[Cellular immunity evaluation of five mycobacterium tuberculosis recombinant proteins and their compositions].

Objective: The cellular immunity of 5 Mycobacterium tuberculosis recombinant proteins and their compositions was evaluated. Method: A total of 88 fresh venous blood from peripheral heparin anticoagulant population, 42 of which were from tuberculosis patients treated by The Tuberculosis Prevention and Treatment Center of Changping District, Beijing, and 46 of healthy volunteers were provided by the Infection Diseases of Chinese Center for Disease Control and Prevention. Healthy volunteers without a history of tuberculosis exposure and any clinical signs and symptoms. Using the Mycobacterium tuberculosis standard strain H37Rv DNA as a template, complete genes of the selected 5 recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c by PCR amplified; 5 recombinant proteins were cloned, expressed and purified as stimulants by genetic recombination and protein purification techniques, and the effector T cell enzyme-linked immunospot assay (ELISPOT) was used to detect cellular immunity in the population. Results: The recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c were successfully cloned, expressed and purified; And the sensitivities were 50.00%, 71.43%, 69.04%, 73.81% and 76.19%, and the specificities were 86.96%, 76.09%, 71.74%, 39.13% and 36.96%. In addition, the positive predictive value, negative predictive value, area under the curve and Youden index were 52.46% to 77.78%, 62.96% to 74.47%, 0.511 to 0.754 and 0.129 to 0.475, respectively. Except for Rv1411c and Rv3418c, the number of spot-forming cell (SFC) detected by Rv3874, Rv3875 and Rv2031c in tuberculosis patients was higher than healthy volunteers, and the differences were statistically significant (P<0.001). Among the 26 compositions composed of 5 recombinant proteins, the sensitivity was 80.95% to 95.24%, and the specificity was 68.89% to 24.44%. As the number of recombinant proteins in the composition increases, the sensitivity gradually increased, but the specificity decreased. Conclusion: The recombinant proteins of Mycobacterium tuberculosis Rv3874, Rv3875 and Rv2031c have strong ability to stimulate T cells to produce immune response, and have certain antigenicity. The efficacy of Rv1411c and Rv3418c alone as diagnostic antigens is not ideal, and the composition composed of multi-component antigens has certain application value. This article provides experimental evidence for the immune diagnosis of tuberculosis and the preparation of new anti-tuberculosis vaccines.

[Application of ARIMA model in predicting the incidence of tuberculosis in China from 2018 to 2019].

Objective: Autoregressive integrated moving average (ARIMA) model was used to predict the incidence of tuberculosis in China from 2018 to 2019, providing references for the prevention and control of pulmonary tuberculosis. Methods: The monthly incidence data of tuberculosis in China were collected from January 2005 to December 2017. R 3.4.4 software was used to establish the ARIMA model, based on the monthly incidence data of tuberculosis from January 2005 to June 2017. Both predicted and actual data from July to December 2017 were compared to verify the effectiveness of this model, and the number of tuberculosis cases in 2018-2019 also predicted. Results: From 2005 to 2017, a total of 13 022 675 cases of tuberculosis were reported, the number of pulmonary tuberculosis patients in 2017 was 33.68% lower than that in 2005, and the seasonal character was obvious, with the incidence in winter and spring was higher than that in other seasons. According to the incidence data from 2005 to 2017, we established the model of ARIMA (0,1,2)(0,1,0)(12). The relative error between the predicted and actual values of July to December 2017 fitted by the model ranged from 1.67% to 6.80%, and the predicted number of patients in 2018 and 2019 were 789 509 and 760 165 respectively. Conclusion: The ARIMA (0, 1, 2)(0, 1, 0)(12) model well predicted the incidence of tuberculosis, thus can be used for short-term prediction and dynamic analysis of tuberculosis in China, with good application value.

[Cloning expression and serological evaluation on Mycobacterium tuberculosis four new antigens].

