ABSTRACT
We conducted prediction and analysis for secretory proteins from Thelazia callipaeda at Genome Scale based on the previous full genome annotation.The software SignalP,TMHMM,big-PI Predictor,MEME,Protcomp and SecretomeP were combined to process the prediction of the secretome of Thelazia callipaeda.The analyses of secretory proteins by GO function enrichment,KEGG pathway,and statistics of domains were performed.Results showed that totally 259 secretory proteins were found in Thelazia callipaeda genome and the amino acid lengths of secretory proteins were mainly concentrated between 100 to 700 aa exclusively.GO function analysis of secretory proteins indicated that they were enriched in the secreting pathways and in the interactions with host.The results of KEGG metabolism secretory proteins suggested that some of them contributed to drug metabolism and glutathione metabolism.And domain analysis suggested that most of them were glycoside hydrolase,contributing to sugar metabolism.Around 126 secretory proteins had antigenicity of B-cell epitope.In summary,we found that secretory proteins in Thelazia callipaeda were most small proteins,which were involved in sugar metabolism and antioxidative activity,facilitating Thelazia callipaeda to invade the hosts and play a key role in the parasitic course.
ABSTRACT
Objective To investigate the physicochemical properties and immunomodulatory activities of crude polysaccharides and their fractions from Sophorae Flavescentis Radix. Methods The crude polysaccharide (SFP-100) was obtained successively by boiling Sophorae Flavescentis Radix in water, ethanol precipitating, dialyzing and freeze drying. SFP-100 was separated withDEAE-cellulose column to obtain three fractions, and these fractions were fruther separated with Sephadex G-100 column to obtain their sub-fractions. The sugar content was determined by phenol-sulfuric acid method, the molecular distribution was determined with gel filtration chromatography, and the monosaccharide composition was analyzed with capillary electrophoresis after PMP derivatization. The immunobiological activities were estimated by measuring the proliferation of mouse spleen lymphocytes as well as the IFN-secretion in mouse splenocytes and the TNF-α secretion in mouse peritoneal macrophages. Results The yield of SFP-100 from Sophorae Flavescentis Radix was 4.83％ and sugar content was 71.62％. SFP-100 was separated into three fractions SFP-100-A, SFP-100-B and SFP-100-C, whose yields were 3.5％, 25.6％ and 16.7％, and the sugar content was 85.99％, 72.09％ and 24.30％, respectively.The monosaccharide composition and their molar ratio for SFP-100-A, SFP-100-B and SFP-100-C were Ara∶Glc∶Gal=7.16∶91.02∶1.82, Xyl∶Ara∶Glc∶Rha∶Gal∶GalA=0.05∶1.00∶0.85∶0.04∶0.35∶0.43, and Xyl∶Ara∶Glc∶Rha∶Gal∶GlcA∶GalA=0.20∶1.00∶0.33∶0.36∶0.45∶0.55∶14.37, respectively. SFP-100-B was further separated into two sub-fractions SFP-100-B-a and SFP-100-B-b with Sephadex G-100, and the other two sub-fractions SFP-100-C-a and SFP-100-C-b were also obtained with the same Sephadex G-100 from SFP-100-C. SFP-100-B-a showed a main wide peak, which had the relative molecular weight 1.02×105 and monosaccharide composition Ara∶Glc∶Rha∶Gal = 1.00∶0.06∶0.02∶0.29. Meanwhile, the relative molecular weight and monosaccharide composition were 5.43×104 and Xyl∶Glc =1.00∶3.81 for the main peak of SFP-100-C-a, and 2.75×104 and Ara∶Glc∶Rha∶Gal∶GlcA∶GalA=1.00∶2.10∶0.57∶0.74∶1.09∶33.75 for the main peak of SFP-100-C-b, respectively. The crude polysaccharides SFP-100 and its fractions, SFP-100-B and SFP-100-C, as well as the sub-fractions, SFP-100-B-a, SFP-100-B-b and SFP-100-C-a, increased the proliferation of spleen cells, all in a dose-dependent manner. Further, SFP-100 could improve the secretion of IFN-γ and TNF-α in spleen cells, while SFP-100-B, SFP-100-C, SFP-100-B-a and SFP-100-B-b could stimulate the secretion of IFN-γ. Conclusion The crude polysaccharides of Sophorae Flavescentis Radix and their fractions showed a good immunomodulatory activity, which may be related to the clinical use of Sophorae Flavescentis Radix for the anti-HBV and anti-inflammatory therapy.
