金黄色葡萄球菌N315T7噬菌体cDNA展示文库的构建  

Construction of T7 phage display cDNA library from methicillin-resistant Staphylococcus aureus N315

作　　者：于俊媛 周莹冰[2] 张雯庆 祁琳[1] 李萌[1] 姜北[1] 张骁鹏 汪正清[1] 饶贤才[1] 黎庶[1] 胡晓梅[1] 

机构地区：[1]第三军医大学基础医学部微生物学教研室,重庆400038 [2]重庆市渝中区疾病预防控制中心,重庆400010

出　　处：《第三军医大学学报》2014年第13期1350-1353,共4页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(CSTC2011jjA10050;CSTC2011BB5026)~~

摘　　要：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1.335×106 pfu，扩增文库的噬菌体滴度为3.26×1010 pfu/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 T7 phage display cDNA library from methicillin-resistant Staphylococcus aureus （MRSA） N315 in order to found a basis for screening and identification of regulatory factor of N315 staphylococcal cassette chromosome gene （scc）. Methods Total RNA was extracted from MRSA N315 by Tripure reagents and digested with DNase Ⅰ to get rid of the contamination of genomic DNA. Then, the total RNA was reverse-transcribed into double-stranded cDNA with random hexamer primers, and subsequently treated with end modification, adaptors ligation, and EcoRⅠ/HindⅢ digestion. After eliminated excess adaptors and small fragments （less than 300 bp）, the cDNA fragment was ligated into T7Select 10-3 vector. The MRSA N315 phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR amplification were used to evaluate the library. Results The total number of primary recombinants was about 1.335×106 pfu, and the phage titer of the amplified library was 3.26×1010 pfu/mL. Insert ratio of 24 random plaques was proved to be 100% by PCR amplification, and the length of insert DNA ranged from 300 bp to 1 500 bp. Conclusion A T7 phage display cDNA library from MRSA N315 is successfully constructed.

关 键 词：噬菌体展示 CDNA文库 耐甲氧西林金黄色葡萄球菌 N315 

分 类 号：Q785[生物学—分子生物学] Q939.48