Extra cut-off value of aldosterone-to-renin ratio for screening primary aldosteronism in hypertensive patients with diabetes mellitus.

Primary aldosteronism (PA) and diabetes mellitus (DM) may coexist. We previously found that DM and impaired glucose tolerance (IGT) may decrease the efficiency of the aldosterone-to-renin ratio (ARR) for screening PA. Thus, we wanted to determine appropriate ARR cut-off values for screening PA in patients with hypertension with DM and IGT. Data from 736 patients with hypertension were collected. They were divided into PA (77 cases), PA with DM (27 cases), PA with IGT (44 cases), hypertension without PA (353 cases), hypertension with DM (without PA, 127 cases), and hypertension with IGT (without PA, 108 cases). Receiver operating characteristic (ROC) curves were used to identify the appropriate ARR cut-off values in different conditions. Screening efficiencies of these cut-off values were evaluated across different groups. ARR cut-off values for screening PA in hypertensive patients without DM and IGT, with DM, and with IGT were 29.65, 23.15, and 26.9, respectively. All cut-off values demonstrated high sensitivity and specificity: 92.2% and 88.7%, 92.6% and 79.5%, and 88.6% and 85.2%, respectively, and areas under the ROC curves were 0.941, 0.904, and 0.909, respectively. Our results suggest that extra ARR cut-off values may be necessary for effective screening PA in hypertensive patients with DM and IGT, particularly in those with DM.

Online prediction model for primary aldosteronism in patients with hypertension in Chinese population: A two-center retrospective study.

Background: The prevalence of primary aldosteronism (PA) varies from 5% to 20% in patients with hypertension but is largely underdiagnosed. Expanding screening for PA to all patients with hypertension to improve diagnostic efficiency is needed. A novel and portable prediction tool that can expand screening for PA is highly desirable. Methods: Clinical characteristics and laboratory data of 1,314 patients with hypertension were collected for modeling and randomly divided into a training cohort (919 of 1,314, 70%) and an internal validation cohort (395 of 1,314, 30%). Additionally, an external dataset (n = 285) was used for model validation. Machine learning algorithms were applied to develop a discriminant model. Sensitivity, specificity, and accuracy were used to evaluate the performance of the model. Results: Seven independent risk factors for predicting PA were identified, including age, sex, hypokalemia, serum sodium, serum sodium-to-potassium ratio, anion gap, and alkaline urine. The prediction model showed sufficient predictive accuracy, with area under the curve (AUC) values of 0.839 (95% CI: 0.81-0.87), 0.814 (95% CI: 0.77-0.86), and 0.839 (95% CI: 0.79-0.89) in the training set, internal validation, and external validation set, respectively. The calibration curves exhibited good agreement between the predictive risk of the model and the actual risk. An online prediction model was developed to make the model more portable to use. Conclusion: The online prediction model we constructed using conventional clinical characteristics and laboratory tests is portable and reliable. This allowed it to be widely used not only in the hospital but also in community health service centers and may help to improve the diagnostic efficiency of PA.

An online alpha-thalassemia carrier discrimination model based on random forest and red blood cell parameters for low HbA2 cases.

BACKGROUND: Since screening of α-thalassemia carriers by low HbA2 has a low positive predictive value (PPV), the PPV was as low as 40.97% in our laboratory, other more effective screening methods need to be devised. This study aimed at developing a machine learning model by using red blood cell parameters to identify α-thalassemia carriers from low HbA2 patients. METHODS: Laboratory data of 1213 patients with low HbA2 used for modeling was randomly divided into the training set (849 of 1213, 70%) and the internal validation set (364 of 1213, 30%). In addition, an external data set (n = 399) was used for model validation. Fourteen machine learning methods were applied to construct a discriminant model. Performance was evaluated with accuracy, sensitivity, specificity, etc. and compared with 7 previously published discriminant function formulae. RESULTS: The optimal model was based on random forest with 5 clinical features. The PPV of the model was more than twice the PPV of HbA2, and the model had a high negative predictive value (NPV) at the same time. Compared with seven formulae in screening of α-thalassemia carriers, the model had a better accuracy (0.915), specificity (0.967), NPV (0.901), PPV (0.942) and area under the receiver operating characteristic curve (AUC, 0.948) in the independent test set. CONCLUSION: Use of a random forest-based model enables rapid discrimination of α-thalassemia carriers from low HbA2 cases.

