PK-15细胞酵母双杂交三框cDNA文库构建及鉴定  

Construction and identification of yeast two-hybrid three-frame cDNA library of PK-15 cells

作　　者：李珣[1] 陈思宇[1] 胡传活[1] 王晓晔[1] 

机构地区：[1]广西大学动物科学技术学院,南宁530005

出　　处：《南方农业学报》2016年第5期726-730,共5页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31502079;31402151);广西自然科学基金项目(2015GXNSFBA139067;2015GXNSFBA139077)

摘　　要：【目的】构建PK-15细胞酵母双杂交三框eDNA文库，为深入研究猪伪狂犬病病毒（PRV）与宿主细胞间的相互作用机制打下基础。【方法】提取PK-15细胞总RNA，采用SMART和LD-PCR合成全长的双链cDNA（dscDNA），并对其进行均一化和SfiI酶切处理。，处理后的dscDNA分别连接3种阅读框pGADT7-SfiI载体，将连接产物转化至大肠杆菌BH5α构建三框cDNA初级文库，取三框cDNA初级文库进行混合扩增，通过滴度测定和PCR鉴定其库容量和基因重组率，最后收集文库细菌提取文库质粒。【结果】3种读码框cDNA文库的库容量分别为3．0×10^6,2．0×10^6和2．0×10^6CFU，文库实际扩增基数〉150万CFU；一号和三号读码框的基因重组率均为100％，二号读码框的基因重组率大于93％。cDNA文库外源基因插入片段长度在500～3000bp。cDNA文库质粒浓度约1．0mg/mL，质粒收获量约3．0mg。【结论】构建的PK-15细胞酵母双杂交三框cDNA文库具有较高的重组率和库容量，符合酵母双杂交筛选要求，可用于研究PRV与PK-15细胞间的相互作用机制。【Objective】Yeast two-hybrid three-frame c DNA library of PK-15 cells was constructed, in order to lay the foundation for investigating interaction mechanism between pseudorabies virus（PRV） and its host cells. 【Method 】Total RNA was extracted from PK-15 cells, and the full-length double-strand c DNA（ds c DNA） was synthesized using SMART and LD-PCR, which was ligated to three-frame expression vector p GADT7-Sfi I after nomalization and Sfi I digestion,respectively. The ligation product was transformed into Escherichia coli to construct primary three-frame expression c DNA library. Three primary c DNA libraries were mixed and amplified. The capacity and recombination rate of library were determined by titer test and PCR identification. Finally, all bacteria were collected to extract library plasmid. 【Result】The capacities of three reading frame libraries were 3.0×10^6, 2.0×10^6and 2.0×10^6CFU, respectively. The practical amplification base of library was more than 1.5 million CFU. The recombination rates of three frame libraries were 100%, more than93% and 100%, respectively. The inserted fragment of exogenous gene was 500-3000 bp in length. The plasmid concentration of c DNA library was about 1.0 mg/m L, the plasmid yield was about 3.0 mg. 【Conclusion】The constructed yeast two-hybrid three-frame c DNA library of PK-15 cells has high capacity and recombination rate, so which meet the requirements of yeast two-hybrid screening, can be used to research interaction mechanism between PRV and PK-15 cells.

关 键 词：PK-15细胞 酵母双杂交 三框cDNA文库 均一化 猪伪狂犬病病毒(PRV) 

分 类 号：S852.65[农业科学—基础兽医学]