The Preparation Of Cholesterol Sensitive Structure SSD And Its Application In Chinese Traditional Lipid Screening

Objective:In order to make SSD higher recombinant expression and more effective,for it as one of the most potential drug targets in high cholesterol treatment.In the same time,it’s importan to establish the molecular interaction platform for screening Chinese traditional medicine monomer components,which can control the change of sterol.Methods:This study selected SSD as the target segments,In order to make the higher recombinant expression and easier to be soluble in water,for the SSD is across the membrane area,we designed it with a label of 6 x his,at the same time added a GST label;The study of screening positive transformed colonies using medium containing 100μg/mL Zeocin antibiotics YPD,and identified by PCR through the extraction of yeast genome.Screen high copy recombinant yeast colonies with different concentrations of Zeocin YPD.The high copy positive recombinant yeast strain induced by methanol and enlarging culture,supernatant was collected and induced its expression,total protein extraction in the supernatant and identification of recombinant protein GSTHisSSD.Recombinant protein GSTHisSSD by GST affinity column purificatied,then purified protein HisSSD,collected and concentrated protein and SDS-PAGE gel electrophoresis test protein purity.Cholesterol domain SSD as a target,we use the biomolecular interaction analysis system to screen sixteen Chinese traditional medicine monomer(Curcumin/Astragalus vitexin/Ferulic acid/Methyl glucoside/Emodin/Soybean isoflavone/Glycyrrhizic acid/Ammonium/Hesperidin/Big leaf madder/Cortex moutan/Resveratrol/Rutin/Ginkgo in lipid/Ginkgo lactone C and A/Aydroxy safflower yellow pigment A),which can control the change of sterol,with reference to the cholesterol.Then make analysis for them screened by the computer and SSD protein.Results:The recombinant prokaryotic expression plasmid pPICZ-GSTHisSSD was successfully constructed and transformed into a laboratory yeast strain by electrical stimulation of 300V 15ms.High copy positive recombinant colonies were screened out;then the His-tagged protein SSD was purified.The SSD protein was binded with cholesterol protein with a concentration gradient,which was fixed on the carboxyl chip by microarray.Conclusion:SSD protein recombinant eukaryotic Pichia expression system is successfully using recombinant DNA technology for the first time in this study.After application of biomolecular interaction analysis system of preliminary screening Chinese Medicine monomer components,a high-throughput screening method and drug screening platform of Chinese medicine monomer as the target of a SSD protein were established.There is an important significance to explore new cholesterol-lowering Traditional Chinese Medicine,which provides a reliable experimental basis for pharmacodynamic preclinical studies.