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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。
OBJECTIVE: To construct a cDNA expression library of osteosarcoma 9607 cells and to create conditions for the screening of specific antigens of osteosarcoma. Finally, it will improve the prognosis of patients and open up a new way of rehabilitation for patients. METHODS: Total RNA was isolated from 9607 cells, reverse transcribed into double-stranded cDNA, blunt-ended and ligated with EcoRI adapter. Phosphorylation of EcoRⅠ aptamer 5 ’end, Sephacryl-S400 column removal of less than 400bp cDNA fragment, lambda gt11 (vector), packaged protein in vitro packaging primary cDNA library. After taking the appropriate dilution, E. coli Y1090 was infected and the number of clones and the recombination rate were determined. The size of cDNA inserts was determined by PCR. Results The cDNA expression library of osteosarcoma 9607 cells containing 1.3 × 106 recombinant was constructed. The recombination rate was 93.5%. The recombinants were inserted into the foreign DNA fragments of not less than 0.5 kb in length and the average length was about 1.3 kb. Conclusion The quality standard of good library is reached, which is suitable for further screening of cDNA clones of interest so as to open up ways of improving patient rehabilitation.