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Objective@#To investigate the comparative study of serum hepatitis B virus (HBV) large protein (HBV-LP) , HBV-DNA, and Pre S1 antigen (Pre S1-Ag) detection in the evaluation of serum HBV replication in patients with chronic hepatitis B.@*Methods@#A total of 482 patients infected with chronic hepatitis B virus (CHB) were enrolled and the serums were collected in a hospital of Hefei city in Anhui province from June 2013 to March 2015. The serum HBV-LP, HBV markers(HBV-M) and Pre S1-Ag were detected using ELISA, and HBV-DNA were quantified using quantitative real-time PCR. The positive detection rate difference of HBV-DNA, HBV-LP and Pre S1-Ag were compared, the correlation between the logarithm of HBV-DNA copies number and the absorbance value of HBV-LP was analyzed using Spearman rank correlation.@*Results@#The positive rates of HBV DNA, HBV-LP, and Pre S1-Ag were 67.22% (324/482), 73.86% (356/482), and 37.34% (180/482), respectively (P<0.01). The positive rates of the three markers were 54.57% (185/339), 64.90% (220/339), and 27.73% (94/339), respectively, in 339 HBeAg-negative CHB patients (P<0.01). In HBeAg negative patients, the positive rate of HBV-LP in 185 cases of HBV-DNA positive samples was 90.81% (168/185), which was higher than that of Pre S1-Ag with rate of 39.46% (73/185) (P<0.01).The positive rate of HBV-LP was 33.77% (52/154) in 154 cases of patients with negative HBV-DNA whose positive rate was higher than Pre S1-Ag with positive rate of 13.64% (21/154)(P<0.01). The positive rates of HBV-DNA, HBV-LP and Pre S1-Ag in HBsAg, HBeAg and HBcAb positive groups were 97.04% (131/135), 94.81% (128/135), and 60.00% (81/135), (P<0.01); The positive rates of three indexes of HBsAg, HBeAb, HBcAb positive group were 53.74% (122/227), 63.88% (145/227), and 27.31% (62/227); The positive rates of three indexes of HBsAg and HBcAb positive group were 55.79% (53/95), 67.37% (64/95), and 28.42% (27/95) (P<0.01). The absorbance value of HBV-LP was positively related with the logarithm of HBV-DNA copies number (Spearman rank correlation coefficient was 0.908, P<0.01).@*Conclusion@#There was a good correlation between HBV-LP and HBV-DNA load value, and could be used as an effective complement for the detection of HBV-DNA and HBV-M. Compared with Pre S1-Ag.
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A rapid and sensitive method of loop-mediated isothermal amplification(LAMP) was established to detect Pseudomonas aeruginosa(P.aeruginosa).Three pairs of LAMP primers(inner,outer and ring primers) were designed according to the gbca gene of P.aeruginosa.Since adding hydroxy naphthol blue(HNB) to the reaction system, a positive reaction was indicated by a colorchange before and after the reaction,and was verified by agarose gellectrophoresis.Both LAMP and PCR were applied to detect clinical specimens, the sensitivity and specificity of the detection method were evaluated,and were compared with those of conventional PCR.A LAMP method for detecting P.aeruginosa was successfully established.The LAMP method showed specificity for P.aeruginosa without other bacteria amplification.The established LAMP method in this study enables rapid,sensitive and specific detection of P.aeruginosa,and can be applied for grass roots and small scale laboratories as well as field surveillance.
