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
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 evaluate the efficacy of anti-HB levels among health workers vaccinated with DNA recombinant Hepatitis B vaccine derived from Chinese Hamster ovary cells [CHO-cells]. A study of 20 subjects protected with anti-HB levels [>/= 10m 1 U/ml] were assessed. Setting: Department of Medicine, Lahore General Hospital. No non-responder and major adverse effects were identified in this study. CHO cells derived DNA recombinant Hepatitis B vaccine are effective in standard dose regime.However, further work is required to check the efficacy of low dose hepatitis B vaccination regimens using different routes of administration within the population. To decrease the cost of vaccines and screening tests, efforts should be made at national and international levels so that mass Hepatitis B immunisation programmes are initiated
Assuntos
Humanos , Masculino , Feminino , Vacinas contra Hepatite B/imunologia , Antígenos de Superfície da Hepatite B , Células CHO/imunologia , Vacinas contra Hepatite B , DNA Recombinante/biossíntese , DNA RecombinanteRESUMO
1. A new cloning procedure is described for cDNA synthesis from mRNA released by in vitro translation of polysomes in a cell-free amino acid incorporating system. The usefulness of the method lies in the feasibility of employing nanogram amounts of mRNA. 2. Complementary DNA is synthesized derectly in the translation mixture simply by adjusting the concentration of some components and removing ribosomes by boiling and centrifugation. 3. As an example, we report here the construction and characterization of cDNA clone corresponding to chick alfa (1) procollagen starting from a collagen-synthesizing polysome fraction obtained from chick embryos