Objective: To evaluate the serological diagnostic value of Mycobacterium (M.) tuberculosis four new antigens Rv0432, Rv0674, Rv1566c and Rv1547. Methods:Rv0432, Rv0674, Rv1566c and Rv1547 were amplified from M. tuberculosis strain H37Rv genomic DNA by using PCR, among which Rv1547 was divided into two segments for amplification (Rv1547-1 and Rv1547-2). The segments were cloned into expression vector PET-32a while the recombinant proteins were purified by affinity chromatography. Serums were incubated with BL21 (DE3) proteins. Antibodies IgG against M. tuberculosis were tested with 151 serum samples (41 healthy people and 110 TB patients) by using ELISA. The diagnostic efficiency of antigens was analyzed by means of receiver operating characteristic curve. Difference of the objective proteins in TB patients and healthy controls was compared by t-test. Results: Recombinant antigens Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2 were successfully expressed and purified. Results from ELISA showed that the sensitivity, specificity, positive predictive value, negative predictive value, Youden index and area under the curve of Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2, as 43.64%-92.73%, 80.49%-92.68%, 0.92-0.94, 0.38-0.80, 0.363-0.732 and 0.649-0.915. All the objective proteins showed significantly higher antibody levels in TB patients, when compared to the healthy controls (P<0.000 1). Conclusion: The newly identified antigens Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2 all performed well when being used for TB serological diagnosis, thus were expected to be new candidate antigens used for TB diagnosis.

[Identification and evaluation of T cell epitopes of Rv0585c from Mycobacterium tuberculosis].

Objective: To investigate the human T cell epitopes of Mycobacterium (M.) tuberculosis Rv0585c protein antigen and their immunogenicity and provide evidence for the development of specific tuberculosis immune diagnostic techniques and tuberculosis vaccine. Methods: We synthesized peptides from M. tuberculosis Rv0585c protein antigen predicted by TE-predict and IEDB human T cell epitope prediction tool. The cellular immunoreactivity of the predicted peptides was evaluated through ELISpot assay with the peripheral blood monouclear cells (PBMC) of clinical tuberculosis patients. In animal experiments, BALB/c mice were respectively immunized with high dose (100 µg/mice) and low dose (50 µg/mice) of the peptides of Rv0585c, at the same time, high dose (50 µg/mice) and low dose (20 µg/mice) of Ag85B protein were used in positive control group. The levels of IFN-γ, IL-2, IL-4 and IL-10 were tested with ELISA kit respectively. Results: By means of bioinformatics technique, 66 human T cell epitopes of Rv0585c were predicted, from which9 peptides concentrated epitopes were synthesized for the animal immune experiments. Peptides P10110, P10112 and P10117 were confirmed to be antigenic. The sensitivity and specificity of P10110, P10112 and P10117 were 14.00%, 12.00%, 6.00% and 100.00%, 100.00%, 97.96% respectively when they were used as diagnostic reagents of tuberculosis. The sensitivity and specificity were 22.00% and 97.96% when the epitopes were combined together. The results of animal immunity test showed that high levels of cytokines IFN-γ, IL-2, IL-4 and IL-10 were induced by high and low dose of P10110, and high levels of IFN-γ、IL-2 and IL-10 were induced by high and low dose of P10112, which were much higher than that in negative controls, respectively (P<0.001). Conclusion: Rv0585c, including its human T cell epitopes, has good immunogenicity and immunoreactivity, stimulating the body to produce a stronger cellular immune response and has better potential application value in cellular diagnosis of tuberculosis and the development of new type of tuberculosis vaccine.

[Diagnostic value of carotid atherosclerosis score for ischemic stroke].

