人骨肉瘤9607细胞cDNA表达文库的构建和鉴定

Construction and evaluation of cDNA library of osteosarcoma 9607 cells

目的构建骨肉瘤9607细胞cDNA表达文库,为筛选骨肉瘤特异性抗原创造条件,最终从临床角度增加患者预后质量和开辟患者康复的新途径。方法从9607细胞中提取TotalRNA,分离mRNA,反转录合成双链cDNA,末端削平,和EcoRⅠ适配子连接,磷酸化EcoRⅠ适配子5’端,Sephacryl-S400柱除去小于400bpcDNA片段,和噬菌体λgt11(载体)连接,包装蛋白体外包装后形成初级cDNA文库。取适量倍比稀释后感染E.coliY1090,测定文库克隆数、重组率,用PCR法测定cDNA插入片段大小。结果建成含1.3×106重组子的骨肉瘤9607细胞cDNA表达文库,重组率93.5%,重组子插入外源片段不小于0.5kb,平均长约1.3kb。结论达到良好文库的质量标准,适合进一步筛选目的cDNA克隆,以开辟提高患者康复的途径。

Aim To construct the cDNA expression library of human osteosarcoma 9607 cells for screening the specific antigen of osteosarcoma. Methods Total RNA was extracted from human osteosarcoma cell line 9607, purifying mRNA. First and second strand cDNA were synthesized through reverse transcription. After scraping the distal end, the cDNA fragments were connected with EcoR Ⅰadapters, and the end of adapters was phosphorylated.The cDNA < 400 bp was removed by Sephacryl) S400 spin column, and the remaining were ligated with the dephosphorylation arms of λgt11. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E.coli Y1090 for titration. The size of cDNA inserts and the diversity of library were evaluated through PCR.Results The osteosarcoma 9607 cell line cDNA library consisting of 1.3×106 recombinants bacteriophages was constructed,and reconstruction rate was 93.5%. The exogenous insert of the recombinants was over 0.5kb with an average exogenous insert of about 1.3 kb.Conclusion The cDNA library is eligible to screen target clones for improving the rehabilitation of patients.