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OBJECTIVE@#To conduct eukaryotic expression of the leucine-rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope.@*METHODS@#The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy-PortParam and Protean. The full-length splicing primers were designed using PCR-based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4-LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.@*RESULTS@#The recombinant pcDNA3.4-LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy-PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C3073H4942N846O953S28 and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T-cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T-cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4-LRRC15 plasmid into HEK293 cells, SDS-PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15-His fusion protein was purified from the cell culture medium, and SDS-PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein.@*CONCLUSIONS@#The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.
Subject(s)
Animals , Humans , Amino Acids , Antigens, Helminth/genetics , Cysticercus/genetics , Epitopes/genetics , Eukaryota , HEK293 Cells , Leucine-Rich Repeat Proteins , Membrane Proteins , Taenia solium/geneticsABSTRACT
Objective To investigate the effect of Toxoplasma gondii excretory-secretory antigens (ESA) on CD4+ CD25+ Foxp3+ T (Treg) cells in mice carrying Lewis lung carcinoma, and examine the inhibitory effect of T. gondii ESA on tumor growth. Methods C57BL/6 mice were randomly assigned into the PBS group (n = 14) and the Lewis group (n = 34). Mice in the Lewis group were subcutaneously injected with 2 × 105 Lewis lung carcinoma cells in the right axilla, while animals in the PBS group were injected with the same volume of sterile PBS. On day 7 post-injection (D7), mice in the PBS group were further divided into the PBS2 group and the PBS2 + ESA group, of 7 mice in each group, and mice in the Lewis group were further divided into the Lewis2 group and the Lewis2 + ESA group, of 17 mice in each group. Then, mice in the PBS2 + ESA group and the Lewis2 + ESA group were intraperitoneally injected with 100 μL of ESA. The mouse spleen coefficient was calculated in each group 7 days post-injection with ESA, and the changes of Treg cell counts and the long-term tumor growth were measured in tumor-bearing mice. Results The spleen coefficient was significantly greater in the PBS2 + ESA group and the Lewis2 + ESA group than in the PBS2 (0.66% ± 0.09% vs. 0.30% ± 0.02%, P < 0.05) and Lewis2 groups (0.69% ± 0.07% vs. 0.33% ± 0.03%, P < 0.05) 7 days post-treatment with ESA, respectively, and the percentage of splenic Treg cells in splenocytes was significantly lower in the PBS2 + ESA group and the Lewis2 + ESA group than in the PBS2 (1.28% ± 0.14% vs. 2.06% ± 0.07%, P < 0.05) and Lewis2 groups (1.58% ± 0.14% vs. 2.44% ± 0.23%, P < 0.05), respectively. T. gondii ESA treatment caused a delay in tumor growth, and the tumor size was significantly smaller in the Lewis2 + ESA group than in the Lewis2 group (P < 0.05). Conclusion T. gondii ESA may reduce the proportion of splenic Treg cells in splenocytes and inhibit tumor growth in mice carrying Lewis lung carcinoma.
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Objective To investigate the effect of Toxoplasma gondii excretory-secretory antigens (ESA) on CD4+ CD25+ Foxp3+ T (Treg) cells in mice carrying Lewis lung carcinoma, and examine the inhibitory effect of T. gondii ESA on tumor growth. Methods C57BL/6 mice were randomly assigned into the PBS group (n = 14) and the Lewis group (n = 34). Mice in the Lewis group were subcutaneously injected with 2 × 105 Lewis lung carcinoma cells in the right axilla, while animals in the PBS group were injected with the same volume of sterile PBS. On day 7 post-injection (D7), mice in the PBS group were further divided into the PBS2 group and the PBS2 + ESA group, of 7 mice in each group, and mice in the Lewis group were further divided into the Lewis2 group and the Lewis2 + ESA group, of 17 mice in each group. Then, mice in the PBS2 + ESA group and the Lewis2 + ESA group were intraperitoneally injected with 100 μL of ESA. The mouse spleen coefficient was calculated in each group 7 days post-injection with ESA, and the changes of Treg cell counts and the long-term tumor growth were measured in tumor-bearing mice. Results The spleen coefficient was significantly greater in the PBS2 + ESA group and the Lewis2 + ESA group than in the PBS2 (0.66% ± 0.09% vs. 0.30% ± 0.02%, P < 0.05) and Lewis2 groups (0.69% ± 0.07% vs. 0.33% ± 0.03%, P < 0.05) 7 days post-treatment with ESA, respectively, and the percentage of splenic Treg cells in splenocytes was significantly lower in the PBS2 + ESA group and the Lewis2 + ESA group than in the PBS2 (1.28% ± 0.14% vs. 2.06% ± 0.07%, P < 0.05) and Lewis2 groups (1.58% ± 0.14% vs. 2.44% ± 0.23%, P < 0.05), respectively. T. gondii ESA treatment caused a delay in tumor growth, and the tumor size was significantly smaller in the Lewis2 + ESA group than in the Lewis2 group (P < 0.05). Conclusion T. gondii ESA may reduce the proportion of splenic Treg cells in splenocytes and inhibit tumor growth in mice carrying Lewis lung carcinoma.
