a-the cysM, expression in E. coli BL21 (DE3) induced by 1 mmol / L IPTG, 37 ℃, 6 h, expression of the target protein in the amount of more than 25% of total bacterial protein expression. Through the Ni-Agrose affinity column supernatant purified cysE and cysM fusion protein purified from the bacterial lysates. SDS-PAGE results show that up to 90% purity of the fusion protein affinity column purification method, the recovery efficiency of 60%. (3) New Zealand white rabbits were immunized with the purified cysE cysM protein, polyclonal antibody. Detection antibody titer by enzyme-linked immunosorbent assay (ELISA), antibody titers reached 1:102.4 K, showed that purified polyclonal antibody prepared to have stronger immune binding activity. Has a specific reaction to rabbit serum as an anti-HRP-labeled goat anti-rabbit IgG secondary antibody, Western blot analysis of the the cysE and cysM fusion protein, indicating that the test antibodies against the prokaryotic expression cysE cysM fusion protein . Established through this study the of E. coli cysE and cysM gene cloning, prokaryotic expression and polyclonal antibody preparation methods and techniques. For further study of the cysteine ??synthase transgenic cashmere goat technical foundation, has important theoretical value of breeding practices.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Goat
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