ST细胞酵母双杂交cDNA文库的构建及鉴定

Construction and Characterization of the Yeast Two-hybrid cDNA Library of ST Cells

为了筛选与猪细小病毒(porcine parvovirus,PPV)结构蛋白VP2相互作用的宿主蛋白,深入研究病毒入侵及致病的分子机制,本试验建立了ST细胞酵母双杂交cDNA文库。采用RNeasy Min Kit提取ST细胞的总RNA,反转录合成cDNA第一链之后,通过SMART技术合成了双链cDNA,并利用同源重组的方法,构建了ST细胞的酵母cDNA文库。结果显示,文库滴度为8.1×108 CFU/mL,插入的双链cDNA片段大小为250~1 000bp,平均大小约为500bp,文库的重组率为96%。此文库可用于筛选与病毒相互作用的ST细胞宿主蛋白,为进一步阐明病毒的致病机制奠定了基础。

To seek out proteins interact with VP2 structural protein of porcine parvovirus（PPV）in ST cell and study the molecular mechanisms of viral infection and pathogenicity,a ST cDNA yeast hybrid library was created.Firtly,the total RNA of ST was extracted using RNeasy Min Kit,and double strands cDNA was synthesized by SMART technique.Then,the ST cDNA library was successfully created by homologous recombination in yeast,the titer was 8.1×10^8 CFU/mL,the inserts varied from 250 to 1 000 bp,the average of inserted fragments was about 500 bp,and the recombination rate was 96%.These datas indicated that the yeast twohybrid cDNA library was successfully constructed and might be useful for screening the host proteins interacting with structure protein of PPV.