RESUMO
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 lambdaZAP 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 x 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.
Assuntos
Humanos , Bacteriófagos , Genética , DNA Complementar , DNA de Neoplasias , DNA Recombinante , Biblioteca Gênica , Vetores Genéticos , Leucemia Promielocítica Aguda , Genética , Metabolismo , Patologia , DNA Polimerase Dirigida por RNA , Metabolismo , Transcrição Gênica , GenéticaRESUMO
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 lambdaZAP 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 x 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.
Assuntos
Bacteriófagos/genética , DNA Complementar/biossíntese , DNA de Neoplasias/biossíntese , DNA Recombinante/biossíntese , Biblioteca Gênica , Vetores Genéticos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , DNA Polimerase Dirigida por RNA/metabolismo , Transcrição Gênica/genéticaRESUMO
To provide an evidence for clinical application, the distribution and radioimmunoimaging of 131I-labeled angiostatin in tumor-bearing mice were studied. Angiostatin was labeled with 131 I by the conventional chloramines T (Ch-T) method and injected into C-57 mice bearing Lewis lung cancer. The biodistribution of 131 I-angiostatin and whole body ECT imaging were studied at various intervals after injection. The average percentage of ID/g was 12. 48, 18. 56 and 23. 17 for tumor and 7. 04 ,5. 47 and 1. 73 for liver at 48, 96, 144 hours postinjection, respectively. The T/NT ratios were 1. 77, 3. 39 and 13. 39 for liver at 48 ,96 and 144 hours postinjection, respectively. The tumor was showed clearly at 96 hours after injection . The quality of tumor imaging was relevant to the T/NT ratio . The results demonstrated that the 131I-angiostatin binds specifically to the receptor on endothelial cells in the tumor and may also possess the capability of locating lung cancer tissue.