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Objective To investigate the immunodiagnostic valueofrecombinant granulocvte protein 5(rGRA5) protein in prokaryotic Toxoplasma gondii.Methods The PCR was used to ampliify GRA5 gene,then insert the target gene into the prokaryotic expression vector pET28a,and identified by double digestion and sequencing, then transfecte the correct targert gene into BL21 competent cells, SDS-PAGE and Western blot were used to detect the expression of the recombinant protein in BL21.ELISA was used to detect the serum of 100 cases of serologically Toxoplasma-positive individuals.Results The products of GRA5 gene of 363 bp in length was successfully amplified by PCR,the prokaryotic expression vector pET28a-GRA5 was constructed successfully, the result of double digestion showed that the insert fragment size was correct, and DNA sequencing results showed that the homology of GRA5 gene with GenBank was 100%.Also the expression of GRA5 protein was successfully detected in BL21 by Western blot(about 14 ku).ELISA method was used to detect 100 cases of patients with 73 cases showed positive results, the positive diagnosis rate was 73.0%.Among them, the positive detection rate of IgG positive samples was 72.5% in 40 cases,the positive detection rate of IgM positive samples was 53.3% in 30 cases,the positive detection rate of IgG and IgM positive samples was 93.3% in 30 cases.Conclusion The prokaryotic expression vector pET28a-GRA5 is constructed successfully, and the recombinant protein has potential for immunological diagnosis of toxoplasmosis.
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Objective To explore the clinical value of five methods commonly used for the detection of clinical syphilis antibody.Methods A total of 160 confirmed syphilis cases were chosen as the experimental group while 200 non-syphilis cases were set as the control group.Serum specimens were detected by methods as Treponema pallidum particle agglutination assay (TPPA),chemiluminescent microparticle immune assay (CMIA),enzyme linked immunosorbent assay (ELISA),emulsion method (TP-AD) and toluidine red unheated serum test (TRUST).Sensitivity and specificity were evaluated on five methods.Titers of syphilis antibody in different stages and pre/post on treament among syphilis patients were compared and analyzed under the five methods.Results The sensitivity vs.specificity of TPPA,CMIA,ELISA,TP-AD and TRUST appeared as 100.00％ vs.99.50％,99.38％ vs.99.00％,98.12％ vs.99.00％,94.38％ vs.94.50％ and 85.62％ vs.95.50％,respectively.Among the patients at primary or latent stages,the syphilis antibody positive rate detected by TRUST appeared lower than that detected by ELISA,TPPA,CMIA or TP-AD,and the differences were statistically significant (P＜0.01).There were no statistical differences in the syphilis antibody positive rate of syphilis patients in the secondary or tertiary stages detected by five methods (P＞0.05).In each stage of the syphilis patients,the syphilis antibody positive rate detected by ELISA or of CMIA combined with TRUST both reached 100.00％.Before and after treatment in 121 cases of confirmed syphilis,there was statistically significant difference in the syphilis antibody positive rate detected by TRUST method (P＜0.05).There was no statistical significance in the syphilis antibody positive rate detected by other four methods (P＞0.05).Conclusions The sensitivity and specificity of TPPA,CMIA and ELISA methods were better.Methods as ELISA or as CMIA combined with TRUST both appeared reliable for syphilis screening in every stage of the disease.TRUST was suitable for the determination of active stage syphilis and monitoring the effects after treatment.
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Objective To explore the clinical value of five methods commonly used for the detection of clinical syphilis antibody.Methods A total of 160 confirmed syphilis cases were chosen as the experimental group while 200 non-syphilis cases were set as the control group.Serum specimens were detected by methods as Treponema pallidum particle agglutination assay (TPPA),chemiluminescent microparticle immune assay (CMIA),enzyme linked immunosorbent assay (ELISA),emulsion method (TP-AD) and toluidine red unheated serum test (TRUST).Sensitivity and specificity were evaluated on five methods.Titers of syphilis antibody in different stages and pre/post on treament among syphilis patients were compared and analyzed under the five methods.Results The sensitivity vs.specificity of TPPA,CMIA,ELISA,TP-AD and TRUST appeared as 100.00％ vs.99.50％,99.38％ vs.99.00％,98.12％ vs.99.00％,94.38％ vs.94.50％ and 85.62％ vs.95.50％,respectively.Among the patients at primary or latent stages,the syphilis antibody positive rate detected by TRUST appeared lower than that detected by ELISA,TPPA,CMIA or TP-AD,and the differences were statistically significant (P＜0.01).There were no statistical differences in the syphilis antibody positive rate of syphilis patients in the secondary or tertiary stages detected by five methods (P＞0.05).In each stage of the syphilis patients,the syphilis antibody positive rate detected by ELISA or of CMIA combined with TRUST both reached 100.00％.Before and after treatment in 121 cases of confirmed syphilis,there was statistically significant difference in the syphilis antibody positive rate detected by TRUST method (P＜0.05).There was no statistical significance in the syphilis antibody positive rate detected by other four methods (P＞0.05).Conclusions The sensitivity and specificity of TPPA,CMIA and ELISA methods were better.Methods as ELISA or as CMIA combined with TRUST both appeared reliable for syphilis screening in every stage of the disease.TRUST was suitable for the determination of active stage syphilis and monitoring the effects after treatment.
