自身免疫性肝炎患者外周血淋巴细胞全长cDNA文库的构建

Construction of a full-length cDNA library from peripheral blood lymphocytes of autoimmune hepatitis

目的成功构建自身免疫性肝炎患者外周血淋巴细胞cDNA文库,同时进行文库的鉴定。方法分离自身免疫性肝炎患者外周血淋巴细胞并提取总RNA,利用SMART技术构建全长cDNA文库并进行文库质量的鉴定,最后扩增文库并随机挑取10个噬菌斑鉴定插入片段大小。结果构建的cDNA文库滴度为3.4×108 pfu/mL,重组率>97%,文库经过扩增后滴度达6.6×10^(11)pfu/mL,插入片段长度在0.5～2.0kb之间,平均长度为1.25kb。结论本文构建的自身免疫性肝炎患者外周血淋巴细胞cDNA文库符合建库要求,为进一步筛选、克隆其发病相关基因、探索发病机制奠定基础。

Objective To construct a full length cDNA library from peripheral blood lymphocytes of autoimmune hepatitis and identify the quality of the library. Methods The lymphocytes were abstracted from autoimmune hepati tis fresh blood and total RNA was extracted. The full length cDNA library was constricted and identified by SMART （Switching Mechanism At 5＇ end of RNA Transcript） technique. At last, to amplify the library and picked ten phage plaques randomly to identify the size of the inserts by using PCR reaction. Results The titer of the cDNA library was 3.4 × 10^8 pfu/mL in which the percentage of recombinant clones were above 97%, and the titer of am plified library was 6.6 × 10^11 pfu/mL. The insert sizes ranged from 0.5 to 2.0 kb and the average length was 1.25 kb. Conclusion The data show that this library meets the requirement of a standard cDNA library, and it will lay a solid foundation for further screening and cloning autoimmune hepatitis responsive genes.