Developing an ultra-performance liquid chromatography-tandem mass spectrometry for detecting aldosterone in human plasma.

BACKGROUND: Accurately measuring plasma aldosterone concentration is difficult but meaningful for primary aldosteronism (PA) diagnosis. METHODS: In this study, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for plasma aldosterone detection, evaluated its performance according to guidelines issued by CLSI, including detection limit, linearity, precision, and compared it with chemiluminescence immunoassay. Then, a reference range of plasma aldosterone in young people was established by using this method. RESULTS: The lower limit of quantitation (LOQ) was 10 pg/ml. The mean recovery rates of analyte added to serum were 100.07-102.05% in different concentrations. The linearity range was 20-2000 pg/ml. Inter-assay CVs were 2.20-3.97% at aldosterone concentrations of 65.66-854.75 pg/ml. The regression equation of UPLC-MS/MS (x) and chemiluminescence immunoassay (y) was y = 1.002x + 65.854 (r = 0.9456, n = 237). The reference range of plasma aldosterone detected by UPLC-MS/MS was 11.30-363.82 pg/ml in young people in South China, and there was no statistically significant difference in plasma aldosterone concentration between two genders. CONCLUSION: In conclusion, UPLC-MS/MS can rapidly and accurately detect plasma aldosterone and is appropriate for clinical application.

High Efficiency and Problems of Chemiluminescence Assay-Detected Aldosterone-To-Renin Ratio in Practical Primary Aldosteronism Screening.

Primary aldosteronism is a main cause of secondary hypertension which can be effectively treated. The screening test for primary aldosteronism is benefit for minimizing damage to the patient. In the previous retrospective study, we obtained the optimal cutoff value of aldosterone-to-renin ratio detected by chemiluminescence assay, a newly developing method, and prompted its high efficiency in primary aldosteronism screening in upright position. In this study, we want to evaluate its efficiency in practical work. We used this ratio to continuously screen 238 patients, and 58 patients were finally diagnosed with primary aldosteronism. We found it had 86.13% accuracy rate in the upright position compared with the final clinical diagnosis. False negative and positive rates were 13.79% and 13.89%. Diagnostic sensitivity and specificity were 86.21% and 86.11%, which are slightly different from results in our previous study. False negative rate can be improved by combining the aldosterone-to-renin ratio with aldosterone concentration. We also found impaired glucose tolerance may be a reason for high false positive rate. Besides, chemiluminescence assay may be interfered in aldosterone detection. Although it has some shortcomings, chemiluminescence assay-detected aldosterone-to-renin ratio is a highly effective index for screening primary aldosteronism in practice.

High efficiency of the aldosterone-to-renin ratio in precisely detecting primary aldosteronism.

The aldosterone-to-renin ratio (ARR) is extensively used for primary aldosteronism detection. Chemiluminescence immune assay (CLIA) is newly applied in aldosterone and renin detection for calculating the aldosterone-to-renin ratio. The performance of new ARR in aldosteronism detection is poorly evaluated. We aim to estimate the diagnostic value of this new aldosterone-to-renin ratio by highly standardized and clinically based protocol. Four hundred and forty-two patients were enrolled in our retrospective study. They went to the first affiliated hospital of Sun Yat-sen University with difficult-to-control hypertension. Primary aldosteronism diagnosis was based on clinical criteria, including a saline infusion test and other necessary inspections. ARR was calculated from plasma aldosterone and renin measured by CLIA. The cutoff value was determined and the diagnostic value was evaluated. The cutoff value of ARR for primary aldosteronism diagnosis was 28.3, with a sensitivity of 87.6%, specificity of 100%, negative-predictive value of 96.4%, and positive-predictive value of 100%. Then, we found that Age was weakly correlated with ARR. The cutoff values of ARR for primary aldosteronism diagnosis in 26-45-, 46-65-, and 66-85-year-old patients were, respectively, 29.45, 27.95, and 28.4, with sensitivities of 87.5%, 87.7%, and 87.5%, specificities of 100% for all, negative-predictive values of 97.7%, 94.3%, and 96.3%, and positive-predictive values of 100% for all. ARR generated by CLIA is a good diagnostic test for primary aldosteronism without making a false-positive diagnosis. Although ARR is correlated with age, ARR cutoff values for different ages are not more efficient than that for total sample in primary aldosteronism diagnosis.

