Cloning Of Full Length Of CDS Encoding For MT1, MT2 And MT3 Genes And Prokaryotic Expression Of MT2 Gene In Rabbit

Total RNA was extracted from muscle tissue,brain tissue,gastric tissue and liver tissue of ten rabbits. The CDS sequence of rabbit MT1,MT2 and MT3 genes was cloned by RT-PCR using the primer that was designed according to CDS sequence of human, musculus bovine and rat in GenBank. The CDS sequence were 186bp,186bp and 201bp in length,encoding 61,61 and 66 amino acids of MT1,MT2 and MT3 genes separately. It's 82.42% homologous of MT1,MT2 and MT3 genes.It's 78.28% amino acid homologous of MT1,MT2 and MT3.genes.Analysis of the rabbit MT1 gene CDS sequence showed that it was highly homologous to other species such as Canis familiaris, Homo sapiens, Rattus norvegicus and Mus musculus. MT2 gene CDS sequence showed that it was highly homologous to other species such as Bos taurus, Homo sapiens, Rattus norvegicus,Mus musculus and Pongo abelii. MT3 gene CDS sequence showed that it was highly homologous to other species such as Bos taurus,Canis familiaris,Homo sapiens,Rattus norvegicus,Sus scrofa,Mus musculus and Ovis aries.It Construct phylogenetic tree of MT1,MT2 and MT3.Analysis of the MT1,MT2 and MT3 genes by bioinformatics methods.It predicted that protein molecular weight were 6.8 kDa,7.81 kDa and 4.25kDa and isoelectric point were 5.12,5.34 and 5.32 separately.There are five main hydrophobicity region,three potential phosphorylation sites,ten disulfide bonds separately of MT1.There are five main hydrophobicity region,three potential phosphorylation sites,ten disulfide bonds separately of MT2.There is one N-linked glycosylation site at N-199, one main hydrophobicity region,two potential phosphorylation sites,Ten disulfide bond separately of MT3.Analysis protein secondary structure of the MT1,MT2 and MT3.The protein secondary structure formed by random coli whit amount of 96.72% that composed by 59 amino acid residues of MT1.The protein secondary structure formed by random coli whit amount of 95.08% that composed by 58 amino acids residues of MT2.The protein secondary structure formed base on a a-helices by 6 amino acids whit amount of 9.09%,the interval amonga-helices standed by random coli which formed by 54 amino acids residuces with amount of 81.82% of MT3.The CDS of MT2 gene is constructed into prokaryotic expression vector pET-32a, and induced to express in E.coli BL21(DE3).The result indicated that MT2 was an 27kDa fusion protein with Trx-tag,His-tag and S-tag.Optimizing the expressive condition of fusion protein, we found the optimal induction concentration was 0.6 mmol,the best time for induction was 8h.We found two expressive forms of fusion protei, soluble fusion protei and insoluble fusion protein, by analysised its solubility for this protein.