ABSTRACT
Objective To construct a recombinant plasmid pGEX-TSO45W-4B-TSOL18 of Taenia solium,to obtain purified recombinant protein TSO45W-4B-TSOL18 and to prepare rabbit antiserum against the recombinant fusion protein.Methods TSO45W-4B and TSOL18 encoding genes were connected with hydrophobic (Gly4Ser)3 linker.TSO45W-4B-TSOL18 fusion gene was synthesized and cloned into an expression vector pGEX-1λT to construct a recombinant plasmid pGEX-TSO45W-4B-TSOL18.The positive recombinants were transformed into Escherichia(E.) coli ArcticExpress (DE3),and the expression of recombinant protein was induced with isopropylβ-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).The expressed recombinant fusion protein was purified through affinity chromatography.The purified recombinant protein was mixed with complete Freunds adjuvant at a dosage of 0.5 mg.Rabbit was intramuscularly and subcutaneously immunized in the hind leg and the back,respectively.After 2 weeks the rabbit was boosted with purified recombinant protein which was mixed with incomplete Freunds adjuvant at the same dosage.Rabbit can be boosted every 10 days until an adequate response was achieved.At the 6th day after the last immunization,blood was collected from the rabbit heart.Serum was separated to purify and prepare the antiserum.ELISA was applied to determine the titer of the antiserum and Western blot assay was used to determine the specificity of the antiserum.Results The size of the synthesized fusion gene TSO45W-4B-TSOL18 was 789 bp.The results of restriction enzyme digestion showed that the recombinant plasmid pGEX-TSO45W-4B-TSOL18 was successfully constructed.DNA sequencing showed that the size of the fusion gene TSO45W-4B-TSOL18 was 789 bp,which was consistent with expected result.As demonstrated by SDS-PAGE,relative molecular mass of the expressed recombinant fusion protein was approximately 55 × 103,and its purity was 85％ after purification with affinity chromatography.Titer of the antiserum against the recombinant protein was 1 ∶ 512 000 in ELISA assay and the specific rabbit antiserum against the purified recombinant protein TSO45W-4B-TSOL18 could specifically bind to the recombinant fusion protein in Western blot assay.The relative molecular mass of the specific band was about 55 × 103,which was consistent with expected size.Conclusions The recombinant plasmid pGEX-TSO45W-4BTSOL18 of Taenia solium is successfully constructed.High quality recombinant fusion protein TSO45W-4B-TSOL18 and high titer rabbit antiserum are successfully prepared.
ABSTRACT
Objective To explore the enterovirus infection status among healthy children under 15 years old in the border areas of Yunnan province that connecting Myanmar.Methods A total of 319 stool samples were collected from healthy children in the 10 entrance ports.Enterovirus was isolated from these stool samples and then poliovirus and adenovirus were serotyped by neutralization test using specific anti-sera.All the non-polio enteroviruses(NPEVs)were identified by partial sequencing of VP1 gene.Results All 53 enterovirus were isolated from 319 stool samples and 16.6% of them carried the virus.23 polio virus(PVs)and 30 NPEVs were isolated with rates of carrying the virus were 7.2% and 9.4% respectively.4 adenovirus were also isolated with a rate as 1.25%.1 isolate could not be amplified by any Pan-enterovirus primers or by RT-PCR so was not able to be sequenced.The results of NPEVs sequencing showed that:1 isolate(3.3%)was classified into 1 serotype of HEV-A while 20 isolates(66.7%)were classified into 11 serotypes of HEV-B and 8 isolates(26.7%)were classified into 3 serotypes of HEV-C.However,we could not isolate any viruses that belong to HEV-D.nt.Result from the aa identify calculation showed that the nt and aa identification between isolates and corresponding standard strains were more than 75% and 85% respectively.The findings were similar to the international standards.Conclusion Our results showed that the rate of carrying the enterovirus especially poliovirus in some areas of Yunnan province that bordering Myanmar was higher than that of rate through the routine acute flaccid paralysis detection system.Of the enterovirus isolated,HEV-B group appeared the predominant with the wide spread of enterovirus serotype.Some newer enterovirus were also detected such as EV73(2 strains),EV75(1 strain),EV80(1 strain)and EV96(4 strains).
ABSTRACT
Molecular typing was conducted according to the reported methods for 2 enteroviruses that were isolated from healthy children in the border areas of Yunnan Province with Myanmar. RT-PCR and sequencing were performed with 292/222 primers according to the Oberste's methods. The resulting sequences were blasted against the Genbank database and compared with all available enterovirus database. Analysis of homology at nucleotide and amino acid level identically suggested that the two enteroviruses are human enterovirus 73.
