| Researches have showed that NGAL (neutrophil gelatinase-associated lipocalin) might be a new gene related to tumor, which is over-expressed in the whole malignant transformation of the esophageal epithelium cell. But its regulatory mechanism is still a debatable issue. The analysis of 5' -flanking sequence of NGAL gene disclosed that there is a typical NF-K B binding site " GGGAATGTCC " between the -180bp~ -171bp region of 5'-flanking sequence of NGAL, that suggested NF-K B play a role in the regulation of NGAL during the whole process of malignant transformation.As one of the universal transcription factors, many members of nuclear factor kappa B family have been identified and cloned, which involved the expression of more than 150 genes controlling the immune response, cell apoptosis, inflammation, and oncogenesis, invasion and metastasis, endurance of drug etc. However, which factor plays the role at all and whether or not there are some new factors unidentified during the malignant transformation are kept unknown. This study took advantage of the yeast one-hybrid technology to screen the cDNA library of yeast hybrid by designing the k B element as the bait, aiming to elucidate the reason of the NGAL over-expression, and provide a theoretical base for the regulatory mechanism of NGAL.Main procedures and methods are as follows:1. Insert the designed bait into DNA-BD vector in proper orientation by means of genetic engineering. The target-reporter constructs were identified by restriction endonucleases digesting and DNA sequencing correctly.2. The appropriate target-reporter constructs were introduced into host stain YM4271 by the chemical transformation methods. The ideal host strain used in the yeast one-hybrid system could be chosen by the 3-AT inhibited experiment stringently.3. Screen the SHEEC cDNA library by large-scale transformation methods. The putative positive clones bind to kappa B element could beobtained through selecting repeatedly on DO supplement selection media lacking in the appropriated nutrients and by raising the selecting pressure on 3-AT containing medium.4. Obtain the single plasmid of cDNA library after the transformation of the positive plasmid isolated from yeast putative clones into E. coli. The single purified plasmid was introduced into host stain YM4271 of yeast one-hybrid system to verify if the gained yeast positive clone will promote the expression of the reporter in vivo.5. Sequence the verified positive clones.6. Identify and analysis the results of DNA sequencing by the bioinformatical methods.The results are:1. Synthesized three tandem copies carried the target binding site of kB element with two antiparallel oligonucleotides: one represented the sense strand and the other antisense complement, and inserted in the upstream of the reporter gene of DNA-BD vector after annealed, gained the target-reporter constructs after the screen and identification.2. Stringently screen the yeast host cell integrated with the bait element in the genome after integrated the appropriate target-reporter constructs into the genome of the yeast host cell YM4271 under 3-AT selecting pressure.3. 351 putative positive clones were obtained after screening the cDNA library of the human esophageal cancer cell line SHEEC.4. 37 positive clones were gained after the plasmid extracting and restriction endonucleases digesting against the 351 putative positive clones.5. 31 positive clones which may promote the expression of the reporter gene were confirmed, through the clone by clone approach in vivo towards the 37 positive clones.6. The 31 yeast positive clones were sequenced, identified and classified by preliminary analysis of bioinformatic, and found they are different in the structure of p65 or p50 factors from that reported before.Conclusion are:1. The yeast one-hybrid operational system was constructed successfully.2. It showed that there were some new factors might play a role in the esophageal cancer cell by the bait designing, ta... |
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