Cloning, Prokaryotic Expression Of E.coli CysE, CysM And Preparation Of Their Antibodies

The major component of goat cashmere fiber is keratin, which is a variety of combination of amino acids. The highest contents of keratin are glutamic acid and cystine, and cystine is a sulfur-containing amino acids. Goat and other mammals can only synthesis cysteine indirectly from methionine viatrans-sulphuration. Using genetic engineering, the expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the goat. It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. This study focuses on cloning serine acetyltransferase gene (cysE) and O-acetylserine sulfhydrylase-B gene (cysM) which encoding cysteine synthetase in Escherichia coli, prokaryotic expressing the enzymes and preparing their antibodies, which as preliminary works for the production of transgenic cashmere goat.(1) The cysE and cysM genes are amplified by PCR from Escherichia coli genomic DNA. The cysE gene is 822 bp, which encoding 274 amino acids, and cysM gene is 912 bp, which encoding 304 amino acids. Each one of them is a complete open reading frame. Sequences alignment between sequencing results and published Genbank cysE (8177668) and cysM (8176794) sequences shows 100% homology. Prokaryotic expression vectors pET32a-cysE and pET32a-cysM are successfully constructed and efficiently express in BL21 (DE3) after induced.(2) Induce by 1 mmol/L IPTG, 37℃, 6h, prokaryotic expression vectors pET32a-cysE and pET32a-cysM efficiently express in Escherichia coli BL21 (DE3), the expression of target protein over all the bacterial protein by 25%. The cysE and cysM proteins are purified from Bacterial lysate by Ni-Agrose affinity purification. SDS-PAGE results show that over 60% of the proteins are recovered, and the purity of purified proteins is 90%.(3) Using purified cysE and cysM proteins as antigens immunize New Zealand white rabbits to create polyclonal antibodies. Enzyme-linked immunosorbent assay (ELISA) detection shows that antibodies titers reach 1:102 400, indicates polyclonal antibodies have a strong immune binding activity. At the same time, using rabbit serum as primary antibody, HRP labeled goat anti-rabbit IgG as secondary antibody, the Western blott result shows that the antibodies have specific responses to cysE and cysM proteins. Cloning, prokaryotic expression and polyclonal antibody preparation methods and techniques of Escherichia coli cysE and cysM gene were successfully established. Identified and purified cysE and cysM protein from prokaryotic expression. The preparation of recombinant cysE, cysM and their polyclonal antibodies has provided reliable tools for the future study in the transgenic sheep of cysteine biosynthesis gene.