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作 者:张洁[1] 孙秀珍[1] 颜虹[2] 张妮[3] 冯向莉[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 [2]西安交通大学医学院公共卫生系 [3]西安交通大学医学院第二附属医院康复医学科,陕西西安710004
出 处:《细胞与分子免疫学杂志》2011年第5期504-506,510,共4页Chinese Journal of Cellular and Molecular Immunology
基 金:卫生部临床学科重点项目(2007)
摘 要:目的:构建藜草花粉变应原λ噬菌体cDNA表达文库,并进行初步鉴定。方法:用TRIzol试剂抽提藜草花粉总RNA并分离mRNA。以纯化的mRNA为模板,以含有XhoI内切酶位点和18 bp的Poly(dT)序列的引物,用ZAP Express cDNA合成试剂盒反转录合成cDNA。将cDNA修饰、分级纯化后,获得大于400 bp的cDNA片段。将带有黏性末端的双链ds cDNA与Uni-ZAP XR载体连接,采用ZAP Express cDNA文库合成试剂盒利用λ噬菌体构建藜草花粉λ噬菌体cDNA表达文库,用包装蛋白对合成的ds cDNA进行体外包装,以包装产物感染宿主菌XL1-Blue MRF即获得cDNA表达文库。用PCR方法检测cDNA表达文库的滴度和插入片段的大小。结果:构建的藜草花粉变应原λ噬菌体表达文库的滴度为9.7×108pfu/L,重组率为100%,库容量为4.85 pfu。插入片段的长度平均约为1.0 kb。结论:构建的cDNA表达文库的容量及插入片段的大小合适,适用于藜草花粉变应原cDNA克隆的筛选,为制备基因重组藜草花粉变应原疫苗奠定了基础。AIM: To construct and identify the express library of album pollen allergens cDNA.METHODS: Total RNA were extracted from the album pollen with TRIzol reagent and the mRNA was isolate for the amplify followed.A double stranded cDNA(ds cDNA) was synthesized using primers containing Xho I and Poly(dT) sequence by ZAP Express cDNA synthesis kit.The ds cDNA was modified and purified by gel chromatography,and then the cDNA fragment with the length of more than 400 bp containing sticky ends was obtained.The cDNA fragment was ligated with Uni-ZAP XR vector and subsequently treated with in vitro packaging using phage by ZAP-cDNA express GigapackⅢ Gold cloning kit.The express library of album pollen cDNA was constructed by in vitro packaging.The recombination rate and the lengths of fragments inserted of the cDNA library were detected by polymerase chain reaction.RESULTS: The titer and the recombination rate of cDNA expression library constructed were 9.7×105 and 100%,respectively.The capacity of the library was 4.85 Pfu.The average length of cDNA fragments inserted was about 1.0 kb.CONCLUSION: Based on the capacity of cDNA expression library constructed and the length of cDNA insertion fragments,the cDNA expression library constructed is qualified to screening target cDNA clone,laying the foundation for preparation of gene recombinant allergen pollen vaccine.
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