【摘要】 应用SMART技术构建猪肾上皮细胞(PK-15)酵母双杂交cDNA文库,为进一步筛选与副猪嗜血杆菌(HPS)细胞致死膨胀毒素(CDT)不同亚基蛋白相互作用蛋白奠定基础。提取PK-15的总RNA,经LD-PCR合成双链cDNA,运用SMART技术,再通过同源重组的方法,构建了PK-15细胞的酵母cDNA文库。经检测,文库滴度达到了2.8×108CFU·mL-1,插入的双链cDNA片段范围为300～1 000 bp,片段平均大小为500 bp。此文库可用于筛选与HPS CDT不同亚基蛋白相互作用的蛋白,这将为治疗靶点的选择、基因缺失弱毒活疫苗的设计提供科学依据。更多还原

【Abstract】 The yeast two-hybrid cDNA library of porcine renal epithelial cells(PK-15)was constructed by SMART technique,which laid a foundation for further screening of different subunit protein interacting proteins with Haemophilus parasuis(HPS) cytolethal distending toxins(CDT). Firstly,the total RNA of PK-15 was extracted and synthesized cDNA by LD PCR. Next,the PK-15 yeast two-hybrid c DNA library was constructed by using SMART technique and the homologous recombination method. The results showed,the titer of built library reached 2.8×108 CFU·mL-1,insertion element located between 300-1 000 bp,the average insert size of the cDNA library was 500 bp. The library could be used to screen proteins interacting with different subunit proteins of HPS CDT,which would provide scientific basis for the selection of therapeutic targets and the design of weakly toxic live vaccines with gene deletion.更多还原