To Study The Efficacy Of PCH Optical Isomers And Pharmacological Profiles Of PH Optical Isomers, Construction And Expression Of Recombinant Vector PGEX-6p-2-Notch3～(41-656) And PGEX-6p-2-Notch3～(228-656), And Preparation Of Anti-Notch3 Protein Monoclo

phencynonate hydrochloride (PCH) and penehyclidine hydrochloride(PH) are novel anti-cholinerigic drugs invented by the Institute of Pharmacology and Toxicology, AMMS. phencynonate hydrochloride has been developed as a new medicine for motion sickness (MS), But the side effects of phencynonate hydrochloride were dry mouth and drowsiness. There is one chiral carbonic atom in the molecular structure of PCH. Thus, there were two optical isomers of PCH with R(-)-PCH and S(+)-PCH, S(+)-PCH was less active in comparison to R(-)-PCH and its racemate, R(-)-PCH had the highest affinity to central muscarinic receptors among the three compounds. Thus, to comparative evaluate potency of R-phencynonate hydrochloride [R(-)-CPG] with its optical isomers in prevention and treatment of motion sickness (MS). In the rabbit model , The ED50 values of R(-)-CPG and CPG in inhibiting adverse circling syndrome were 4.01mg/kg and 8. 94mg/kg, respectively, both ED50 value ratio is 1:2.23, but S(+)-CPG did not show significant effect on anti-motion sickness at the dose of 34mg/kg(p>0.05); In the cat model, The ED50 values were 0.07mg/kg and 0.58mg/kg respectively, both ED50 value of R(-)-CPG and CPG ratio is 1:8.29, S(+)-CPG also did not show significant function of anti-motion sickness at the dose of 3. 0mg/kg(p>0. 05) ; The anti-motion sickness effect of R(-)-CPG is more potent than its racemate and S(+)-CPG. R(-)-CPG maybe develop as an anti-motion sickness drug in clinic in the future.Two chiral carbons existed at the structure penehyclidine hydrochloride (PH) compound, so there are four optical isomers for PH, There absolute configurations are RR', SR' ,RS' and SS' , RR' and SR' have the higher affinity to muscarinic receptors than RS' and SS' . Subtype selectivity of muscarinic acetylcholine receptors was investigated in radioligand binding experiments in Chinese hamster ovary(CHO) cells stably transfected with the genes encoding the Ml, M3, M4 subtypes of muscarinic receptors. The order of affinity of SR' -PH to M1,M3 and M4: M4(Ki=9.1nM)>Ml (Ki=11.412nM)>M3(Ki=53. 668nM) ; The order of affinity of RR' -PH to M1, M3 and M4: M3(Ki=0.022nM)>M1(Ki=0.091nM)>M4(Ki=0.136nM). SR' -PH had far greater selectivity for M4 over M1and M3; RR' -PH had far greater selectivity for M3 over M1and M4.Cerebral autosomal dominant arteriopathy with subcortical infarcts and lecukoencephalopathy (CADASIL) is caused by mutations in the Notch3 gene located in the short arm of chromosome 19. Notch3 is a type I transmembrane protein mainly expressed in human arterial vascular smooth muscle cells (VSMCs). it is obviously laborious to sequence the 33 exons of NOTCH3 gene although 60-70% mutations occur in exons 3 and 4. Notch3^41-656 and Notch3^228-656 gene fragment after Polymerase Chain Reaction(PCR) were ligated into BamHI and SalI restrict site of pGEX-6p-2 vector to generate recombinant vector pGEX-6p-2-Notch3^41-656 and pGEX-6p-2-Notch3^228-656, recombinant plasmids encoding GST fusion protein were transformated into Escherichia coilBL21(DE3).The production of GST fusion proteins were induced by adding IPTG( isopropylβ-D-thiogalactoside)to the culture medium, followed by an additional incubation at 20°C for 14h. Insoluble fusion proteins were purified by reduced glutathione (GSH). The GST fusion protein was assayed by SDS-PAGE and polyarcrylamide gel electrophoresis. Fusion proteins were used to immunize BABL/C mice, Our successful cloning and expression of pGEX-6p-2-Notch3^41-656 and pGEX-6p-2-Notch3^228-656 gene and purification of GST fusion protein .preparing cell fusion, production of anti-Notch3 protein monoclonal antibody.