Fusion Expression Of NGAL Gene In Prokaryote, Antibody Preparation And Its Expression Pattern In The Esophageal Cancer

NGAL (neutrophil gelatinase-associated lipocalin) protein was found at first in neutrophil granulocyte in 1993 by Kjeldsen et al. And it involved in inflammation, embryonic development, immunity response, chemotaxis phenomena, signal transduction, occurence and progression procedure of many cancers. We had found NGAL gene was overexpressed in esophageal cancer cell line(SHEEC)by cDNA microarray screening different expression genes between immortal esophageal epithelial cell line(SHEE) and SHEEC transformed from SHEE, but not known its mechanisms. Furthermore research indicated that NGAL might be involved in tumor's invasion and metastasis with unknown mechnisms, too. Recently some researches have shown that NGAL gene is associated with cell modulation and differentiation. So it can be predicted that NGAL maybe have some more important functions. Obviously, so many research results indicate NGAL may be an important disease marker, especially on cancers. So intensive research of NGAL function on physiology and pathology has important scientific significance and application merits. Now NGAL protein itself and its antibody both were not commercilized, which limited intensive application of NGAL on clinical research, at the same time restricted intensive understanding of NGAL function, too. So to construct the expression vectors of NGAL gene and to express NGAL protein, then to prepare anti-NGAL antibody, at last to detect NGAL expression level and discuss its expression pattern with the anti-NGAL antibody by IHC, Western blotting and ELISA experimental techniques have an important significance for furthermore research of NGAL function. Main Procedures and Methods:1. To construct four NGAL gene fusion expression vectors and to analyze comparatively their expressed proteins in E.coli.2. To purify NGAL protein with Ni2+- metal chelate affinity chromatography and to prepare anti-NGAL polyclonal antibody.3. To detect the expression level of NGAL in the esophageal cancer by IHC, Westernblotting and ELISA experimental techniques and to discuss the expression pattern of NGAL in the esophageal cancer, which serves for furthermore reseach work of NGAL gene function. Main Results:1. Four NGAL gene fusion expression vectors had been successfully constructed in prokaryote.2. Four NGAL fusion proteins were successfully expressed in prokaryote and their productivity,solubility as well as subcellular localization were all analysed, at last the pDsbA2.0 - NGAL expression vector which can be suitable for large-scale expression was screened.3. Purification and identification of NGAL protein had been done successfully, at last the NGAL protein with some extent purity and abundance had been gained.4. Anti-NGAL polyclonal antibody with satisfied titer ,sensitivity and specificity was prepared successfully and was suitable for IHC Western blotting as well as ELISA.5. Expression pattern of NGAL in the esophageal cancer was discussed primarily by IHC Western blotting and ELISA.Conclusion and significance:1. Different fusion partner has different effect on productivity, subcellular localizition and solubility of target protein.2. Ni2+- metal chelate affinity chromatography is a protein purification technique with very high efficacy.3. Detecting techniques for NGAL expression pattern were established successfully:IHC, Western blotting and ELISA.The expression pattern of NGAL in the esophageal cancer was shown through different aspect. IHC experiment manifested positive staining located in the cytoplasm or cellular membrane of the central esophageal carcinoma nest with high-grade differentiation. Western blotting experiment displayed that expression level of NGAL in some esophageal carcinomas were higher than those of nomal esophageal mucosas,but many more samples must be detected to verify this point. And ELISA experiment manifestedprimarily that NGAL expression can be detected in the normal plasm/serum. All of these had built up a good...