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S-adenosylmethionine (SAM) is an important intermediate in vivo metabolite , the 40 kinds of biochemical reactions involved , from the gene expression , cell membrane , etc. flows into the peptide synthesis . Clinical studies have shown , SAM for arthritis , depression , liver function disorders have a good effect , but also the prevention of cancer , cardiovascular disease and anti-aging senior health care drugs. A considerable number of patients with hepatitis , and the faster pace of life , depression and arthritis patients have continued to increase , S- adenosylmethionine broad market presence in the country . So far, S-adenosylmethionine domestic research has not yet industrialized , although some foreign supply of goods , but the price is not expensive and popular. Therefore, the domestic development of high-yielding low-cost S-adenosylmethionine production process is extremely urgent. In this thesis, completed the Saccharomyces cerevisiae S-adenosylmethionine synthetase gene (sam2) Cloning and prokaryotic expression . 2B-41 in Saccharomyces cerevisiae chromosomal DNA as a template , according to the GenBank landing S-adenosylmethionine synthetase gene ( accession number M23368) primers were designed for PCR amplification of a DNA fragment of about 1200bp . By TA cloning technique, the PCR product was cloned into pUCm-T vector to construct the recombinant plasmid pUCm-T-sam2, was transformed into E. coli DH5 α, by blue-white screening , colony PCR and plasmid digested two positive clones were screened out . Select a positive clones were sequenced and sequence analysis using Blast software . The results showed that the sequence registered in Genbank sam2 gene homology of 99% to 100% amino acid sequence identity . With EcoR I / XhoI digested by sequencing the correct recombinant plasmid pUCm-T-sam2 and expression vector pET-28a (), and purified sam2 connected to the pET-28a (), construct the expression vector pET-sam2. The pET-sam2 transformed E.coli BL21 (DE 3 ). Recombinant bacteria induced by IPTG , SDS-PAGE electrophoresis express exogenous protein molecular weight of about 47KD, consistent with the expected molecular weight . While the effects of different IPTG concentrations and different induction temperature effects on protein levels found IPTG concentration on the expression of SAM little effect , low temperature is not conducive to the expression . Enzyme activity was measured as 0.0237U/mL.
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