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Objective To research the immuno-protection of SJIR-2 DNA vaccine with nanometer microspheres a-gainst Schistosoma japonicum infection in mice. Methods To construct eukaryotic expression plasmid pEGFP-SJIR-2, identified by double digestion and sequenced delivery. The recombinant plasmid pEGFP-SJIR-2 was ex-tracted and was encapsulated into PLGA nanometer microspheres which were modified by CHS. 40 female BALB/c mice were randomly divided into 4 groups (n=10), each group of mice were injected with PBS, empty pEGFP plasmid, CHS-PLGA nanometer microspheres and CHS-PLGA-pEGFP-SJIR-2 nanometer microspheres 100 μg, re-spectively. Two weeks after the last immunization, each mouse was infected by cercaria of Schistosoma japonicum, sera of mice in each group were collected before each immunization and challenge infection. ELISA was used to de-tect the change of IgG in each group of micesera. 42 days later, all mice were sacrificed. The adult worms and eggs were collected and counted, and the worm and egg reduction rates were calculated as well. Results The recombi-nant plasmid pEGFP-SJIR-2 was successfully constucted, and there was significant difference in the numbers of worm and egg between CHS-PLGA-pEGFP-SJIR-2 group and PBS group ( P<0. 01 ) . The worm andegg reduction rates in CHS-PLGA-pEGFP-SJIR-2 group were 37. 36% and 46. 82% respectively. The IgG levels in mice sera of CHS-PLGA-pEGFP-SJIR-2 group were remarkably higher (P<0. 01) compared with PBS group. On the contrary, there was no significant difference between both pEGFP plasmid group and CHS-PLGA group in the numbers of worm and egg compared with PBS group. Conclusion SJIR-2 nanometer microspheres nucleic acid vaccine has some immuno-protection against Schistosoma japonicum infection in BALB/c mice,while it is worth further studying for it’ s potential value to be a candidate antigen molecule of Schistosoma japonicum vaccine.
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Objective To determine the viability of Schistosoma japonicum cercariae by staining. Methods Schistosoma japonicum cercariae were stained by 0.4%trypan blue 0.5%methylene blue?eosin?borax M.E.B 0.5%eosin 0.5%methy?lene blue and 0.05% neutral red respectively for 5 min then they were observed under a stereoscopic microscope. Results The dead cercariae were stained in the trypan blue M.E.B eosin and neutral red but unstained in the methylene blue. The vi?tal cercariae were unstained in all the five kinds of dyes. Conclusion The staining methods by using 0.4% trypan blue 0.5%M.E.B 0.5%eosin and 0.05%neutral red can be used to determine the viability of S. japonicum cercariae.
