Mannose-binding Lectin Inhibits Human Cytomegaloyirus Infection Of Human MD-DC Department Of Pediatrics

IntroductionHuman cytomegalovirus (HCMV) is a member of herpes virus family, with a lipid enveloped, double-stranded DNA as229kb chromosome. HCMV infection is common in children. The viral factors produced by HCMV can escape from the host immune, so the body can not completely remove the virus. Studies have shown that HCMV envelope glycoprotein is necessary for HCMV replication and infection and play an important role in in the host cell receptor recognition, absorption and penetration of host cells. HCMV infection starts from the viral envelope glycoprotein binding with the cell surface receptor. Then HCMV enters cells by membrane fusion or receptor-mediated endocytosis. So how to inhibit HCMV glycoprotein binding with the target cell membrane is an important entry point for controlling HCMV infection.DC cell surface-specific intercellular adhesion molecule3with non-integrin molecules (dendritic cell-specific ICAM-3-grabbing nonintegrin, DC-SIGN) was one type of Ⅱ Membrane protein with the addition of a C-type lectin. It can identify a variety of pathogens such as viruses, bacteria, worms and fungi, and play an important role in the DC cell recognition.Innate immune molecule mannose-binding lectin(MBL) is a member of C-type lectin superfamily member, and can highly efficiency combined with a variety of sugar-based composition as the body's first defense line against viruses and involved in the regulation of the infection process. No research on MBL applied to HCMV infection is reported.In this study, we purified natural MBL (hMBL) and recombinant MBL (rMBL), cultured from human peripheral blood mononuclear cells derived DC cells (monocyte-derived dendritic cell, MD-DC); explore the different concentrations of hMBL/rMBL inhibit HCMV infection MD-DC and explore its mechanism.Methods:1. The establishment of the expression of human wild-type MBL in Chinese hamster ovary cells. We establish the wild-type MBL in pNOW/CMV-A expression vector by the reference of improved Ohtaniet al methods. Then use this vector to transfect CHO cell line with transfection reagents, screen stably transfected cell line with G418, analysis the MBL gene expression in stably transfected cell line fluorescence quantitative PCR, detected MBL protein concentration by ELISA.2. The purification, quantification and SDS-PAGE identification of rMBL and hMBL. CHO/MBL cells are cultured under20%fetal calf serum MEDM medium,37℃,5%CO2and saturated humidity. Take the supernatant and human fresh plasma to purify with the mannan-Sepharose4B affinity chromatography. Use the SDS-PAGE to identify the purity and the Coomassie brilliant blue to the protein quantification.3MD-DC cells obtained. MD-DC cells were isolated and cultured from human peripheral blood mononuclear cells (PBMC) stimulated by the GM-CSF and EL-4. In the day3we change the half amount of cytokines culture medium. In the day6MD-DC cells obtained.4. MBL inhibit the ability of MD-DC capture HCMV. At day6, the cloned MD-DC Were harvested. Then MD-DC were pre-exposed to several dilutions of the hMBL/rMBL for30min, then HCMV suspensions were added to MD-DC for2h to compare the inhibitory effect of hMBL/rMBL on the HCMV infection of MD-DC. We use immunofluorescence confocal microscopy to observe the ability of MD-DC to capture HCMV, fluorescence quantitative PCR to analyze HCMV DNA in MD-DC, flow cytometry to analyze HCMV pp65in MD-DC. The percentage of HCMV DNA copy number captured by MD-DC is the ratio about HCMV DNA copy number and initial HCMV DNA copy number added. According to the instructions,Hcmv wer marked with PKH26, and DC-SIGN on MD-DC surface were stained with CD209-FITC. Under Immunofluorescence confocal imcroscopy obserce the inhibitory of MBL on the ability of MD-DC to capture HCMV particles by analysis of virus quantitative on the unit cell area.5. MBL inhibit the ability of HCMV diffusion between MD-DC. At day6, the cloned MD-DC were harvested. Then MD-DC infected by HCMV co-culture with hMBL/rMBL for three days to compare the inhibitory effect of hMBL/rMBL on the HCMV diffusion between MD-DC. In24hours,48hours,72hours, We use fluorescence quantitative PCR to analyze HCMV DNA in MD-DC. In72hours ues flow cytometry to analyze HCMV pp65in MD-DC.Results:1. The results of targeted gene expression in stable transfected cell are two screened stable cell lines by RT-Q-PCR.The gene expression in CHO-MBL is1000times more than that in empty vector.2. We obtain447.86ug rMBL from800ml CHO/MBL supernatant and420.92ug hMBL from200ml human plasma.Identified by SDS-PAGE. The SDS-PAGE identification of purity hMBL and rMBL, The non-reduced hMBL from human plasma has monomer form M:Marker(26×103)and polymer(2dimer52×103,3dimer82×103); The reduced hMBL from human plasma has polymer(>170×103); The non-reduced rMBL has monomer (32×103); The reduced rMBL has polymer(>170×103)3. PBMC stimulated by the GM-CSF and IL-4,then in the day6MD-DC cells obtained. MD-DC. morphology was observed under light microscope as visible cell clusters suspended growth, irregular shape, showing pseudopodia. Characteristics dendritic like morphology under electron microscope. We detect the characteristics DC signs as MHC-Ⅱ, CD80, CD86, CD40by flow cytometry. DC-SIGN with The anti-CD209-FITC monoclonal antibody by flow cytometry shows the percentage of DC-SIGN is88%.4. In hMBL/rMBL inhibition the ability of MD-DC capture HCMV experiments, fluorescent quantitative PCR show that compared to the control group,1μg/ml of hMBL group and1μg/ml rMBL have no significant differences, and5μg/ml and10μg/ml hMBL/rMBL groups all have significant differences.10μg/ml concentrations of hMBL/rMBL have significant inhibition on the ability of MD-DC capture HCMV by immunofluorescence confocal microscopy and flow cytometry.5. In hMBL/rMBL inhibition the ability of HCMV diffusion between MD-DC experiments, fluorescent quantitative PCR show in the first24hours and48hours of co-culture, each experimental group has no significant difference with the control group, until72hours,1μg/ml hMBL/rMBL groups have no significant difference while5μg/ml and10μg/ml hMBL/rMBL groups all have significant differences. Flow cytometry in72hours10μg/ml hMBL/rMBL groups are significantly lower than the control group. Conclusion:1,Successfully establish the CHO/rMBL and obtain a large number of high-purity rMBL and hMBL;2Successfully isolate and culture MD-DC with high expression of DC-SIGN from human peripheral blood mononuclear cells3MD-DC can capture HCMV and HCMV can be spread between the MD-DC.4The hMBL/rMBL in physiological concentration range (5-10μg/ml) can significantly inhibits human cytomegalovirus infection of human MD-DC cells, and the hMBL is more ffective than rMBL...