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Objective To compare the immune protective effects of three antigens of Trichinella spiralis encapsulated larvae on mice.Methods The mice were immunized with Trichinella spiralis encapsulated larvae somatic antigen,encapsulated larva excretory-secretory antigen and encapsulated larva surface antigen,3 times with a 7-day interval,and the adjuvant control and normal control group were set up.Seven days after the final immunization,each mouse was orally challenged with 200 Trichinella spirais larvae.The intestinal adult worms and muscle larvae of Trichinella spiralis of each group were recoveried and examined on Day 7 and Day 30 post-challenge,respectively.The level of 8eruln IgG to antigens of Trichinella muscle muscle larvae wa8 detected by ELISA.Results The intestinal adult worms were reduced by 84.89%.89.73%,85.65%.2.57% in the encapsulated larva somatic,excretory-secretory and surface antigen groups,respectively.The muscle lalwae were reduced by 71.71%,80.98%,73.66%,5.60%, respectively.Adtlltwornl reduction rates(P<0.05) and musclelarva reduction rates(P<0.01) of the encapsulated larva excretory-secretory antigen group and surface antigen group were higher than those of encapsulated larva somatic antigen group.The antibody titers in all the immunized groups increased significantly.and the GMRT values of the encapsulated larva somatic,excretory-secretory and surface antigen groups were 32 798.89,3 474.51,2 984.83,respectively,and were 6.09,7.56,6.50 times higher than those of the normal control group(459.32).Conclusions Trichinella spiralis encapsulated larva antigens can induce strong resistance of host to a subsequent challenge infection.Among these antigens,excretory-secretory antigen is more immunogenic.
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BACKGROUND: Direct wet mount examination of vaginal secretion, widely applied for the diagnosis of Trichomonas vaginalis infection in woman patients, is rapid and economical. However, the sensitivity of this technique is not so high. In this study enzyme-linked immunosorbent assay(ELISA) was employed for the detection of serum anti-T. vaginalis IgG antibodies from vaginal trichomoniasis patients. METHODS: Eighty sera from trichomonoasis patients who visited a Dr. Yoon Kyong's Obstetric & Gynecologic Clinic in Songnam and 30 non-infected healthy men were tested for detection of anti-T. vaginalis IgG antibody. Soluble lysate and excretory-secretory antigen prepared by mixing of six isolates of T. vaginalis, and lysate from one isolate(KT4) were used as antigen for ELISA. RESULTS: The sensitivity of ELISA using lysate of six isolates was 95.0%, and the sensitivity of the lysate from KT4 and mixed excretory-secretory antigen from 6 isolates were 86.4% and 76.3%, respectively. Specificities of ELISA by three 93.3%, 96.3% and 92.0%, respectively. CONCLUSION: It is suggested that ELISA using mixed lysate of T. vaginalis six isolates could be useful tools for the diagnosis of trichomoniasis.
Subject(s)
Female , Humans , Male , Antibodies , Diagnosis , Enzyme-Linked Immunosorbent Assay , Immunoglobulin G , Trichomonas vaginalis , TrichomonasABSTRACT
Objective To find out the specific diagnostic antigens in excretory-secretory (ES) products from muscle larvae of Trichinella spiralis. Methods The ES antigens (ESA) of Trichinella spiralis muscle larvae cultured in vitro at 18 h and 30 h were analyzed by SDS-PAGE and Western blotting. Results At different times after cultivation, the protein components of ESA of T. spiralis muscle larvae were similar. SDS-PAGE revealed that the molecular weight(MW) of the major bands of 2 ES antigens were 112, 110, 108, 97, 53, 49, 45, 42, 35, 23 and 16 kDa. Western blotting showed that the protein bands with 102, 97, 95 and 53 kDa in 18 h ESA and the protein bands with 53, 49, 45 and 43 kDa in 30 h ESA cross-reacted with sera from the patients with paragonimiasis, clonorchiasis, schistosomiasis, and cysticercosis, respectively. The protein component with 23 kDa in ESA only reacted with sera from the rats and mice infected with T. spiralis and the patients with trichinellosis, but not reacted with sera from animals and patients infected with other parasites, and sera from normal rats, mice and persons. Conclusion The protein component with 23 kDa in T. spiralis ESA is the specific antigen of T. spiralis muscle larvae and it could be applied to the serodiagnosis and seroepidemiological survey of trichinellosis.
