3 ?g/well.All BALB/c mice immunized with pJME by im.and gene gun injection,and JE inactivated vaccines by ip.injection survived for 21 days.BALB/c mice immunized with pJE by im.injection and gene gun injection partially survived for 21 days.Titres of neutralization antibody produced with pJME were higher than pJE.Protective immunity and titre of neutralization antibody produced by im injection was the same as gene gun injection(im/gene gun injection: 1∶320/1∶320) at day 21.The antibody from BALB/c mice sera after twice pJME immunization only reacts with JEV-E protein. Conclusions Expression efficacy of proteins encoded by pJME and pJE in transfected cells is different.Expression level of related proteins was dependant on recombinant amount for transfection in a certain degree.Immunity effect induced with pJME was higher than pJE.The efficacy of DNA immunization produced by gene gun injection was higher than im.injection.Titres of neutralization antibody induced by DNA immunization were correlated to efficacy of protective immunity.Neutralization antibody from BALB/c mice sera produced by pJME immunization contained anti-E antibody against JEV-E protein.

Objective To study the epidemic characteristics,distribution of pathogen and clinical characteristics of hand,foot and mouth disease in Shenyang area,2014.Methods Swab specimens was collected from 5 070 cases of hand,foot and mouth disease caces,the F-PCR method was adopted for entero-virus 71 (EV71 ),coxsackievirus A 16(CA16)and universal enteroviruses(EU)detection,and combining the relevant clinical data for comparative analysis .Results June to August was the peak epidemic period.5 070 nasopharyngeal swab specimens were detected positive 3 715 intestinal virus nucleic acid,and the detection rate was 73.27%,including CA16 positive 1 481 (39.87%),EU positive 1 148 (30.90%),EV71 positive 1 086(29.23%).The proportion of EU positive was highest in June(29.71 %).The proportion of EV71 pos-itive(24.78%)and CA16 positive(33.27%)were highest in July,respectively .The proportion of nervous system symptoms in EV71 infection group(88 /1 12,78.57%)was higher than those in EU infection group (97 /147,65.99%)and CA16 infection group (44 /78,56.41 %).The proportion of abnormal myocardial enyzme in CA16 infection group (37 /78,47.44%)was higher than those in EU infection group (48 /147, 32.65%)and EV71 infection group(34 /1 12,30.36%).Conclusion CA16 is the major pathogen causing hand,foot and mouth disease in Shenyang area,2014.Severe hand,foot and mouth is still dominated by EV71 .

Objective Construction and expression of recombinant plasmid fusing encoding prME protein derived from Japanese encephalitis virus （JEV） and GM-CSF of BALB/c mouse. Methods GM-CSF gene was ampli/ied by nested-RT-PCR from BALB/c spleen cells. JEV prME protein gene was eluted by the digestion with restrict/on endonucleases BamH I/EcoR I from pJME piasmid. Genetic fusion of prME protein and GM-CSF are subcloned info pcDNA3.1 （＋） eukaryotic vector, and named as pJME/GM-CSF.The recombinant was confirmed by restriction enzymes digestion and DNA sequencing, and then transfected into China hamster ovary （CHO） cells by Lipofectamine2000. pJME/ GM-CSF expression in CHO cells was examined by Western blot. Results We observed 2001 bp and 2472 bp DNA fragments when pJME/GM-CSF was digested with BamHI/EcoRI and BamHI/NotI respectively as expected. The estimated molecular weight of the fusion protein was 85kD. Conclusion The recombinant pJME/GM-CSF was constructed and transfected into CHO cells successfully with pJME/GMCSF stably expressed.

AIM: To investigate the correlation of hepatitis B virus X protein (HBx) with renal tubular epithelialcell apoptosis in hepatitis B virus-associated glomerulonephritis (HBVGN) and the possible signaling mechanism. METHODS: The activation of JAK2/STAT3 signal pathway and the expression of apoptosis -related proteins in humankindey proximal tubular epithelial cells (HK-2 cells) were determined by Western blotting after transfection with HBx eukaryoticexpression vector.The cell proliferation was observed by CCK-8 assay.The cell apoptosis was analyzed by the imagingof HO33342 staining, transmission electron microscopy and flow cytometry with Annexin V /PI double staining.RESULTS:After transfection of the target gene HBx, the expression levels of both p-JAK2 and p-STAT3 were significantly increased.At the same time, the cell proliferation was obviously inhibited, and the apoptotic rate was increased.After incubationwith AG490, the JAK2/STAT3 signal pathway was partially blocked, and the cell apoptosis induced by HBx was reduced. CONCLUSION: HBx up-regulates the activation of JAK2/STAT3 signal pathway to induce renal tubular epithelialcell apoptosis, which is possibly involved in the pathogenic mechanism that HBV directly damages nephridial tissue .

