Prokaryotic Expression And Polyclonal Antibody Preparation Of Human NGAL

Objective: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 transduc-tion, occurrence and progression procedure of many cancers.Emerging evidence has demonstrated that NGAL is up-regulated in multiple malignancies, including breast cancer,esophageal cancer,colon carcinoma,gastric cancer and chronic myeloid leukemia.and play a critical role in tumorigenesis and progression. NGAL is thought to be a protective factor in renal, and it has natural resistance to the enzyme.NGAL is a 25KD glycoprotein that generated earlier in the urine. NGAL was found to be a sensitive early biomarker for diagnosing AKI and evaluate the occurrence and development of acute renal failure.The paper to construct the expression vector of NGAL gene and to expression NGAL protein,then to prepare anti-NGAL polyclonal antibody,at last to detect NGAL expression level and discuss its expression pattern with the anti-NGAL polyclonal antibody by IHC, Western blotting experimental techniques have an important significance for furthermore research of NGAL function.1 To construct the prokaryotic expression vector pET28a-NGALNGAL was amplified by RT-PCR from human leukemia cell K562. The fragment and pET28a were cutted by double enzyme(HindIII,BamH I),and then recycled the goal fragments and pET28a were ligated by T4 ligase,it is pET28a-NGAL,and then the pET28a-NGAL was transducted into Rosetta DE3.After selecting the positive colony and extracted the plasmid, we indentified the plasmid by double enzyme cut and sequencing experiment. the sequence result and the NGAL gene ORF sequence carried on the homologous comparison.2 Expression of NGAL fusion proteinThe Plasmids pET28a and pET28a-NGAL were transformed into Rosetta DE3.After transformed,The recombinant Plasmid pET28a-NGAL were identified by restrietive enzymes assays and automatic DNA sequeneing respectively. The fusion protein were induced by IMm IPTG for about 3 hours.15%SDS-PAGE identified the fusion pretein.15%SDS-PAGE dyeed by coomassie brilliant blue staining.Protein expression level was analyzed by Quantity One.3 pET28a-NGAL polyclonal antibody preparationThe fusion protein was analyzed by 15%SDS-PAGE.The arget band was cut off from banding gelatin including the goal protein. The banding gelatin fully milled and freezied by PBS.The expressed protein with equal volume of Complete Freund's Adjuvant were used to immunize male white rabbits.The goal protein with equalwas Incomplete Freund's Adjuvant were used to immunize male white rabbits every two weeks, for a total of 4 times immune.Ten days after the final immunization, the serum was separated from blood by Carotid artery.The NGAL Polyclonal antibody was skimpily purified by saturated ammonium sulfate method and precise purified by anti-rabbit affinity column.The purified polyclonal antibody was then subpackaged and stored at -20℃.4 The NGAL polyclonal antibody specificity detectionIn order to detect the specifity and sensitivity of self-made NGAL Polyclonal antibody,The fusion and purified protein were collected to detect by Western blotting. The self-made antibody was alse evaluated by immuno-histochemical techniques.Results:1 To construct the prokaryotic expression vector pET28a-NGALAfter RT-PCR, about the purpose of the 625bp fragments detected by Agrose gel electrophoresis,with expected results are consistent.Restrictive enzymes assay and automatic DNA sequencing confirmed the successful construction of plasmid pET28a-NGAL.1.5% Agarose gel showed the specific band at 5369bp and 625bp after restrictive enzymes assay.2 Expression of NGAL fusion proteinpET28a and Recombinant plasmid pET28a-NGAL was expressed in E.coli after induction with IPTG. The NGAL fusion protein was identified by SDS-PAGE, but not showed in protein of uninduced by IPTG. with the increase of IPTG induction time, protein expression quantity also increases, after Three hours later, the expression of the fusion protein reached the peak.Experimental proof:fusion protein pET28a-NGAL was expressed in Rosetta DE3 bacteria. Just fusion protein expression rate was analysised up to 12.2% by Quantity One.3 NGAL polyclonal antibody preparation and purificationAfter the fusion protein was analyzed by 15%SDS-PAGE,and purified the goad protein was immuned rabbit.Serun against human was concentrated and simply purified by SAS precipitation method and precise purified by anti-rabbit affinity column. The result showed that self-made polyclonal antibody can be used for further study.4 Identification of NGAL-pAbTo further study the specificity and sensitivity of our self-made NGAL-pAb, The total fusion and purified protein were collected to detect by NGAL-pAb. Western blot results showed that the recombinant pET28a-NGAL protein could be detected by NGAL-pAb, This antibody could specifically recognize the NGAL protein expressed in the E.coli system.Immuno-histochemical staining indicated that NGAL was expressed in breast cancer.Conclusion:constructed the prokaryotic expression vector pET28a-NGAL.and Inducted NGAL fusion protein expression.the purification fusion protein was injected to preparate NGAL polyclonal antibody.It can be used for further research in the function of NGAL.