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目的构建MRSA N315菌株的T7噬菌体cDNA展示文库,以筛选和鉴定影响N315 ccr基因表达的调节因子。方法采用Tripure试剂提取N315总RNA,逆转录合成双链cDNA,经末端修平、接头连接、酶切后柱洗脱等处理后,连接T7Select 10-3载体,体外包装扩增获得N315 T7噬菌体cDNA展示文库;通过双层平板实验鉴定所建原始文库及扩增文库的库容;PCR鉴定文库插入片段质量。结果原始文库重组克隆数为1.335×106pfu,扩增文库的噬菌体滴度为3.26×1010pfu/mL。随机PCR扩增显示文库阳性重组率为100%,插入片段在300~1 500 bp之间。结论 MRSA N315的T7噬菌体cDNA展示文库构建成功。
Objective To construct a T7 phage cDNA display library of MRSA N315 strain for the screening and identification of regulatory factors that affect the expression of the N315 ccr gene. Methods Total RNA of N315 was extracted by Tripure reagent and double-stranded cDNA was reverse transcribed. T7Select 10-3 vector was ligated after being trimmed, ligated and digested with enzyme. The cDNA of T7Select 10-3 was amplified and packaged in vitro. Library; The library of the original library and the amplified library was identified by double-layer plate experiment; The mass of the inserted library was identified by PCR. Results The number of recombinant clones in the original library was 1.335 × 106 pfu, and the phage titer in the amplified library was 3.26 × 1010 pfu / mL. Random PCR amplification showed that the library-positive recombination rate was 100% and the inserted fragment was between 300 and 1500 bp. Conclusion The T7 phage cDNA display library of MRSA N315 was successfully constructed.