Gene cloning and prokaryotic expression of glycosyltransferase from Ligustrum quihoui / 中国中药杂志

According to the previous results from transcriptome analysis of Ligustrum quihoui, a glycosyltransferase gene(xynzUGT) was cloned by rapid amplification of cDNA ends(RACE). The full length cDNA of xynzUGT was 1 598 bp, consisting of 66 bp 5'-UTR, 1 440 bp ORF and 92 bp 3'-UTR. The ORF encoded a 480 amino-acid protein(xynzUGT) with a molecular weight of 54 826.67 Da and isoelectric point of 5.82. The structure of enzyme was analyzed by using bioinformatics method, the results showed that the primary structure contained a highly conserved PSPG box of glycosyltransferase, the secondary structure included α helix(38%), sheet(12.1%) and random coil(49.9%), and tertiary structure was constructed by peptide chain folding to form two face-to-face domains(often referred to as a Rossmann domains), between which a substrate binding pocket is sandwiched. The phylogenetic tree analysis indicated that xynzUGT might catalyze glycosylation of phenylpropanoids, such as tyrosol. Further simulation experiment of molecular docking between enzyme and tyrosol showed that Gly138 and Ser285 located in the binding pocket interacted with tyrosol by hydrogen bonding. SDS-PAGE analysis exhibited that the prokaryotic expression system successfully expressed recombinant xynzUGT with molecular weight of 58 370.57 Da, but it exists in the form of non-soluble inclusion bodies. Using the molecular chaperone and enzyme co-expression method, the soluble expression was promoted to some extent. The above works laid the foundation for further studying on enzymatic reaction and clarifying the functional mechanism of enzyme.

A study of the effect of ligustrum quihoui carr (LQC)on the immune function of B lymphocytes and NK iymphocytes in mouse with impaired immune function / 国际中医中药杂志

ObjectiveTo explore the effect and mechanism of LQC on the immune function of B lymphocytes and NK lymphocytes in mouse with impaired immune function. Methods Mice with impaired immune function led by Cyclophosphamide (Cy)were used as experimental animmals, and divided into five groups randomly, twelve mice in every group, which is NS control group, Cy control group, Cy+ LQC minor and major dose group, and Cy + LQC decoction group. NS control group was given NS 0.2 ml/d subcutaneously 10 d, and the rest groups were given Cy 0.8 ml/d subcutaneously (20 mg/kg · d-1) 10d, to establish a mouse model of immune dysfunction. Since the 11th day each group was given NS, Cy, LQC minor dose, major dose and fructus ligustri lucidi decoctoin. Mice of each group were killed after 7 days. The percentage of B lymphocytes (CD3 - CD19 + ) and NK lymphocytes (CD3 - CD 16 + CD56 + ) in the peripheral blood of the experimental mice were detected by flow cytometer. ResultsIn comparison with LQC major dose group [ (20.44± 1.78)and(19.12± 1.70) ], fructus ligustri lucidi group[ (19.90± 1.42) and (20.17± 1.66) ], CD3-CD19+and CD3-CD16+CD56+ cells of LQC minor dose group[ (11.54±0.98) and (12.46±0.08)]were decreased significantly (P＜0.01), which were increased significantly compared with Cy group[ (4.53± 1.70) and (5.03 ±1.22) ] (P＜ 0.01), but they had no significant difference with NS [ ( 11.84 ± 0.99) and ( 12.90± 0.28) ] (P ＞ 0.05).In comparison with Cy group and NS group, CD3-CD19+ and CD3-CD16+CD56+ cells of LQC major dose group were increased significantly (P＜0.01), which had no significant difference with fructus lignstri lucidi group (P＞0.05). Conclusion The immune functions of the mice with impaired immune function were improved by LQC, it also could increase the quantity ofCD3-CD19+ cell and CD3-CD16+CD56+ cell. The dose of LQC was positively correlated with the modulation effect of LQC on the immune function.