Subject(s)
Child , Female , Humans , Male , Cell Line , China , DNA Primers , Genetics , Enterovirus , Classification , Genetics , Enterovirus Infections , Virology , Molecular Sequence Data , PhylogenyABSTRACT
<p><b>OBJECTIVE</b>This report presented an overview on the epidemiology of enterovirus in Yunnan province, the People's Republic of China.</p><p><b>METHODS</b>A total of 210 strains of non-polioviruses isolated under acute flaccid paralysis surveillance during a 5-year study period from 1997 to 2000 and 2004 were examined. Of the 210 non-polioviruses strains, a total of 12 strains of adenoviruses were serologically identified. The remaining 198 isolates were used for molecular typing, and the viral genomes of 195 nonpolio enteroviruses (NPEVs) were translated to corresponding amino acid sequences and compared with those of the prototype strains.</p><p><b>RESULTS</b>Based on molecular typing, 5 isolates were classified into 5 serotypes of human enterovirus A species while 158 isolates into 34 serotypes of B and 32 isolates into 6 serotypes of C species. However, we did not isolate any viruses which belonged to human enterovirus D species. Thus, under acute flaccid paralysis surveillance, human enterovirus B species accounted for 75.2% of the 210 isolates and was considered as the predominant one, followed by human enterovirus C (12.2%), adenovirus (5.7%), and human enterovirus A (2.4%).</p><p><b>CONCLUSION</b>Although the epidemiological characteristics of NPEVs from Yunnan province remained "unknown", the molecular typing method had provided us a breakthrough to understand the epidemiology of these viruses.</p>
Subject(s)
Humans , China , Epidemiology , Enterovirus , Classification , Genetics , Enterovirus Infections , Epidemiology , Genes, Viral , Molecular Epidemiology , SerotypingABSTRACT
Objective To study the inhibition effects of antibacterial proteins from Musca domestica larvae on JEC and A375 tumour cells. Methods Antibacterial proteins with concentrations of 0.02%,0.1%,0.5%,2.5% and 12.5% were supplied in the culture of JEC and A375 tumour cells in vitro.The cell cycles and apoptosis of JEC and A375 tumour cells were detected by flow cytometry,and the apoptosis index(AI) was measured.The morphology of apoptotic cells was observed with HE stainings and AO staining.The culture without antibacterial proteins was served as control. ResultsThe ratio of apoptosis index/proliferation index(AI/PI) of JEC cells increased with the concentration of antibacterial proteins.The PI and apoptosis rate of 2.5% antibacterial proteins group and 12.5% antibacterial proteins group significantly increased,and G0/G1 significantly decreased.For A375 cells,there were significant differences in G2/M, S,G0/G1,G2/M,AI/PI and PI between 12.5% antibacterial proteins group and control group(P
ABSTRACT
<p><b>BACKGROUND</b>To develop a rapid and more sensitive TaqMan RT-PCR assay specific for Banna virus.</p><p><b>METHODS</b>Total RNA of strains of Banna virus and other arboviruses were extracted and reverse transcribed to cDNAs. With the cDNAs as templates, the TaqMan RT-PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Banna virus. The sensitivity, specificity and the possibility of this assay to detect Banna virus in pools of mosquitoes. Meanwhile, human sera samples added with different dilutions of Banna virus culture medium supernatant were evaluated.</p><p><b>RESULTS</b>All the collected 12 Banna virus strains in our country were detected as positive, while the results of other arboviruses such as Japanese encephalitis virus (JEV), Batai virus, Sindbis virus and Orbiviruses were negative. This assay was more than 100 times sensitive than that of traditional PCR. About 0.5 CCID-50 of Banna virus in 50 microl samples could be detected with this assay. The sensitivity decreased by about 10 times when the dsRNA was denatured in 65 degrees C instead of 99 degrees C, and also when the virus seeds were kept at room temperature for 1 day or 1 week at 4 degrees C. There was no significant difference in the results of Banna virus detection between artificial human serum samples and positive control virus. Six were detected as positive for Banna virus-specific nucleic acids in 112 pools of mosquitoes, while 3 was positive with virus isolation.</p><p><b>CONCLUSION</b>The Banna virus-specific TaqMan RT-PCR assay was proved to be highly sensitive, specific and showed a good reproducibility; the assay may be applied for surveillance of this virus in clinical samples and pools of mosquitoes screening.</p>
Subject(s)
Animals , Humans , Coltivirus , Genetics , Culicidae , Virology , RNA, Viral , Genetics , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Methods , Taq Polymerase , MetabolismABSTRACT
<p><b>BACKGROUND</b>To develop a rapid, specific and sensitive method for detecting Sindbis virus (SINV) with SYBR GREEN I real time PCR.</p><p><b>METHODS</b>Total RNA of strains of Sindbis virus and a related virus were extracted and reverse transcribed to cDNAs. With the cDNAs as template, the SYBR GREEN I real time PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Sindbis virus, and the sensitivity, specificity and reproducibility were evaluated.</p><p><b>RESULTS</b>For the PCR, 55 degrees C was chosen as the optimal anneal temperature and 0.5 micromol/L as the optimal primer concentration. Using this method, all the selected SINV were detected as positive, while the results of control arboviruses such as Geta virus, Japanese encephalitis virus (JEV), Batai virus, Seadornavirus Orbiviruses and synthesized WEEV cDNA were negative. With this system, 0.1 PFU/ml SINV cDNA could be detected; The sensitivity of this assay was about 100 times higher than standard RT-PCR. All the results were reproducible within two compatible tests, and the stability of the detection system was very good. The test results of simulated infection human serum samples showed that human serum had no obvious interference with this system. With this system, 6 of 151 clinical samples with unknown fever or encephalitis were determined as positive.</p><p><b>CONCLUSION</b>The developed SYBR GREEN I real time PCR assay for detecting Sindbis virus was highly sensitive, specific and showed a good reproducibility and stability. It is our belief that the present method can be further used in clinic sample to verify its stringency.</p>