小鼠巨噬细胞cDNA酵母表达文库的构建与鉴定  

Construction and identification of yeast expression cDNA library of mouse macrophage

作　　者：王顺清[1] 林蔡弟[1] 毛平[1] 张玉平[1] 许艳丽[1] 

机构地区：[1]广州医学院附属市一人民医院血液科,510180

出　　处：《中华生物医学工程杂志》2010年第4期292-297,共6页Chinese Journal of Biomedical Engineering

基　　金：基金项目：国家自然科学基金面上项目（30871101）;广州市医药卫生科技重点项目（2006-ZDi-02）

摘　　要：目的采用Gateway技术构建适合酵母表达的小鼠巨噬细胞cDNA文库并进行鉴定。方法将小鼠巨噬细胞的mRNA分离纯化后，以生物素标记的寡聚胸苷酸Oligo（dT）为引物反转录后连接attB衔接子（attBAdapter），层析柱纯化，通过BP重组反应将500bp以上的片段克隆到含attP衔接子的pDONR^TM222载体，电转化人ElectroMAX^TMDHIOB^TM T1 Phage Resistant Cells，构建Gateway入门cDNA文库，并完全随机挑取单菌落，提取质粒酶切鉴定重组子插入片段大小。构建Gateway目的载体，通过LR重组反应将入门文库转入此目的载体成为酵母表达文库，挑取单克隆鉴定重组子插入片段大小。结果经鉴定，入门文库平均滴度为（6．80±0．10）×10^5cfu／ml，文库总容量为7．48×10^6cfu，平均插入片段为（2．20±0．20）kb，重组率为100％。酵母表达文库平均滴度为（3．24±0．10）×10^6cfu／ml，文库总容量为3．89×10^7cfu，平均插入的片段为（2．27±0．15）kb，重组率为95．83％（23／24）。结论构建的小鼠巨噬细胞cDNA入门文库和酵母表达文库都符合高质量文库的要求，可用于进一步的研究。Objective To construct and identify the cDNA library suitable for yeast expression of mouse maerophage by Gateway technique. Methods The mRNA extracted from mouse macrophage was purified. Moreover, eDNA were synthesized by biotin-conjugated Oligo （dT） primer. The double-strand eDNA was bound to attB Adapter and then purified by the eDNA size fractionation columns. BP recombination reaction between the attB- flanked cDNAs （〉500 bp） and attP- containing donor vector pDONR^TM222 was performed, and the products were transformed into ElectroMAX^TM DH10B^TMTI Phage Resistant Cells to generate a Gateway entry eDNA library. Colonies were randomly selected from the plating assay plates. Plasmid DNA was isolated and the cDNA library qualified by plasmid digestion. A reading frame cassette to pGADT7 vector was ligated to construct a Gateway destination vector. The cDNA entry library was transformed into yeast expression library after LR recombination reaction with destination vector and the Gateway entry cDNA library. Results An entry cDNA library was constructed with a titer of （6.80±0.10）× 10^5 cfu/ml, total clones of 7.48×10^6 cfu, an average insertion size of about （2.20±0.20） kb and the percentage of recombinant clones was 100%. A yeast expression library was constructed with a titer of （3.24±0.10）× 10^6 cfu/ml, total clones of 3.89 × 10^7 cfu, a mean insertion size about （2.27 ±0.15） kb and the percentage of recombinant clones was 95.83% （23/24）. Conclusion The entry cDNA library and the yeast expression cDNA library meet the requirements of standard library, thereby can be used in further study.

关 键 词：巨噬细胞 基因文库 DNA 互补 GATEWAY技术 酵母菌 

分 类 号：Q78[生物学—分子生物学]