OBJECTIVE: To discuss the diagnostic value of carotid atherosclerosis score for ischemic stroke. METHODS: In the study, 151 patients with ischemic stroke were enrolled, who were diagnosed by cranial CT scan or cranial MRI scan, and examined with carotid duplex ultrasound, and 151 healthy check-up cases matched by age and sex were chosen as control group, who were excluded ischemic stroke by cranial CT scan or cranial MRI scan. All the control cases were examined with carotid duplex ultrasound also. Intima-media thickness (IMT), the number of carotid plaques, the size of each plaque, the location of the plaque and each plaque's echo, texture, surface regularity were estimated by carotid duplex ultrasound. RESULTS: The IMT of the case group and the control group were (0.946±0.185) mm and (0.863±0.148) mm, and there were significant differences (P<0.001); The parameters of arterial plaque correlated with ischemic stroke were plaque's echo, texture and surface regularity, however the plaque size and location were not correlated with ischemic stroke. The median and quartile of carotid artery plaque score were 3 and 2 respectively in case group, 1 and 2 respectively in control group, and there were significant differences (P<0.001); The parameters of carotid arterial atherosclerosis associated with ischemic stroke were carotid artery plaque score,carotid stenosis degree and IMT, but not the number of carotid plaques. The median and quartile of carotid arterial atherosclerosis score were 5 and 4 respectively in case group, 2 and 4 respectively in control group, and there were significant differences (P<0.001); The area under the curve (AUC) for IMT, the number of carotid plaques, carotid artery plaque score and carotid arterial atherosclerosis score were 0.679, 0.677, 0.704 and 0.805,respectively (P<0.001). The accuracy of carotid atherosclerosis score was the highest. CONCLUSION: Carotid artery plaque score and carotid atherosclerosis score can be used for the diagnosis of ischemic stroke, and the accuracy of carotid atherosclerosis score is higher.

Characterization of grass carp spleen transcriptome during GCRV infection.

The aim of the study was to investigate the grass carp hemorrhagic infection pathway and its key-related genes. Grass carp reovirus (GCRV) might cause hemorrhagic disease in grass carps. Healthy grass carp fingerlings (N = 60) were divided into control and infected groups. Fish in the control group were intraperitoneally (ip) injected with 0.6% fish physiological saline; the infected group received 5,000,000 50% tissue culture infective doses of GCRV 873 standard strain, a double-stranded RNA (dsRNA) virus strain, ip, in 0.5 mL. Illumina HiSeqTM 2000 was used for transcriptome sequencing, and real-time polymerase chain reaction (PCR) used to detect complement factors II (C2), III (C3), and V (C5); profibrinolysin (PLG); and coagulation factor II (F2) expression. A total of 2,722,223 reads were detected in the control group, and 2,751,111 in the infected group. Among 11,023 unigenes obtained after transcriptome assembly, 10,021 unigenes were significantly differentially expressed. Gene ontology and KEGG analysis, a collection of databases dealing with genomes and biological pathways, were performed to classify unigenes into functional categories, to understand gene function and identify regulatory pathways. Real-time PCR analysis showed that C2, C3, C5, PLG, and F2 expression levels were down-regulated, confirming results of pathway-enrichment analysis. This is the first application of high-throughput sequencing technology to investigate the in vivo effects of GCRV, on genes and pathways involved in the immune response to infection in grass carp.

Cloning, characterization and expression analysis of coagulation factor II gene in grass carp (Ctenopharyngodon idella).

Here, we characterized the structure and function of the coagulation factor II (FII) gene in grass carp and determined its role in coagulation mechanisms. The FII gene EST was obtained using a constructed splenic transcriptome database; the full-length FII gene sequence was obtained by 3' and 5' RACE. The open reading frame (ORF) of FII was cloned and the full-length gene was found to be 1718 bp, with an ORF of 1572 bp; the gene contained a 25 bp 5'-untranslated region (UTR) and 108 bp 3'-UTR. The ORF encoded 524 amino acids, including 74 alkaline amino acids (arginine and lysine) and 69 acidic amino acids (aspartic acid and glutamic acid). The theoretical pI was 6.22. The calculated instability index (II) was 39.81, indicating that FII was a stable protein; the half-life period was predicted to be approximately 30 h. Amino acid sequence comparisons indicated that grass carp FII showed most similarity (71%) to FII of Takifugu rubripes, followed by Oplegnathus fasciatus (48% similarity) and Larimichthys crocea (47% similarity). A real-time reverse transcription PCR analysis showed that under normal circumstances, FII was most highly expressed in the liver, followed by the gill, spleen, thymus, and head-kidney (P < 0.001). After injection of the grass carp reovirus 873 (GCRV873), the pattern of FII expression was significantly altered (P < 0.001); gene expression was high after injection, suggesting a response involving the initiation of the coagulation system and defense of the body in combination with the platelet and complement system.