AchR特异性TsF cDNA文库的构建及筛选

Construction and Screening of TsF cDNA Library Specific for AchR

目的 构建 Ach R特异性 Ts F c DNA文库并对该文库进行筛选。方法 从建立的分泌 Ach R- Ts F的特异性 Ts细胞系中直接提取了 Ts F Poly A+ m RNA,反转录合成全长 c DNA,以脱磷酸化的λgt11Eco RI臂为载体 ,体外包装成噬菌体颗粒并转染 E.coli Y 10 90 hsd R,得到 c DNA文库 ,并用 TCR-α单克隆抗体膜上免疫印迹法对该文库进行筛选。结果 重组噬菌体的滴度约为 1.4× 10 7pfu/ m l,阳性重组率为 86 .9% ,重组噬菌体 DNA的酶切电泳分析证实插入片段大小在 0 .8～ 4 .0 kb之间。初步筛到 2 7个阳性重组噬菌体 ,其插入片段大小约为 1kb左右。结论 该文库的建立有望获得持续高效表达 Ach R特异性 Ts

Objective To construct the T suppress factor (TsF) cDNA library specific for AchR and screen out the positive clones of TsF. Methods The full length cDNA was synthesized by the reverse transcripting from the TsF PolyA +mRNA, which had been extracted from the AchR specific suppressor lines (ARSL). The cDNA library was obtained using Lambda gt11 vector. Finally, this cDNA library was screened by Western blotting using monoclonal antibody specific for TCR αchain. Results Analysis of this cDNA library showed that titer of the recombinant bacteriophage was 1.4×10 7 pfu/ml, the rate of positive recombinant was 86.9%. The recombinant bacteriophage DNA were digested by EcoRI and electrophoresis analysis found that the main size of insertion was 0.8～4.0 kb . As a result of screening, 27 positive clones were obtained. The positive recombinant bacteriophage DNA were digested by EcoRI, and electrophoresis analysis found that the size of insertion was 1 kb. Conclusion The results suggested that this cDNA library is available , the recombination phages can express TsF specific for AchR effectively and could be used to prepare the products of gene engineering of TsF.