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Objective To study the role of autophagy in the replication process of Toxoplasma proliferation. Meth-ods As the experimental groups, these cells were infected by Toxoplasma gondii tachyzoites at given MOI (2 ∶ 1, 4 ∶ 1 ,8 ∶ 1 ,16 ∶ 1 ) . Host cell autophagy was detected through acridine orange staining and MDC fluorescence stai-ning at different time points (1, 2, 4, 8, 24, 48, 96 hs post infection). Detect the condition of HEF cells autoph-agy with acridine orange fluorescence staining and MDC fluorescence staining, and detect the replication kinetics of Toxoplasma gondii infection at different time points using Giemsa staining. Results The results of acridine orange and MDC fluorescence staining showed that autophagy inhibitors and inducers could inhibit and promote the autoph-agy of HEF cells respectively. From the results of Giemsa staining, it was found that the proliferation of Toxoplasma gondii in HEF cells could be promoted with autophagy inducers and be inhibited with autophagy inhibitors. Conclu-sion The regulation on autophagy of host cell could regulate the proliferation and replication of Toxoplasma gondii.
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Objective To determine the correlation between mouse maerophage metalloelastase (MME)and vascular endothelial growth factor(VEGF)expression involved in angiogenesis of colon cancer.Methods A eDNA fragment coding for domainsⅠandⅡof MME was transfected into murine CT-26 colon cancer cells that were MME deficient.The enzymatic activity of recombinant MME was confirmed by cleavage of native substrate in vitro.An orthotopic implantation model was established by using MME-transfected cells and control cells.Tumor samples were subjected to in situ hybridization (ISH)and immunohistochemical staining(IHC)to detect expressions of MME and VEGF.The microvessel counting was used to assess angiogenesis of murine colon tumors.Results It was demon- strated that the tumor growth was significantly inhibited in MME-transfected group compared with pcDNA3.1 transfected and nontransfected groups(P＜0.001).It was also found that,compared with pcDNA3.1-transfected and nontransfected groups,the microvessel formation in MME transfected group was significantly reduced(P＜0.001).The expression of VEGF mRNA and protein was significantly lower in MME-transfected group than those in the controls,as demonstrated by ISH(MME-transfected group versus pcDNAa.1-transfected group,P=0.028;and versus nontransfected group,P=0.003) and by IHC(MME-transfected group versus pcDNA3.1-transfected group,P=0.025;and versus non- transfected group,P=0.008).Conclusions The MME gene transfected into murine colon cancer cells can effectively suppress the growth of orthotopic tumors by inhibition of vaseularity.Both MME and VEGF gene expression is highly associated with the vascularity of tumors,which may depend on a hal- ance between MME and VEGF expression.
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Objective To clone and express the cell signaling protein 14-3-3 gene from Toxoplasma gondii RH strain. Methods Toxoplasma RH strain tachyzoites, which maintained by mouse passage, were harvested from ascites of mice and genomic DNA was prepared. A pair of primers were designed and synthesized based on the sequence of Toxo 14-3-3 cDNA. A specific fragment of Toxo 14-3-3 gene was obtained by RT-PCR amplification from Toxoplasma genomic DNA. The PCR products were ligated to pGEM-T. The EcoRI / Xho I restricted fragments, confirmed by PCR and EcoRI / XhoI digestion, were cloned into expression vector pET28a and the recombinants were transformd into E.coli BL21. Fusion expression was induced by isopropyl-beta-D-thiogalactoside (IPTG) and confirmed by Western blotting with rabbit anti-Toxoplasma sera. Results The molecular size of Toxo 14-3-3 was 803 bp, which is highly homologous to the previous report cloned from the parasites of intestinal epithelial stage in cat. High expression was obtained in pET28a/ Toxo 14-3-3/E.coli BL21 when confirmed by Western blotting. Conclusion The recombinant construction of Toxo 14-3-3 was generated and expression was induced.
