Cloning And Prokaryotic Expression Of Lipoic Acid Synthase Lias Gene Of Porcine

Lias Acid Synthetase (Lias) is the enzyme that is involved in theendogenous synthesis of lipoic acid and exist widely in a variety of organisms Lipoicacid is a potent mitochondrial antioxidant and involved in energy and amino acidmetabolism. Lipoic acid is one of the most potent natural antioxidants and is vital forcellular function. At present, people have cloned a variety of biological lipoic acidsynthetase gene.There have been a lot of research on the biological functions of lipoicacid, while there are quite few research on the lipoic acid metabolism on anmals, andless on the lipoic acid synthetase.So far no paper about the Lias gene of pig have beenreported.In order to further study the function of the porcine Lias Acid Synthetase (Lias),the primer was designed based on the silico sequences. The Lias Acid Synthetase(Lias) gene was cloned by RT-PCR, and its tissue distribution was detected. Specificprimers of Lias were designed. Then production of the PCR connected with theexpression vector pET-28a(+),after the PCR of the plasmid, restriction enzymedigestion and sequencing, the recombinant plasmid was transferred into theexpression bacterial BL21. To determine the optimum conditions for proteinexpression, the recombinant plasmid was induced with different concentration ofIPTG and different times.The protein was identified by Western blot. The molecular weight is41.58kDa.The results show that:1. The cloning, sequence analysis and tissue distribution of the porcine LiasThe cloned Lias gene contains a complete open reading frame (ORF), and itssequence length is1119bp, and the coding protein include372amino acid residues,which contains72acidic amino acids and65basic amino acids, the molecular weightis41.58kDa and the isoelectric point is9.05. Identity analysis shows that the Liaspredicted peptide sequence in sus scrofa shared91.5%,79.4%,89.4%,88.2%homology with Mus musculus, Rattus norvegicus,Homo sapiens, Bos taurusrespectively. Semi-quantitative RT-PCR analysis showed that the expression level ofLias is the highest in heart,liver, kidney and adipose tissue, spleen, skin, rectum, inlung, duodena jejunum and ileum, muscle.the result showed that lias expressed in all these tissues, but it is higher in the kidney and fat. The phylogenetic tree demonstratesthat pig is the closest to Mus musculus,Rattus norvegicus.2. The prokaryotic expression and identification of the porcine LiasThe ORF of the porcine Lias was cloned into the expression vector pET28a(+),the recombinant expression pasmid was identifified by restriction enzyme digestingand sequencing, the result shows that pET-28a（+）-Lias has been expressedsuccessfully. Then the recombinant expression pasmid pET-28a（+）-Lias wastransformed into Escherichia coli BL21(DE3). The protein molecular weight of thetarget protein is41.58kDa by SDS-PAGE. The fusion protein was mainly expressedin the inclusion body. The obtained inclusion bodies a single target band identified byWestern blot. The band was purposed protein Lias.