Antibodies against Schistosoma japonicum lactate dehydrogenase B enhance enzyme active.

Lactate dehydrogenase (LDH) is a key enzyme in glycolysis process. It catalyzes the interconversion between pyruvic acid and lactic acid. Schistosoma japonicum adult worms largely rely on glycolysis for energy production when they parasitize in human. S. japonicum may be killed if energy production is suppressed. So, we wonder whether antibody against S. japonicum LDH is a harmful factor for S. japonicum surviving. In this study, we cloned and characterized S. japonicum lactate dehydrogenase B (SjLDHB) to evaluate its role in parasite survival. We found SjLDHB was highly similar to S. japonicum lactate dehydrogenase A (SjLDHA) which is another LDH subtype in S. japonicum in amino acid sequence. The optimal temperature of SjLDHB catalytic activity was 37 °C, the optimal pH values for pyruvate reduction and lactate oxidation were 7.0 and 6.0 and Km values of pyruvate and lactate were 0.2752 and 0.2339 mM respectively. Then, we identified SjLDHB expression in male and female S. japonicum. Finally, we evaluated the influence of antibodies on SjLDHB enzymatic activity. Interestingly, we found anti-SjLDHA antibody suppressed SjLDHB enzymatic activity, while anti-SjLDHB antibody and mixed antibody enhanced SjLDHB enzymatic activity in vitro. Although further investigation is needed, we suggest that anti-SjLDHB antibody may be not a negative factor, but a valuable compensation for S. japonicum adult worm surviving and pathogenicity.

ULK1 phosphorylates Sec23A and mediates autophagy-induced inhibition of ER-to-Golgi traffic.

BACKGROUND: Autophagy is an inducible autodigestive process that allows cells to recycle proteins and other materials for survival during stress and nutrient deprived conditions. The kinase ULK1 is required to activate this process. ULK1 phosphorylates a number of target proteins and regulates many cellular processes including the early secretory pathway. Recently, ULK1 has been demonstrated to phosphorylate Sec16 and affects the transport of serotonin transporter at the ER exit sites (ERES), but whether ULK1 may affect the transport of other cargo proteins and general secretion has not been fully addressed. RESULTS: In this study, we identified Sec23A, a component of the COPII vesicle coat, as a target of ULK1 phosphorylation. Elevated autophagy, induced by amino acid starvation, rapamycin, or overexpression of ULK1 caused aggregation of the ERES, a region of the ER dedicated for the budding of COPII vesicles. Transport of cargo proteins was also inhibited under these conditions and was retained at the ERES. ULK1 phosphorylation of Sec23A reduced the interaction between Sec23A and Sec31A. We identified serine 207, serine 312 and threonine 405 on Sec23A as ULK1 phosphorylation sites. Among these residues, serine 207, when changed to phospho-deficient and phospho-mimicking mutants, most faithfully recapitulated the above-mentioned effects of ULK1 phospho-regulation. CONCLUSION: These findings identify Sec23A as a new target of ULK1 and uncover a mechanism of coordinating intracellular protein transport and autophagy.

Antibodies against Clonorchis sinensis LDH could cross-react with LDHB localizing on the plasma membrane of human hepatocarcinoma cell SMMC-7721 and induce apoptosis.

