法国梧桐花粉变应原cDNA表达文库的构建和初步鉴定  

Construction and primary identification of c DNA expression library of Platanus acerifolia pollen allergen

作　　者：刘昀[1] 孙秀珍[1] 徐晶[1] 吴媛媛[1] 史红阳[1] 方萍[1] 

机构地区：[1]西安交通大学医学院第二附属医院呼吸内科,西安710004

出　　处：《山西医科大学学报》2015年第4期327-330,共4页Journal of Shanxi Medical University

基　　金：国家自然科学基金资助项目(81102252);卫生部重点课题基金资助项目(2007353)

摘　　要：目的构建法国梧桐花粉变应原c DNA表达文库并初步鉴定。方法利用Trizol法抽提法国梧桐花粉总RNA,经过Oligo(d T)纤维素柱纯化后分离mRNA,反转录合成ds c DNA,连接EcoRⅠ接头,末端磷酸化,XhoⅠ消化,电泳分离并凝胶回收大于500 bp以上的c DNA片段,与Uni-ZAP XR Vector连接,经过体外包装,感染宿主菌,构建c DNA文库,检测文库容量和重组率,并采用PCR方法对插入的c DNA片段大小进行分析。结果成功构建了一个文库容量为3.5×106pfu/ml,重组率为94%,平均插入片段长度约为0.7 kb的法国梧桐花粉变应原c DNA表达文库。结论所建文库容量及插入片段大小合适,适用于筛选目的 c DNA,为进一步研究法国梧桐花粉主要变应原基因并制备重组标准化法国梧桐花粉主要变应原疫苗奠定基础。Objective To construct a c DNA expression library of Platanus acerifolia pollen allergen and identify its characteristics.Methods Total RNA of Platanus acerifolia pollen was extracted with Trizol and the mRNA was purified with Oligo（ d T） Spin column.The ds c DNA was synthesized using reverse transcription.EcoR I adapters were ligated to the blunt ends and the ds DNA ends were phosphorylated.The ds c DNA was digested by Xho Ⅰ.The c DNA fragments were separated by electrophoresis and the fragments longer than 500 bp were extracted from gel.The fragments were ligated to the Uni-ZAP XR vector.The lambda library was packaged in vitro and was plated on the E.coli cell line XL1-Blue MRF＇.The titer and the effectivity of the obtained c DNA library were tested.The length of the inserted c DNA fragments was analyzed by PCR.Results The c DNA expression library of Platanus acerifolia pollen allergen was constructed successfully.The titer of obtained c DNA library comprised 3.5 × 106 pfu / ml in average and the effectivity of c DNA fragment insertion was 94%.In average the length of c DNA insertion was about 0.7 kb.Conclusion The c DNA expression library contains appropriate contents and the size of c DNA fragments is suitable for screening the target c DNA clone.This expression library provides the basis for screening the major Platanus acerifolia pollen allergen gene and producing recombination allergen vaccine.

关 键 词：法国梧桐花粉 变应原 CDNA表达文库 

分 类 号：Q75[生物学—分子生物学]