Cloning,Bioinformatics Analysis and Eukaryotic Expression Vector Construction of GSTA2 Gene in Sheep
To study the function of glutathione S-transferase alpha 2(GSTA2)gene in sheep,primers were designed according to bovine GSTA2 gene sequence,and total RNA were extracted from sheep skin tissue.GSTA2 were cloned using RT-PCR and eukaryotic expression vector was constructed.The sequence of GSTA2 was analyzed using bioinformatics analysis.Sheep fibroblasts were transfected with GSTA2 eukaryotic expression vector,and the expression of GSTA2 mRNA was detected by qPCR technique.The results showed that CDS of sheep GSTA2 gene(GenBank accession number:OR440604.1)was cloned.The length of GSTA2 CDS was 672 bp,and encoded 223 amino acids.Its molecular weight was 25.39 ku.Amino acid comparison and phylogenetic tree analysis showed that sheep GSTA2 had the highest similarity to Bos taurus,reaching 87.39%.The secondary structure of GSTA2 was mainly composed of α-helix and random coil.The recombinant expression vector pcDNA3.1-GSTA2 was constructed,and the expression level of GSTA2 gene was significantly up-regulated after transfected into sheep fibroblasts(P＜0.001).It was indicated that a new gene(GSTA2)CDS of sheep was successfully cloned and its eukaryotic expression vector was correctly constructed.
万方数据
维普
