摘要
目的构建霜天蛾cDNA表达文库,为进一步筛选霜天蛾主要变应原、制备重组变应原奠定基础。方法利用Trizol法抽提霜天蛾RNA,经过Oligo(dT)纤维素柱纯化后得到mRNA,反转录合成dscDNA,连接EcoRⅠ接头,末端磷酸化,XhoⅠ消化,将双末端黏性化的双链dscDNA经过凝胶柱层析,收集大于400bp的cDNA片段,加工后与UniZAPXRVector连接,经过体外包装,感染宿主菌,构建cDNA文库,检测文库容量和重组效率,并采用PCR方法对插入的cDNA片段大小进行分析。结果成功构建了一个含有5×105重组子的霜天蛾表达文库,重组率为67%,平均插入片段长度约为1.49kb。结论所建文库容量及插入片段大小合适,适用于筛选目的cDNA克隆。
Objective To construct a cDNA expression library of psilgramma menephorn and provide the basis for screening the major allergen and producing recombination allergen of psilgramma menephorn. Methods Total RNA was extracted from the whole body of psilgramma menephorn with TRIZOL mRNA purified with Oligo (dT) Spin-Column. ds cDNA was synthesized through reverse transcription. EcoRI adapters were ligated to the blunt ends and the ends were phosphorylated. The XhoI digestion released EcoRI adapter. The fragments smaller than 400 bp were removed by GHROMA SPIN-400 column and the fragments longer than 400 bp were ligated to the Uni-ZAP XR vector. The lambda library was packaged in vitro and is plated on the E.coli cell line XL1-Blue MRF. The content and recombination rate of cDNA library were tested. The length of the inserted fragments was analyzed by PCR. Results The cDNA expression library contained 5×10 5 recombinants and the recombination rate was 67%. The average length of inserted cDNA fragments was about 1.49 kb. Conclusion The constructed cDNA expression library contains appropriate contents and size of cDNA fragments for screening target cDNA to clone.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2005年第3期244-246,279,共4页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.30271160)
关键词
霜天蛾
CDNA表达文库
鉴定
psilgramma menephorn
cDNA expression library
characterization
