伊氏锥虫微管结合蛋白p15基因的分子克隆与表达

MOLECULAR CLONING AND EXPRESSION OF TRYPANOSOMA EVANSI MICROTUBULE-ASSOCIATED PROTEIN P15 GENE

根据报道的布氏锥虫(Trypanosoma brucei)微管结合蛋白p15(Tb-MAPp15)基因及其3′非翻译区保守序列设计引物,采用PCR法从伊氏锥虫云南水牛株(Trypanosoma evansi stock YNB)基因组DNA中克隆得到伊氏锥虫微管结合蛋白p15(Te-MAP p15)基因。克隆片段长273bp,编码90个氨基酸,预测分子量9.0kDa。经同源性比较,所得基因与Tb-MAP p15基因同源率达到94%。抗原性分析Te-MAP p15基因表达的氨基酸序列比T.brucei微管结合蛋白p15多5个,均参与组成其中1个抗原决定簇,并且此抗原决定簇序列形成在蛋白中常作为识别位点的α螺旋。将该基因亚克隆到pGEX-6P-1原核表达载体,转化大肠杆菌E.coli RS21宿主菌,经IPTG诱导,可成功表达。重组融合蛋白大小为35kDa,与预期大小一致,经SDS-PAGE和Western-blot鉴定为重组伊氏锥虫微管结合p15蛋白。

On the basis of the nucleotide sequence of Tb-MAP p15 gene and its 3′conserved untranslated region from Trypanosoma brucei, a MAP p15 gene defined as Te-MAP p15 of Trypanosoma evansi YNB was isolated and cloned from the genomic DNA using the PCR method. It is composed of 273 nucleotides which encoded 90 amino-acid residues, the deduced mass being 9.0 kDa. Comparison of the Te-MAP p15 gene of T. evansi with that of T. brucei, the homology of nucleotides was up to 94%. Antigenic analysis showed that the protein translated by Te-MAP p15 gene contained 5 amino acids more than Tb-MAP p15, and the 5 amino acids took part in the formation of one antigenic determinant together with other N-terminal 11 amino acids. This determinant shaped a-helix which had been recognized as one of the important features of proteins. The Te-MAP p15 gene of T. evansi was subcloned into the prokaryotic expressing vector pGEX-6P-1, and expressed in the E. coli RS21. The recombinant protein showed a single band by SDS-PAGE, the size was about 35 kDa. Western-blot analysis showed that the fusion protein could be recognized by anti-GST antibody.