Objective To study the effect of lgG Fc gene on JEV DNA vaccine immunity. Methods Gene encoding IgG Fc was amplified by nested-RT-PCR technique from BALB/c murine spleen cells. JEV prME protein gene was obtained with restriction endonuclease BamH Ⅰ/EcoR Ⅰ from the eukaryotic recombinant named after pJME, which was constructed by us before. Recombinant, named after pJME/IgG Fc, with above two genes encoding JEV prME protein and BALB/c murine IgG Fc was constructed, and was tested by restriction enzymes analysis and DNA sequencing, then was transfected into China hamster ovary (CHO) cells by Lipo-fectAMINE 2000. Distribution and expression of the fusion proteins encoded by JEV prME protein and BALB/c murine IgG Fc genes in transfected CHO cells were detected by immunofluorescence and Western blot. The BALB/c micc were vaccinated with pJME/IgG Fc via intramuscular injection. Then the cytotoxic T lymphocyt (CTL) activity were assessed by lactic dehydrogenase (LDH) and the neutralizing antibody titer were assessed by 80% plaque reduction neutralization test. Results Molecular weights (2001 bp, 2730 bp) of the two in- serts released from pJME/IgG Fc with two group of restriction analysis associated with BamH 1/EcoR I and BamH Ⅰ/Not Ⅰ were correlated to the expected theoretic results respectively. It was estimated that molecular weight (Mr) of the fusion protein was 101 x 103. The expression of the above fusion protein was mainly distribu- ted in endochylema of transfected CHO cells,and not much in membrane of transfected CHO cells. CHO cells transfected with pJME/IgG Fc could express the fusion protein at the 32th cell passage. After immunization, the CTL activity and the neutralizing antibody titer in the pJMF/IgG Fc vaccinated group increased significantly compared with other vaccinated groups(P <0.05). Conclusion The recombinant pJME/IgG Fc was construc- ted and transfected into CHO cells successfully, and CHO cellular lines expressed fusion protein encoded by JEV prME protein and BALB/c murine lgG Fc genes stably were obtained. IgG Fc gene could reinforce the cellular immunity and humoral immunity of JEV DNA vaccine.

Objective To study the adjuvant effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) coding gene on cellular immunity induced by Japanese encephalitis (JE) virus DNA vaccine. Methods GM-CSF coding gene was amplified by nested-reverse transcriptase-polymerase chain reaction (RT-PCR) technique from BALB/c murine spleen cells. Recombinant plasmids pJME/GM-CSF and pGM-CSF were constructed by JE virus (JEV) prM-E protein with GM-CSF coding gene or GM-CSF coding gene only, respectively. The plasmids were transfected into China hamster ovary (CHO) cells by Lipofectamine 2000. The coding protein expressions and distributions were detected by immunofluorescence. The BALB/c mice were vaccinated with indicated immunogens with or without GM-CSF gene. The changes of T lymphocyte subsets in the spleen and levels of intracellular cytokines, such as interferon (IFN)-γ and interleukin (IL)-4 of splenic cells from mice immunized with different immunogens were evaluated by flow cytometry. The cytotoxicity T lymphocyte (CTL) activity was assessed by lactate dehydrogenase (LDH). The data were compared by one-factor analysis of variance and least significant difference. Results The constructed recombinant pGM-CSF and pJME/GM-CSF were confirmed by restrict enzyme digestion and DNA sequencing. The expressions of the above proteins were mainly in the cytoplasm and minor on cell membrane. The percentage of CD4+ T lymphocytes in pJME/GM-CSF vaccinated group was (33.90±0.79)%, which was significantly higher than that of in other groups (t values were 9. 818, 6. 804, 6.594, 10.061, 9.380, and 17.675, all P<0.05). The percentages of CD4+T lymphocytes in pJME +pGM-CSF (0) and pJME+pGM-CSF (-3) vaccinated groups were (29.83±0.61)% and (29.70±0.51)%, respectively, which were both higher than that in pJME+pGM-CSF (+3) vaccinated group of (27.69+0.50)% (t=3.466, t=3.255, both P<0.05). The percentages of CD8+ T cells in pJME/GM-CSF and pJME+pGM-CSF vaccinated groups were both higher than that in empty vector (pcDNA 3.1+) group and JE inactivated vaccine vaccinated group (t values were 3.811, 2.627, 10.537, and 3.811, all P<0.05). The CTL activity in pJME/GM-CSF vaccinated group was (51.48±0.10)%, which was higher than those in other groups (t values were 22.868, 13.823, 5.377, 32.287, 34.632, and 53.795, all P<0.05). The IFN-γ/IL-4 ratios in pJME/GM-CSF, pJME+pGM-CSF (0) and pJME + pGM-CSF (-3) vaccinated groups were (19.13±1.36), (12.32±0.82) and (7.05±0.43), respectively, which were higher than those in other groups (P<0.05). Conclusion GM-CSF coding gene could enhance the cellular immune response induced by Japanese encephalitis DNA vaccine.