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Objective To detect the dynamic changes of antibody and cytokines in the serum induced by single vaccine delivery system (SVDS) of biodegradable microspheres of recombinant glutathione-S-transferase from Schistosoma japonicum (rSjGST) in mice. Methods By using the constructed expression plasmid pET28a(+)-SjGST, rSjGST was expressed by E.coli BL21. After purified through the column of resin,the biodegradable microspheres of rSjGST were prepared with PLGA (Poly lactic-co-glycolic acid, PLGA 75/25).Six-week BALB/c female mice were immunized subcutaneously with a single vaccine, and the blood was collected from eyes on the 6th, 9th, 12th, 15 th, 18th, 21th week respectively. The dynamic levels of serum specific antibody IgG, IL-2 and IFN-? were detected. Results Compared with the control group, there was a significant difference of serum specific antibody IgG of every immune group (P
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Objective To explore the possibility of heterogenous gene to express in juvenile Schistosoma japonicum and the application of electroporation in transformation of schistosomulae. Methods The plasmids of pEGFP-C1 were introduced into mechanically transformed schsitosomula with electroporation. The presence, transcription and translation of the transgene in electroporated schistosomula were confirmed by PCR, RT-PCR and Western blotting analysis respectively using the genomic DNA, total RNA and protein extracted and isolated from schistosomula cultured in vitro for 48 hours. Meanwhile, localization of EGFP within electroporated schistosomula was performed with confocal laser scanning microscope. Results 760 bp and 276 bp amplified products by PCR and RT-PCR were found coincident with the expected size and expression of EGFP gene in elctroporated schistosomula was confirmed by Western blotting. Fluorescence of EGFP was localized in tegument and subtegument of the electroporated schistosomula with confocal microscopy, especially in the anterior part of the worm. Conclusion The heterogenous gene of EGFP has been successfully introduced into juvenile S. japonicum by electroporation and the expression of transgene was confirmed with molecular and microscopical methods.
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Objective To screen a new schistosome vaccine candidate. \ Methods\ Schistosoma japonicum adult cDNA library was screened using sera from immune rabbits vaccinated with irradiated cercariae and monoclonal antibodies against membrane antigen of S.japonicum schistosomula. Three different fragments of S.japonicum cDNA genes were cloned into pGEM-T vector. The sequences of the inserts were determined using an automatic DNA sequencer and were analysed using Blast program. One of the unknown genes (B8) was selected and its ORF sequence (291 bp) was subcloned into eukaryotic expression vector. The recombinant plasmids were identified by restrictive enzymes and PCR amplification. The positive recombinant plasmids (pBK/SjB8) were transformed into host bacteria XL1-blue, and were then induced by IPTG for expression. SDS-PAGE and Western blotting analysis of total cellular protein from the bacteria were performed to detect the gene products. Results The results demonstrated that ORF of SjB8 gene was subcloned into the plasmid pBK-CMV and could express as fusion protein in XL1-blue. The results of SDS-PAGE and Western-blot also showed that the molecular weight of the fusion protein with 3 kDa ?-galactosidase was approximately 13^6 kDa and the actual molecular weights of the SjB8 was 10^6 kDa. The expressed fusion product of pBK/Sj-B8 could be recognized by immune serum and McAb. Conclusion A new gene of S.japonicum vaccine candidate (SjB8) was cloned into eukaryotic expression vector pBK-CMV and could express 10^6 kDa schistosome protein. The results provide foundation for further study of the protein for its posibility as candidate vaccine.
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Aim To observe the effect of astragalosides on proliferation and collagen synthesis of HSC-T6 cells driven using schistosoma japonicum soluble egg antigen-activated macrophage conditioned medium(SEA-MCM).Methods SEA-MCM was prepared by injection of Schistosoma japonicum SEA via mice peritoneal.The proliferation and collagen synthesis of HSC-T6 cells stimulated with SEA-MCM were measured using MTT colorimetric assay and()~3H-proline incorporation respectively.Results The proliferation and collagen production of HSC-T6 cells were significantly promoted by SEA-MCM.They were significantly suppressed after being treated with AST(32.5、65、130 mg?L~(-1)) showing concentration-dependent effect.Conclusion:Astragalosides may inhibit HSC-T6 cells proliferation and collagen production that was driven by SEA activated MCM in vitro.