Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and...Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR Ⅰ adapters. Then the cDNAs were digested with Xho Ⅰ, and less than 400 bp cDNA fragment was 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 was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK CMV phagemid was digested by Xho Ⅰ and EcoR Ⅰ. The results showed that the NB4 cell line cDNA library consisting of 1.65×10 6 recombinant bacteriophages was constructed with the recombinant ratio of 99.6 %. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.展开更多
文摘Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR Ⅰ adapters. Then the cDNAs were digested with Xho Ⅰ, and less than 400 bp cDNA fragment was 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 was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK CMV phagemid was digested by Xho Ⅰ and EcoR Ⅰ. The results showed that the NB4 cell line cDNA library consisting of 1.65×10 6 recombinant bacteriophages was constructed with the recombinant ratio of 99.6 %. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.
