[Study on quality standard for mori cortex].

OBJECTIVE: To establish a new evaluation model and compare the differences of Mori Cortex from different habitats at different harvest time. METHODS: TLC method was used to identify sanggenon D in the sample with silica gel G plate and a mixture of chloroform-methanol-formic acid (5: 1:0. 3) as a developing solvent. UV was used for determination the centent of total flavonoids of Mori Cortex with sanggenon C as the reference substance. HPLC was applied for determination the contents of sanggenons C, D and DNJ of Mori Cortex. RESULTS: In the TLC chromatogram, sanggenon D showed a distinct fluorescence spot under UV 365 nm with good separation. In UV, sanggenon C calibration curve showed a good linear relationship at the range of 146.8 - 734.0 microg/mL, the average recovery was 97.4% (RSD = 2.1%); For the HPLC quantitation method, sanggenons C, D and DNJ showed good linear within the scope of 700 - 4 000 microg, 500 - 3 000 microg and 4.8 - 96 microg, respectively. The average recovery of sanggenons C, D and DNJ were 98.8% (RSD = 2.6%), 99.1% (RSD = 2.2%) and 98.9% (RSD = 1.7%), respectively. The contents of index components in samples from different habitats at different harvest time were different. CONCLUSION: The established method is reliable and accurate. It can be used for quality control of Mori Cortex.

Identification and characterization of inosine 5-monophosphate dehydrogenase in Streptococcus suis type 2.

Streptococcus suis type 2 is a swine pathogen responsible for diverse diseases. Although many virulent factors have been identified and studied, relatively little is known about the pathogenic mechanisms of type 2. The aim of the study was to identify and understand the characterization of Inosine 5-monophosphate dehydrogenase (IMPDH). A 957-bp gene, impdh, was identified in the virulent S. suis serotype 2 (SS2), and analysis of the predicted IMPDH sequence revealed IMP dehydrogenase/GMP reductase domain. The gene encoding for the IMPDH of S. suis was cloned and sequenced. The DNA sequence contained an open reading frame encoding for a 318 amino acid polypeptide exhibiting 23% sequence identity with the IMPDH from Streptococcus pyogenes (YP281355) and Streptococcus pneumoniae (ZP00404150). Using the pET(32) expression plasmid, the impdh gene was inducibly overexpressed in Escherichia coli to produce IMPDH with a hexahistidyl N-terminus to permit its purification. The (His)6 IMPDH protein was found to possess functional IMPDH enzymatic activity after the purification. The impdh-knockout SS2 mutant ( Delta IMPDH) constructed in this study was slower in growth and one pH unit higher than SS2-H after 6 h of culturing, and found to be attenuated in mouse models of infection for 2.5 times and not be capable of causing death in porcine models of infection in contrast with the parent SS2-H.

[Cloning and characterization of the gene encoding IMPDH of Streptococcus suis serotype 2].

Given the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, S. suis serotype 2 was sequenced partly in an effort to identify important virulence factors. Two new open reading frames were found located between orf2 and mrp. One of new open reading frame (2738 - 3694) that encoded a polypeptide of 319 amino acid residues with a calculated molecular mass of 33.5kDa was identified by Western blot. GenBank database search revealed that the derived amino acid sequence shared low homology with sequences of known function from other genes. Second structure was analyzed by InterPro, PHD, DNAstar software, the deduced protein had functional domains typical of IMP dehydrogenase (IMPDH). The PCR product of the open reading frame was transformed into E. coli BL21 and the fusion protein of 48kDa was expressed. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic. IMPDH activity staining confirmed that the protein has IMPDH function and can catalyze the rate-limiting reaction of GTP biosynthesis, the NAD-dependent reduction of IMP into XMP. Flow cytometry (FCM) revealed that the protein had apparent effect on HEp-2 cell cycle.