Construction and evaluation of cDNA library of osteosarcoma 9607 cells
Liao Bo,Ma Baoan,Zhang Huizhong,Long Hua,Zhang Peng,Fan Qingyu,Department of Orthopeadics, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038,Shaanxi Province,ChinaChen Gang,Department of Hematology,Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an 710004,Shaanxi Province, China
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.
【CateGory Index】： R738.1