Lactate dehydrogenase (LDH) is a terminal enzyme in anaerobic glycolytic pathway. It widely exists in various organisms and is in charge of converting the glycolysis product pyruvic acid to lactic acid. Most parasites, including Clonorchis sinensis, predominantly depend on glycolysis to provide energy. Bioinformatic analysis predicts that the LDHs from many species have more than one transmembrane region, suggesting that it may be a membrane protein. C. sinensis LDH (CsLDH) has been confirmed as a transmembrane protein mainly located in the tegument. The antibodies against CsLDH can inhibit the worm's energy metabolism, kill the worm, and may have the same effects on human cancer cells. In this study, we cloned and characterized human LDHA (HsLDHA), HsLDHB, and CsLDH. Semi-quantitative real-time RCP showed that HsLDHB only existed in hepatocarcinoma cell SMMC-7721. Confocal microscopy and Western blot experiments revealed that HsLDHB was localized in the plasma membrane of SMMC-7721 cells, and the antibodies against CsLDH could cross-react with it. This cross-reaction could inhibit the enzymatic activity of HsLDHB. The cancer cells co-cultured with anti-CsLDH sera showed a significant decrease in cell proliferation rate and increases in caspase 9 and reactive oxygen species (ROS) levels. Therefore, anti-CsLDH antibodies can induce the apoptosis of cancer cells SMMC-7721 and may serve as a new tool to inhibit tumor.

Should we abandon quinine plus antibiotic for treating uncomplicated falciparum malaria? A systematic review and meta-analysis of randomized controlled trials.

In this study, we compared the efficacies and adverse effects of quinine plus antibiotics and other anti-malaria drugs on treating uncomplicated falciparum malaria. By systematically searching the major databases PubMed, Embase, and the Cochrane Library, 14 randomized controlled trials (RCTs) including 1996 cases were identified. Then, we performed a systematic review and cumulative meta-analysis on these data. The primary outcome of these treatments was parasite failure at day 28. There was no significant difference between quinine plus antibiotic therapy (QACT) and artemisinin-based therapies (odds ratio (OR) 0.69, 95 % confidence interval (CI) 0.28 to 1.71) or non-artemisinin-based therapies except quinine monotherapy and chloroquine monotherapy (OR 0.56, 95 % CI 0.18 to 1.74). The secondary outcome was the adverse effects within 28 days, including nausea, dizziness, vomiting, diarrhea, abdominal pain, headache, and tinnitus. QACT significantly increased the risk of tinnitus compared with artemisinin-based therapies (OR 111.65, 95 % CI 12.63 to 986.87) and non-artemisinin-based therapies (OR 48.16, 95 % CI 16.23 to 142.92). Vomiting was more frequently reported in QACT compared with non-artemisinin-based therapies (OR 2.02, 95 % CI 1.14 to 3.56). This meta-analysis suggests that almost all regimens have equivalent treatment effect at the 28th day. However, the patients with QACT had a higher chance to suffer from vomiting and tinnitus. Therefore, QACT does not have significant advantage on treating uncomplicated falciparum malaria.

Schistosoma japonicum tegumental protein 20.8, role in reproduction through its calcium binding ability.

Schistosomiasis threatens thousands of millions of peoples' health every year in the world. Schistosoma japonicum, a pathogen of schistosomiasis, is covered by a lipid bilayer membrane which plays an important role in nutrient transport, signal transduction, interaction with host's immune system, etc. Thus, molecules in the tegumental membrane have gained more and more interest for understanding biological and pathological processes of schistosoma. In this study, we found a protein from S. japonicum cDNA library which has a 20.8 KDa molecular weight (SjTP20.8). Recombinant SjTP20.8 was produced and purified from Escherichia coli. The recombinant protein could be detected by S. japonicum-infected mice and human sera, and it had been found localizing in the tegumental membrane of S. japonicum in the section using immunofluorescence assay. In electrophoretic mobility shift assay, the protein could bind calcium iron in neutral condition. Result of cercariae challenge experiment indicates antibody against this protein can protect mice from chronic hepatic fibrosis. Our results indicate the S. japonicum tegumental protein 20.8 is crucial for the parasite's calcium absorbing and reproduction.

