Construction Of Yeast Two-Hybrid System And Identification And Screening Of Proteins That Interaction With NGAL

NGAL (neutrophil gelatinase-associated lipocalin) is a member of lipocalin family. NGAL gene is highly expressed in the esophageal cancer cell line in previous works, and is hypothesized that the NGAL gene may be a noval human oncogene in enhancing the invasion of tumor cell and induction of cell differentiation. But its biochemistry mechanism is unknown. Yeast two-hybrid system was constructed and bone marrow library was screened, from which some candidates were identified. This study is to gain insight to the biochemistry mechanism of NGAL in esophageal cancer cell line and other tumors. Main Procedures and Methods:1. Interaction between the SV40 large T-antigen and the p53 protein as a positive control was established for the yeast two-hybrid system. Some interested questions were probed in the yeast transformation. It contains the transformation efficiency and the culturing yeast and handling yeast. Comparing of three methods, the yeast simultaneous cotransfomation, yeast sequence transformation and yeast mating, is helpful to the next library-screening step.2. The cDNA of NGAL was obtained by PCR in which the pGEX-NGAL plasmid was a template. Then the fragment was inserted into DNA-BD vector pGBKT7 in the proper orientation by gene engineering. The constructed plasmid pGBKT7-NGAL was identified by restriction endonuclease digesting and DNA sequencing.3. The pGBKT7-NGAL was introduced into yeast AH109 by small-scale chemical transformation method. Toxicity of the NGAL protein on the host stain was tested to compare the growth rate in liquid culture of cells transformed with the empty DNA-BD vector to cells with the pGBKT7-NGAL plasmid. The NGAL protein for transcriptional activation was tested by β-gal analysis. The pretransformed bone marrow library was screened by yeast mating. Positive yeast clones were analyzed and verified by restreaking positive clones on QDO plate and β-gal analysis.4. Plasmid was isolated from yeast and transformed into Ecoli DH5a for amplification. The fragments of positive library plasmids were identified byrestriction endonuclease digestion. The protein interactions of NGAL protein andpositive library proteins in yeast were indentified by sequence transfomation.5. The inserted fragments of positive plasmids were sequenced, verified in yeastand analysed in NCBI database.Main Result:1. The yeast two-hybrid system was constructed.2. The shuttle plasmid pGBKT7-NGAL was constructed. It is suggested that the NGAL was inserted in the MCS of pGBKT7 in expected direction and the reading frame was maintained by restriction enzyme digesting and DNA sequencing. AH109[pGBKT7-NGAL] was constructed. The NGAL protein was nontoxic to AH 109 and couldn't activate the reporter gene alone.3. The pretransformed bone marrow library was screened by yeast mating, and the mating cultures were spread on the QDO plates. 850 yeast positive clones were obtained by restreaking on QDO plate and β-gal analysis. The plasmids were isolated from yeast and transformed into Ecoli DH5a, 484 plasmids with inserts were obtained by analysis of restriction enzyme digesting.4. It was 65 plasmids from the interaction of NGAL protein in yeast by randomly selection and verification. 12 plasmids were obtained and sequenced. Conclusion:1. While the yeast two-hybrid system was constructed, the state of yeast growth and transformation regent PEG can affect transformation efficiency. The transformation efficiency was not affected by the purity of plasmid used and heat shock time in transformation.2. The interactions of 12 proteins and NGAL protein were verified in yeast. The results are supplied a hint to the biochemistry mechanism of NGAL in esophageal cancer cell line and other tumors.