Objective To obtain novel vaccine candidate antigens against Schistosoma japonicum. Methods S. japonicum schistosomula cDNA library was screened by using sera of Microtus fortis that was naturally resistant to schistosomiasis. The positive clones were transformated into Escherichia coli BM25.8, E. coli clones containing the plasmid cultured in LB, and then selected for plasmid extraction, the plasmid DNA was digested by EcoRⅠand Hind Ⅲ, and analysed by agarose gel electrophoresis. The positive clones were also sequenced and the data were analysed through the internet Nucleotide BLAST software of NCBI and Expert Protein Analysis system of GeneRunner and HNN. Results Twelve positive clones were obtained after repeatedly immunoscreening the library and their sizes ranged from 300 bp to 1100 bp. Two novel genes (named as Sj-sMf1 and Sj-sMf2) with complete ORF were obtained. The deduced protein of Sj-sMf1 consisted of 93 amino acids while Sj-sMf2 consisted of 61 amino acids. Sj-sMf1 protein predicted containing one cAMP phosphorylation site and Casein kinase C phosphorylation site, respectively. Sj-sMf1 protein predicted containing one Casein kinase C phosphorylation site and two Protein kinase C phosphorylation sites. Conclusion Two novel genes predictably encoding unknown proteins are obtained from immunoscreening of Schistosoma japonicum schistosomula cDNA library by M. fortis sera.

Objective:To investigate the role of mitofusion 2 (Mfn2) in high glucose (HG)-induced endoplasmic reticulum stress (ERS) and apoptosis of podocytes.Methods:(1) Streptozocin was used to induce a diabetes mellitus (DM) rat model. Renal histopathological changes in rats were observed by HE staining. Expression of Mfn2 and CCAAT/enhancer-binding protein homologous protein (CHOP) in glomeruli was observed by immunohistochemistry. Protein levels of Mfn2, protein kinase RNA-like ER kinase (PERK), phospho(p)-PERK, and CHOP in glomeruli were analyzed by Western blotting. (2) Conditionally immortalized human podocytes (HPC) cultured in vitro were divided into control, mannitol (MA) and HG groups. Expression of Mfn2 was observed by immunofluorescence. Protein levels of Mfn2, p-PERK, PERK and CHOP in HPC were analyzed by Western blotting. Podocyte apoptosis in each group was evaluated by flow cytometry with AnnexinⅤ-PE/7AAD double staining method. (3) HPC were divided into control, HG, HG+Mfn2-Myc plasmid-transfected and HG+control plasmid-transfected groups. Protein levels of Mfn2, p-PERK, PERK and CHOP in HPC were analyzed by Western blotting. Expression of CHOP was observed by immunofluorescence. Mitochondrial membrane potential in each group was observed by mitochondrial membrane potential assay kit with JC-1. Podocyte apoptosis in each group was evaluated by flow cytometry with AnnexinⅤ-PE/7AAD double staining method. Results:(1) Compared with the control group, the glomerular mesangial matrix of the DM group rats was significantly proliferated, and the expression of Mfn2 was down-regulated with the expression of ERS-related proteins p-PERK/PERK and CHOP up-regulated (all P<0.05). (2) Compared with the control group, Mfn2 was down-regulated and p-PERK/PERK and CHOP were up-regulated in HPC of HG group (all P<0.05). Apoptosis of HPC was also increased in HG group. There was no significant difference in the above indicators between the control group and the mannitol group (all P>0.05). (3) Compared with the HG group, mitochondrial membrane potential of HPC was alleviated and apoptosis of HPC was decreased in HG+Mfn2-Myc plasmid-transfected group ( P<0.05). P-PERK/PERK and CHOP were down-regulated in HG+Mfn2-Myc plasmid-transfected group (both P<0.05). There was no significant difference in the above indicators between the HG group and the HG+control plasmid-transfected group (all P>0.05). Conclusions:Mfn2 down-regulation in HG-stimulated podocytes may induce ERS to increase apoptosis of podocytes. Up-regulation of Mfn2 can alleviate the HG-induced ERS and apoptosis in podocytes.

Objective To investigate the role of autophagy in high glucose-induced podocyte lipid droplet metabolism.Methods (1) Cultured,conditionally immortalized human podocytes (HPC)were divided into normal control group,high glucose group and mannitol group.Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation;Protein level of SREBP-1 was analyzed by Western blotting.(2) HPC were cultured and divided into normal control group,high glucose group,high glucose+3-methyladenine (3-MA) group,and mannitol group.Acridine orange staining was used to observe the formation of autophagosomes.Western blotting was used to detect the protein levels of beclin-1 and LC3-Ⅱ/LC3-Ⅰ.Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation;Western blotting was used to analyze the expression of SREBP-1.Results (1) Compared with the normal control group,the lipid accumulation in the high glucose group was increased and the lipid metabolism related molecule SREBP-1 was up-regulated (P ＜ 0.05);There was no significant difference between the normal control group and the mannitol group in lipid accumulation (P ＞ 0.05).(2) Compared with the normal group,the number of autophagosomes was increased and autophagy-related proteins beclin-1 and LC3-Ⅱ/LC3-Ⅰ were up-regulated in high glucose group (all P ＜ 0.05).After intervened with 3-methyladenine,a significant decrease in autophagosomes was observed;Protein levels of autophagy-related proteinsbeclin-1 and LC3-Ⅱ/LC3-Ⅰ were decreased (all P ＜ 0.05);The lipid droplets in the high glucose+3-MA group was decreased and lipid metabolism related molecule SREBP-1 was down-regulated (all P ＜0.05).Conclusion Autophagy may be involved in the process of high-glucose-induced podocyte lipid accumulation by affecting SREBP-1 expression,and inhibition of autophagy can alleviate the high-glucose-induced podocyte lipid accumulation.