Schistosoma japonicum calcium-binding tegumental protein SjTP22.4 immunization confers praziquantel schistosomulumicide and antifecundity effect in mice.

A family of platyhelminth tegument-specific proteins comprising of one or two calcium ion binding EF-hand and a dynein light chain-like domain, termed tegumental proteins, are considered as candidates of vaccine. In this study, we cloned and characterized SjTP22.4, a novel membrane-anchored tegumental protein in Schistosoma japonicum with theoretic MW of 22.4. The recombinant SjTP22.4 could be recognized by S. japonicum infected sera. Immunofluorescence revealed that this protein is not only located on the surface of tegument of adult and schistosomulum and cercaria, but also in the parenchymatous tissues and intestinal epithelium. Circular dichroism (CD) measurement demonstrated rSjTP22.4 had Ca(2+)-binding ability. The rSjTP22.4 vaccination without adjuvants produced comparable high level of antibody with that of immunization with adjuvants together indicated it was an antigen of strong antigenicity. The level of IgG1 is much higher than that of IgG2a and IgE is nearly negative in S. japonicum-infected and rSjTP22.4 immunized mice. In cercaria challenge experiment, mice vaccinated with SjTP22.4 showed no reduction in adult burden and egg production, comparing with the control mice, but 41% decrease in egg mature rate and 32% reduction in liver egg granuloma area. However, the SjTP22.4 immunized mice that received praziquantel treatment at 10d post infection caused 26% reduction in adult burden and 53% decrease in egg mature rate, comparing with the control mice only received praziquantel treatment. In conclusion, SjTP22.4 is a valuable vaccine candidate for S. japonicum of anti-pathogenesis and anti-transmission effect and plays a synergetic role in praziquantel to kill schistosomulum.

The topological structure and function of Echinococcus granulosus lactate dehydrogenase, a tegumental transmembrane protein.

Lactate dehydrogenase (LDH), a terminal glycolytic enzyme, is generally considered as a cytosolic protein. We cloned lactate dehydrogenase from Echinococcus granulosus (EgLDH) and predicted it may be a membrane protein with two transmembrane regions through bioinformatics analysis. Intact worm immunofluorescence with antibodies prepared against linear B cell epitopes predicted in the region inside or outside of the membrane demonstrated that EgLDH spans the tegumental membrane twice, with the N terminal and C terminal all outside, just consistent with the putative topological structure. Then, the enzymatic characteristics and kinetic parameters of recombinant EgLDH were surveyed and the results suggested that EgLDH is responsible for catalyzing the reduction of pyruvic acid into lactic acid under physiological conditions. The enzymatic activity of the recombinant protein was inhibited by antibodies directed against the intact protein or against epitopes that contain key residues in the catalytic center or substrate binding sites. EgLDH is a potential target for drugs and vaccines against E. granulosus.

Gene cloning, expression, and localization of antigen 5 in the life cycle of Echinococcus granulosus.

Antigen 5 (Ag5) has been identified as a dominant component of cyst fluid of Echinococcus granulosus and is considered as a member of serine proteases family, which in other helminth, plays an important role in the egg hatch and larva invasion. However, whether Ag5 is expressed and secreted in all life stages is unknown. In this study, according to the sequence in GenBank, we cloned and sequenced the open reading frame (ORF) of Ag5 gene from the protoscolices of E. granulosus isolated from the sheep in Qinhai Province of China, and found several substitutions and a base insert and deletion in a short region near the stop code, leading to a frameshift mutation which is conserved with the homologue of other cestode. The ORF is 1,455 bp in length, encoding 484 amino acids with a secretory signal peptide. Bioinformatics analysis predicted several phosphorylation and myristoylation sites and a N-glycosylation site and a species-specific linear B epitope in the protein. The ORF was cloned into the plasmid pET28a(+) vector and expressed in Escherichia coli . The recombinant protein was purified by affinity chromatography. Anti-rEgAg5 antiserum was prepared in rats and used to analyze the localization of Ag5 in protoscolex and adult worm by immunofluorescence technique. Results demonstrated that the Ag5 is strongly expressed in the tegument of protoscolex and the embryonic membrane of egg and surface of oncosphere; meanwhile, it is also weakly expressed in tegument of the adult. This study showed that Ag5 is expressed in all stages of life cycle, secreted from the surface of the worm and may be anchored in membrane by its myristoylation sites; these characteristics make it a candidate antigen for diagnosis and vaccine for both intermediate and definitive hosts.

