| Neutrophil gelatinase associated lipocalin(NGAL)is a small molecule secrete glycoprotein as well as a very promising biomaker for acute kidney injury(AKI).It is,in normal conditions,produced at low levels by the epithelial cells of the kidney,but it is quickly upregulated in urinary and plasma within 3 hours of kidney injury.Furthermore,the level of NGAL is proportionally to the severity and duration of the injury.Therefore the accurate determination of NGAL has great value for surgeon on the diagnosis,following-up and outcome prediction of kidney injuries.Quality control,which built on the foundation of control products,is an essential part to obtain reliable results for clinical laboratories.Meanwhile during the companies' reagents research development,the control products are needed for valuing the performance of reagents.In conclusion,the development of stable NGAL control products makes big fifference for both companies and clinical laboratories.The aim of this study is to prepare long-term stability NGAL lyophilized control products by constructing the prokaryotic expression vector pCold-NGAL to express recombinant protein NGAL in Escherichia coli,purifying the protein by Ni affinity chromatography to obtain the bio active protein,and screening stabilizers in the freeze-drying process.The cDNA fragment was synthesized by genetic engineering according to the sequence of human NGAL mature peptide(GIn20-Gly197).The fragment was cloned to the expression plasmid pCold ?.The recombinant plasmid pCold-NGAL was identified by restriction enzyme and sequencing,the results showed that expression vector pCold-NGAL was constructed successfully.The recombinant plasmid pCold-NGAL was transformed into Escherichia coli BL21(DE3)plysS,and inducing by IPTG.The expression products analyzed by SDS-PAGE showed that approximately 25 KDa soluble recombinant protein was expressed by pCold-NGAL.The expression yield of target protein was increased to 33.2% by optimizing induction conditions,such as the concentration of IPTG,temperature and time.The purity recombinant protein was up to 97% after purified by Ni affinity chromatography.The concentration of recombinant protein NGAL is 1.1 mg/m L detected by UV detection,and the immunofluorescence reagent revealed the percentage of bioactivity is 47.2%.Screening stabilizers by the stability of activity on reconstitution at 25 oC of freeze-dried NGAL control(The concentration of NGAL: 157.1 ng/m L).Diluted recombinant protein NGAL into three levels,the contentions of which are 157.1 ng/m L,550 ng/m L,1571.4 ng/m L respectively,marked as Low,Middle,High.Evalue the precision and stability of the three level control materials by immunofluorescence reagent,and compare the results difference by using the third party reagents.The results shows that: the stability of activity on reconstitution at 4 oC and 25 oC of freeze-dried NGAL control are both 7 days,and the NGAL control can be stored at 37 oC,50 oC and 4 oC for 14 days,7 days,4 months respectively.The results of NGAL control on third-party platform varies,and also the precision results.The precision results on Abbott platform is less than 5%.In this study,we have successfully constructed the expression vector pCold-NGAL and obtained high purity,bioactive recombinant protein NGAL,and prepared well stabled NGAL control material,which lays a good foundation for the study of NGAL properties during freeze-drying as well as marketing application of NGAL control material. |
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