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Detection of filarial antigen in blood or urine samples would provide an accurate indication of active infection. The absence of a simple, well established animal model and limitations in getting the required amount of parasite material from human sources have been the main obstacles for the diagnosis ofWuchereria bancrofti infection. An inhibition ELISA has been developed for detection of filarial antigen using a partially purified Brugia malayi mf ES antigen (BmE DE1) and its affinity purified antibodies. Filarial antigen was detected in the sera of 88% of microfilaraemic, 60% of chronic filarial, 17% of endemic normal and none of the non-endemic normal subjects. The sensitivity and specificity of the assay were 88% and 89% respectively. Moreover, undiluted urine samples from 82% of microfilaraemic and 17% of endemic normal, but none of the non-endemic normal samples showed the presence of filarial antigen.With the limitations on the availability of sufficient homologous parasite materials, the inhibition ELISA using BmE DE1 and anti BmE DE1 antibodies shows promise for the detection of active infection in bancroftian filariasis in man.Moreover, its detection in urine makes it more possible to test patients in field areas.
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Localization of antigens homologous to excretory-secretory proteins in developing embryos of Setaria digitata has been carried out by indirect fluorescent antibody test, [14C] labelling studies and Western blotting. Indirect fluorescent antibody test showed binding of excretory-secretory antibodies at perivitelline space. The fluorescent antibody binding was almost absent at small morulae stage and increasing in intensity in the successive developmental stages with maximum at coiled microfilaria stage. Hatched microfilaria did not show the presence of antigens by immunofluorescence. Immunocomplex of excretory-secretory antiserum against "amniotic fluid" collected from developing embryos of Setaria digitata labelled with [14C] amino acids showed highest radioactivity at coiled and tadpole stages and differed significantly from small morulae, big morulae and hatched microfilaria. Immunoblot analysis of amniotic fluid showed two proteins, 16·5 and 11 kDa, to be highly antigenic. The antigenic protein (11 kDa) content as seen by immuno blotting increased during embryogenesis and decreased at the stage of hatching.
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Objective To compare the immune protective effect of different antigens of Trichinella spiralis:excretory-secretory(ES) antigen of adult worms, ES antigen of muscle larva and their mixed antigens in mice. Methods ES antigens of Trichinella spiralis adult worms and muscle larva were produced by the physiological saline culture methods. The ES antigens of Trichinella spiralis adult worms, muscle larva and the mix were used to immunize mice 3 times at 7 day interval, adjuvant control and normal control were set up. Seven day after the final immunization, each mouse was orally challenged with 200 Trichinella spiralis larva. Intestinal adult worms and muscle larva of Trichinella spiralis of each group were recoveried and examined on Day 7 and Day 30 post-challenge,respectively. Results The intestinal adult worms of Trichinella spiralis reduced by 87.95%, 69.48% and 84.34% in the adult worm ES antigen group, muscle larval ES antigen group and their mixed antigen group, respectively, while muscle larva reduced by 74.79%, 87.97% and 86.87% respectively. Adult worm reduction rates of the adult worm ES antigen group and mixed antigen group were higher than those of muscle larval ES antigen group (P
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The Wuchereria bancrofti microfilarial excretory-secretory antigens were fractionated into ES1, ES2, ES3 and ES4 by ultra-membrane filtration and evaluated for their diagnostic utility by enzyme linked immunosorbent assay. Three of the four fractions showed antigenic activity (ES2, ES3 and ES4). The antigen fractions ES2 and ES4 were highly active in the detection of filarial IgM antibody in clinical filariasis and microfilaraemia respectively. The chemical characterization of the ES2 and ES4 antigen fractions showed that they were glycoproteins.