环形泰勒虫TRAP-A蛋白酵母双杂交诱饵质粒的构建与鉴定

CONSTRUCTION AND IDENTIFICATION OF THE PGBKT7-TRAP-A PLASMID

为了筛选出环形泰勒虫TRAP蛋白在虫体入侵媒介蜱唾液腺过程中相互作用蜱源蛋白,构建了能够用于酵母双杂交筛选系统的重组诱饵质粒pGBKT7-TRAP-A。本研究以环形泰勒虫裂殖体为材料,根据环形泰勒虫TRAP-A结构域的基因序列设计引物,经过PCR扩增获得576 bp的基因片段,将其连接到线性化载体pGBKT7上。经双酶切鉴定和序列分析以及重组诱饵质粒在酵母双杂交系统中的自激活、细胞毒性及其表达情况检测,结果显示,本研究成功构建了酵母双杂交重组诱饵质粒pGBKT7-TRAP-A,构建的诱饵质粒对Y2HGold酵母菌无自激活活性和细胞毒性,且构建的诱饵质粒pGBKT7-TRAP-A可以在Y2HGold酵母菌内正确表达。表明所构建的诱饵质粒pGBKT7-TRAP-A可以用于筛选小亚璃眼蜱唾液腺酵母双杂交c DNA文库,获得可能与环形泰勒虫TRAP-A蛋白相互作用的蜱源蛋白。

In the present study, the bait plasmid of pGBKT7-TRAP-A were constructed to screen the interactive proteins derived from the salivary glands of Hyalomma anatolicum anatolicum with Theileria annulata TRAP-A protein. The ampliflcation primers were designed based on the sequence of TRAP-A adhensive domain of annulata merozoite. The fragment with the length of 576 bp was amplifled and ligated into the pGBKT7vector. The pGBKT7-TRAP-A plasmid was conflrmed using enzyme digestion and sequence analysis and then transformed into yeast cells Y2HGold. The TRAP-A fusion protein was expressed and examined for its autoactiviation and toxicity to yeast growth. Results indicated that the TRAP-A fusion protein was expressed in yeast cells Y2HGold without interruption of cellular growth. In addition, the recombinant protein had no transcription activity of reporter genes or toxicity to the yeast cells Y2HGold. Therefore, the pGBKT7-TRAP-A plasmid may be used for the screening of the interactive proteins from Hyalomma anatolicum anatolicum in yeast two-hybrid system.