羊泰勒虫主要表面蛋白P32基因的克隆及真核表达载体的构建

Cloning of the Major Surface Protein P32 Gene from Ovine Theileria and Construction of Eukaryotic Expression Vector

提取羊泰勒虫裂殖子总RNA,用特异性引物扩增出其主要表面蛋白P32基因,并成功地将该基因纯化后克隆到pGEM-TEasy载体上,经PCR和EcoRⅠ酶切鉴定均为阳性的重组质粒进行了测序和分析。随后将该基因从克隆载体上双酶切,与毕赤酵母分泌性表达载体pPIC9K相连接,构建重组表达载体pPIC9K-MPSP-P32,转化大肠埃希菌JM109,经测序证实,基因序列完全正确。

The total RNA was isolated from the purified merozoite of ovine Theileria. The major surface protein P32 gene was amplified by RT-PCR and then cloned into pGEM-T Easy vector. Positive clones were identified by PCR screening and EcoR I digestion and sequenced. To construct recombinant eukaryoticexpression vector, the P32 gene was cleaved from the pGEM-T Easy plasmids by double restriction endonuelease digestion, and then subcloned into the Pichia pastoris expression vector pPIC9K, and the recombinant plasmid pPIC9K-MPSP-P32 was transformed into E. coli JM109. The gene sequencing suggested that the result was correct.