The protective effect of the recombinant 53-kDa protein of Trichinella spiralis on experimental colitis in mice.

BACKGROUND: Helminth infection has been proven to reduce the severity of experimental inflammatory bowel disease (IBD). The excretory-secretory proteins of helminths play an important role in the process of immunomodulation. AIMS: In the present study, we aimed to investigate the protective potential of recombinant Trichinella spiralis (TS) 53-kDa protein (rTsP53), a component of excretory-secretory proteins, on experimental colitis in mice. METHODS: BALB/c mice were treated subcutaneously with 50 µg rTsP53 three times at an interval of 5 days. Colitis was induced by intrarectal administration of 5 mg trinitrobenzene sulfonic acid (TNBS). Disease activities and macroscopic and microscopic scores were evaluated. To determine immune response provoked by rTsP53, we measured specific IgG1 and IgG2a values against rTsP53 in sera of mice. We also detected cytokine profiles as well as the markers of alternatively activated macrophages (M2) in mice. RESULTS: RTsP53 ameliorated significantly the disease activity index (DAI) as well as the macroscopic and microscopic scores. IgG1 but not IgG2a was the predominant specific antibody detected in the sera of immunized mice, indicating the potential of stimulating T-helper (Th) 2 bias response by rTsP3. Pre-treatment with rTsP53 decreased serum Th1 cytokines (TNF-a, IFN-γ) and elevated serum levels of serum Th2 cytokines (IL-4, IL-13); it also decreased colonic Th1 cytokines (TNF-α, IL-6) and colonic regulatory cytokines (IL-10, TGF-ß1). RTsP53 increased colonic M2 markers, arginase-1 (Arg-1), and found in inflammatory zone 1 (FIZZ1), compared to mice without rTsP53 pretreatment. CONCLUSIONS: RTsP53 is a potential protective agent for IBD.

Molecular cloning, expression, and characterization of cyclophilin A from Clonorchis sinensis.

This study described the recognization, cloning, and recombinant expression of cyclophilin A-like gene from Clonorchis sinensis adult complementary DNA library (CsCyPA) and its expression and secretion in adult. Western blotting demonstrated the recombinant CsCyPA could be recognized by sera of clonorchiasis patients and a sole protein of the same size in the excretory-secretory antigens of in vitro cultured adult could be recognized by antiserum raised against the recombinant CsCyPA. Immunohistochemistry demonstrated that the CsCyPA was secreted in scattered vesicles from subtegumental parenchyma cells to the surface of tegument and mainly released from the tegument. ELISA showed the serum levels of IgG against CsCyPA in clonorchiasis patients negatively correlated with worm loads. This study suggested that C. sinensis adult in biliary ducts could release CsCyPA without signal peptide through nonclassical secretory pathway into the liver and might play a role in inflammation and biliary epithelium proliferation and adenomatoid hyperplasia.

Cloning and expression of 21.1-kDa tegumental protein of Clonorchis sinensis and human antibody response to it as a trematode-nematode pan-specific serodiagnosis antigen.

