Introduction: The aim of this research project is to elucidate the crosstalk of innate and adaptive immune reactions against the DNA containing bacteria. : This study held in the Core laboratory, Science Technology Center, Mongolian National University of Medical Sciences (MNUMS). Murine aortal endothelial cells, END-D cultured and the cell viability checked by MTT assay. In addition, the NO production, protein and gene expression studied by Griess Reagent assay, R.T-PCR and immunoblotting, respectively. Results: 0.1µM, 1µM and 10µM of TLR9 ligand exhibited no cytotoxic action against the cells by MTT assay. IFN-ү alone induced NO production in END-D cells. In the other hand, TLR9 ligand at 0.1µM, 1µM and 10µM up-regulated IFN-ү induced NO production in dose dependent manner. RTPCR results exhibit that TLR9 ligand up regulates iNOS mRNA. Immunoblotting analysis showed the enhanced iNOS protein expression and phosphorylation of STAT1 in cells pre-treated with TLR9 ligand. Discussion: We have demonstrated CpG DNA, TLR9 ligand, up-regulates IFN-ү induced NO via enhanced IFN-ү signaling. The result of Western Blotting and RT-PCR support the up-regulation of NO. CpG DNA can be used as agent against virus and bacteria. Further research need to be conducted. Conclusion TLR9 ligand, CpG DNA up-regulates IFN-ү induced NO production in time and dose dependent manner. TLR9 ligand augments the expression of iNOS mRNA and STAT1 phosphorylation in response to IFN-ү.

BACKGROUND Bovine colostrums is the milk secreted by cows during the first few days after parturition. It contains many essential nutrients and bioactive components, including growth factors, immunoglobulins, lactoperoxidase, lactoferrin and cytokines ets. Lactoferrin has been reported for its multifunctional properties such as antifungal, antibacterial, antiviral antioxidant and anticancer activities. The aims of this study focused on the isolation and purification of lactoferrin from Mongolian bovine colostrums. Lactoferrin purified using HiTrap DEAE an ion exchange chromatography. Lactoferrin purification efficiency was about 60.5%. The single band of purified lactoferrin has been observed in SDS-PAGE electrophoresis. METHODS Bovine colostrum was collected at a cow farm in the Darkhan province of Mongolia. At first the cream was separated by centrifugation (10000 xg 20 min at 4oC). In order to separate the whey, the samples were precipitated with 1mol/l to pH 4.6 and centrifuged at 10000 g 20 min again. The samples of whey were stored at -18oC to the analysis. Lactoferrin was purified by HiTrap DEAE an ion exchange chromatography using 0.005 M phosphate buffer (pH 7.7) and linear gradient NaCl from 0.25M, 0.5M, 1M. During chromatography, protein in the eluents was monitored by ultraviolet absorbation at 280 nm with the instrument. Purity test done by using polyacrylamide gel electrophoresis under denaturated condition (SDS-PAGE) method by Laemmli (1970). For HPLC determination of the lactoferrin by Shimadzu Nexera X2 HPLC system with UV/ VIS detector were used. Detection was carried out at the wavelength 280 nm. Separation was performed on a chromatographic column Protein R C18 ,2.2 x 150 mm, 5 μm particle size. Linear gradient and flow rate 0.2 ml/min were used. Mobile phase a consisted of water / acetonitrile/ trifluoroacetic acid ( 95:5:0.1). The column temperature was set at 40oC and injection volume was 10 μl. Data were collected and evaluated by software Lab Solution. An external standard method for quantification analytes was used. RESULTS Purified lactoferrin in the present study had a good concentration and purification efficiency was about 60.5 %. Protein fraction from 1M NaCl gradient delivers sharp and clean peak to HPLC chromatogram that fits intensity and retention time of standard bovine lactoferrin. Ammount of lactoferrin in bovine colostrums was 0.6 mg/ml and it`s molecular weight 80 kDa as a standard sample. The retention time of lactoferrin fraction which is purified by SDS-PAGE gel electrophoresis. The peak of fraction same compared to the standard lactoferrin 5.8 minutes by HPLC analysis. CONCLUSION Ion exchange chromatography shows reliable and easy isolation of lactoferrin from Mongol bovine colostrum.