Objective To construct human myeloma cell cDNA expression library as to screen myeloma tumor antigen. Methods Total RNA and purified mRNA were extracted from human myeloma cell line HMy2. First and second strand cDNA ...Objective To construct human myeloma cell cDNA expression library as to screen myeloma tumor antigen. Methods Total RNA and purified mRNA were extracted from human myeloma cell line HMy2. First and second strand cDNA were synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested by Xho I, and smaller than 400bp were removed by Sephacryl-S400 spin column, the remaining were ligated with λZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E.coli XL1-Blue-MRF for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid were excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain , and then the pBK-CMV phagemid were digested by Xho I and EcoR I. Results The HMy2 cell line cDNA library consisting of 1.58×10 6 recombinant bacteriophages was constructed with the recombinant ratio 99.6%. The average length of the recombinant exogenous inserts was about 1.7kb.Conclusion The constructed cDNA library are deserved to screen target clones.展开更多
文摘Objective To construct human myeloma cell cDNA expression library as to screen myeloma tumor antigen. Methods Total RNA and purified mRNA were extracted from human myeloma cell line HMy2. First and second strand cDNA were synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested by Xho I, and smaller than 400bp were removed by Sephacryl-S400 spin column, the remaining were ligated with λZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E.coli XL1-Blue-MRF for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid were excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain , and then the pBK-CMV phagemid were digested by Xho I and EcoR I. Results The HMy2 cell line cDNA library consisting of 1.58×10 6 recombinant bacteriophages was constructed with the recombinant ratio 99.6%. The average length of the recombinant exogenous inserts was about 1.7kb.Conclusion The constructed cDNA library are deserved to screen target clones.