A complete cDNA encoding a 21.1-kDa tegumental protein (CsTP21.1) was recognized from Clonorchis sinensis adult full-length cDNA plasmid library by bioinformatics analysis. Recombinant CsTP21.1 was highly expressed in Escherichia coli, purified by affinity chromatography, and identified by Western blotting. Immunohistochemistry demonstrated that CsTP21.1 is localized in the tegument of the adult worm. The rCsTP21.1-specific IgG1, IgG2, and IgG4 subclasses could be detected in the sera of clonorchiasis patients by ELISA, but their sensitivity was much lower than that of total IgG. The sensitivity and specificity of IgG in 66 serum samples of clonorchiasis patients were 100% and 95.5%, and the sensitivity was independent of worm loads; the cross-reaction rates in 86, 24, and 31 serum samples from patients infected with Fasciola hepatica, Schistosoma japonicum, and nematode were 98.8%, 83.3%, 93.3%, respectively, whereas no cross-reactions with Toxoplasma gondii and sparganum. This study demonstrated that CsTP21.1 is a trematode-nematode pan-specific antigen that is valuable in the development of a universal immunodiagnostic kit for human trematode and nematode infections.

[Expression and characterization of the FABP and Eg95 fusion gene from Echinococcus granulosus].

OBJECTIVE: To construct and express a fusion gene of fatty acid binding protein (FABP) with Eg95 which are protective antigen genes of Echinococcus granulosus, and investigate the immunological characteristics of the recombinant protein. METHOD: Using cDNA fragments encoding FABP and Eg95 genes from E. granulosus Qinghai sheep strain as templates, a fusion gene FABP.Eg95 was amplified by asymmetric polymerase chain reaction th-rough a linking sequence encoding four glycine residues. The PCR products of fusion gene were subcloned into the pET28a (+) vector. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells and induced with IPTG. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography, and its immunogenicity was analyzed by Western blotting. Results The fusion gene length was about 795 bp. Double enzyme digestion analysis and DNA sequencing confirmed that the fusion gene was cloned into pET-28a (+). The recombinant protein (Mr 31,000) was expressed in inclusion body in E. coli expression system, and was purified by Ni-IDA affinity chromatography. Western blotting analysis testified that the recombinant protein could be recognized by sera of cystic echinococcosis patients, but not by sera of healthy persons and schistosomiasis patients. CONCLUSION: The FABP.Eg95 fusion gene has been constructed, and the purified recombinant protein has been confirmed with immunogenicity.

Reverse vaccinology approach identify an Echinococcus granulosus tegumental membrane protein enolase as vaccine candidate.

Applying reverse vaccinology strategy, we employed a sequence encoding an enolase from Taenia asiatica to search its homolog in the expression sequence tag (EST) database of Echinococcus granulosus and found two EST sequences (Access number: CN653186 and CN649593) of a clone Eg_PSGRS_13B09 from E. granulosus protoscolex full-length cDNA library, which are responding for the 5' and 3' partial cds of E. granulosus enolase, respectively. Primers are designed according to the 5' end and 3'end of the putative encoding sequence to amplify the genomic DNA of E. granulosus strain isolated from sheep in Qinghai province of China by polymerase chain reaction (PCR). A sole product of 1,449 bp in length was obtained, which contains two little introns of 78 bp and 69 bp, respectively. The introns were excised by unsymmetrical PCR with combined flank sequences of introns as primers. The structural, functional, and immunological characteristics of putative amino acid sequence were predicted by bioinformatics analysis. The complete coding sequence was predicted to encode 433 residues and contain a transmembrane region aa(104-124), with the N terminus outside and C terminus inside. The inside part is quite the functional domain. SWISS-MODEL modulated its 3D structure as a barrel which constitutes of alternatively arranged alpha helix-beta sheet, with the key sites such as substrate binding region, active sites, Mg(2+)-binding sites closely located at the center. The protein contains a potential nuclear localization sequence aa(190-199) and several linear B cell epitopes and CTL T cell epitopes, of which the outside epitope aa(49-57) and inside epitope aa(228-236) are facultative T cell and B cell epitope, and the linear B cell epitope aa(206-213) contains the active center site Glu(210), suggesting the putative protein is a potential membrane with strong immunogenicity. The complete cds was expressed in Escherichia coli, and the recombinant protein can be recognized by the serum from patient infected with E. granulosus. Reverse vaccinology process identified E. granulosus tegumental membrane protein enolase as vaccine candidate.