OBJECTIVETo explore the effects of hepatitis C virus (HCV) core protein on the activity of double-stranded RNA-dependent protein kinase (PKR).

METHODSThe human hepatoma cell line BEL-7402 was transfected with the HCV core gene-containing eukaryotic expression vector pCMH6K-Core (at various concentrations), or empty vector, or no vector; a group of cells was co-transfected with the luciferase reporter plasmid pGL3-promoter. The cells were treated with interferon (IFN) a-2b to induce the expression and activation of endogenous PKR, or left untreated to serve as controls. The effect of core protein on PKR phosphorylation was detected by western blotting. Luciferase activity was detected to reflect effects of the core protein on the synthesis of cellular proteins. The t-test and F test were used for statistical analyses.

RESULTSIn the case of IFNa stimulation, PKR phosphorylation levels were significantly lower in the HCV core protein expressing cells than in the cells transfected with empty plasmid or with no vector, but the total PKR expression level was not significantly different among these three groups of cells. Cells co-transfected with luciferase plasmid and the core protein expressing vector showed significantly higher levels of luciferase expression than the cells co-transfected with the empty vector. Moreover, the luciferase activity and core protein expression levels increased in a dose-dependent manner, with the luciferase activity of the cells treated with 0.5 mug, 1.0 mug and 1.5 mug pCMH6K-Core being 1.941 ± 0.199 times, 2.868 ± 0.275 times and 3.839 ± 0.338 times higher than that of the empty vector group (all P < 0.05).

CONCLUSIONIn the human hepatoma cell line BEL-7402, the HCV core protein can inhibit the activity of endogenous PKR, thereby promoting cell protein synthesis.

Cell Line, Tumor ; Genes, Reporter ; Genetic Vectors ; Humans ; Phosphorylation ; Protein Biosynthesis ; RNA-Binding Proteins ; metabolism ; Transfection ; Viral Core Proteins ; genetics ; metabolism

The kidney plays a key role in hepatitis B virus (HBV) infection of liver cells. The pathogenesis of HBV-associated glomerulonephritis (HBV-GN) remains unclear, and the mechanism of HBV directly causing the injury of renal tubules has been taken seriously in recent years. HBV can induce the apoptosis of renal tubular cells by regulating cell cycle and activating related signaling pathways including NF-κB and thus lead to the progression of HBV-GN. At present, there are still no drugs targeting the kidney in the treatment of HBV-GN. This article summarizes the research advances in the key factors for HBV infection of cells and the injury of renal tubular cells caused by HBV and elaborates on the possible mechanism of direct infection of renal tubular cells by HBV, so as to provide new ideas for the treatment of HBV-GN.

The liver is closely associated with the kidney, and liver injury in various stages can cause various kidney diseases to varying degrees, which further lead to renal impairment. Such renal impairment in the early stage is often functional and can be reversed by drugs, otherwise it can progress to hepatorenal syndrome, cause acute renal failure, and even threaten human life. The indicators such as serum creatinine and urea nitrogen have a limited effect in the early diagnosis of renal impairment and cannot be used for early monitoring and diagnosis of liver cirrhosis patients with renal impairment. Therefore, early monitoring of liver cirrhosis patients with renal impairment has always been a hot topic in this field. This article summarizes the research advances in the indicators for early diagnosis of renal impairment.

Patients with severe liver diseases are prone to nosocomial infection due to serious liver dysfunction, low immune function, and reduced stress ability to infection, and such infections further aggravate patients’ conditions and directly affect their clinical outcome. This article reviews the clinical features of nosocomial infection in patients with severe liver diseases and related risk factors.