Objective Receptor was screened to find out the interaction between outer membrane protein 25 of Brucella(OMP25) and Brucella infected macrophages.Methods Recombinant OMP25 protein was expressed and purified.The 7 peptide was screened out with OMP25 protein by phage display technology.OMP25-7 peptide interactions were further confirmed by enzyme linked immunosorbent assay(ELISA) after candidate peptides were predicted with bioinformatics software.Four potential 7 peptides were synthetized.Their functions were estimated after Brucella abortus 2308 infected murine macrophages RAW264.7.Results Forty-two cyclic 7 peptides were screened out based on purified OMP25 by the M13 Phage Library Kit.Four peptides,named as ST-7,MT-7,TF-7 and SN-7,were confirmed with ELISA.Colony forming unit(CFU) result demonstrated ST-7,MT-7,TF-7 and SN-7 showed some function for inhibition of intracellular parasite.ST-7 inhibition ratio was 14.4％-51.8％ for Brucella abortus 2308; MT-7 inhibition ratio was 49.6％-69.5％ for Brucella abortus 2308; TF-7 inhibition ratio was 43.2％-74.4％ for Brucella abortus 2308; SN-7 inhibition ratio was 57.5％-82.2％ for 2308.ELISA result showed,that compared to control group,tumor necrosis factor α(TNF-α)increased no statistical significance in each time point of ST-7,MT-7 experiment groups (all P ＞ 0.05).There was no statistical significance in 8 h ago of TF-7,SN-7 experiment groups (all P ＞ 0.05),but there was statistical significance in 12,24 h (all P ＜ 0.05).Conclusions We have obtained four effective OMP25 receptors through phage display technology; TF-7 and SN-7 have inhibited Brucella abortus 2308 invasion and intracellular survival in murine macrophage cells,while ST-7 and MT-7 have only prevented the invasion against Brucella abortus 2308.

We detected the isoniazid resistance in clinical Mycobacterium tuberculosis isolates by high-resolution melting (HRM) curve analysis and assessed the application value of the assay.The isoniazid resistance of 49 M.tuberculosis isolates preserved in laboratory was analyzed by the drug sensitivity test (traditional proportion method).Further analysis was made on the sequencing of the isoniazid resistance determining region in these test strains,and their mutation sites were screened.Specific primers used in the HRM curve analysis were designed based on the screened mutation sites,DNA mutations were assayed in the isoniazid-resistant gene determining region by the HRM curve analysis,and an assessment was made of the detection efficiency of the assay in isoniazid resistance in M.tuberculosis.Results of the drug sensitivity test (proportion method) showed that,of the 49 test strains,there were 20 isoniazid-resistant strains,29 isoniazid-sensitive strains.Results of the sequencing analysis showed that:1) KatG gene had four mutation patterns,i.e.,point mutations at site 234,at sites 234 and 315,at sites 234 and 463,and at sites 234,315 and 463;2) there were three mutations were detected in inhA gene,i.e.,mutations in inhA-8,-15 and-152.Analysis of gene mutation in drug-resistant strains found that of the 20 isoniazid-resistant strains,11 (55 ％) were mutated at codon 315 of KatG gene;6 (30％) were mutated in inhA-15 (4/20),-8 (1/20) and-153 (1/20) of inhA gene;two (10％) were mutated at codon 315 of KatG gene and in inhA-15;in one strain (5％),no mutation was detected in KatG and inhA genes.Through the gene mutation detection,the sensitivity and specificity of isoniazid resistance in M.tuberculosis were 95 ％ and 100 ％,respectively.Results of HRM curve analysis of drug-resistance gene mutations in test strains showed gene mutations were present in 18 strains and absent in 24 ones;referring to DNA sequencing results,the sensitivity and specificity of the assay were 94.7％ and 80％,respectively.Judged by mutations as drug-resistance via the HRM curve analysis,19 resistant and 24 sensitive strains were tested.With the drug sensitivity test results by the proportion method as controls,the sensitivity and specificity of the assay were 95 ％ and 82.76 ％,respectively.Use of the HRM curve in the detection of resistance of M.tuberculosis to isoniazid is characterized by good sensitivity and short time consuming,and has certain value in the rapid diagnosis of isoniazid-resistant tuberculosis.

Animals ; Borrelia burgdorferi ; genetics ; isolation & purification ; China ; Genotype ; Ixodidae ; microbiology

Potential target proteins binding to VirB4 of type Ⅳ secretion system were screened during Brucella infected bovine embryonic trophoblast cells .Brucella VirB4 genes were amplified by PCR with species-specific primers .Expression vector pGBKT7-virB4 was constructed and analysed by sequencing and restriction enzymes ,transforming to the yeast strain Y187 and testing self-activation and toxicity .The cells model and cDNA library of bovine embryonic trophoblast cells infected with Brucella abortus strain were constructed respectively .Utilizing yeast two-hybrid system was employed to screen the target proteins of bovine embryo trophoblastic cells which was conjunctive with virB4 .These proteins were detected by real-time fluo-rescence quantitative PCR .The results suggested that bait plasmid pGBKT7-virB4 was successfully transformed into the Y187 and there was no toxicity and self-activation;the cDNA library of bovine embryonic trophoblast cells infected with Brucella abortus strain was constructed .There screened 13 positive plasmids in which Q10 and SLC3A2 were up-regulated at the mRNA level .In this paper ,we reported the interactions between the VirB4 protein of Brucella and the bovine embryo trophoblastic cells ,which provide an upstream work for further elucidating the pathogenesis of Brucella infection of the host cell .

Background and Objectives: Sheep-induced pluripotent stem cells (siPSCs) have low reprogramming efficiency, thereby hampering their use in biotechnology and agriculture. Several studies have shown that some microRNAs play an important role in promoting somatic reprogramming in mouse and human. In this study, we investigated the effect of miR-200c-141 on somatic reprogramming in sheep and explored the mechanism of promoting the reprogramming. Methods: and Results: The lentivirus system driven by tetracycline (TET)-on carrying Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, hTERT, and SV40LT (OSKMNLST) could reprogram sheep kidney cells into pluripotent cells. Overexpression of miR-200c-141 in combination with OSKMNLST could significantly improve the efficiency of sheep iPSC generation (p＜0.01). Sheep iPSCs derived from miR-200c-141 showed embryonic stem cell (ESC)-like pluripotent properties, were positive for alkaline phosphatase and some pluripotent markers by quantitative real-time PCR (qRT-PCR) and immunofluorescence, and were able to differentiate into three germ layers in vitro. Oar-miR-200c was transfected into HEK293FT cells and was able to target the zinc finger E-box-binding homeobox 1 (ZEB1) 3’UTR using dual luciferase reporting analysis. Overexpression of oar-miR-200c in SKCs significantly reduced the expression of ZEB1, but increased the expression of E-cadherin by qRT-PCR and western blotting analysis. Conclusions These results suggest that miR-200c-141 can promote the reprogramming of sheep somatic cells to iPSCs, and oar-miR-200c targeted ZEB1 3’UTR, significantly decreased expression of ZEB1, and increased expression of E-cadherin. Oar-miR-200c may improve the MET process by affecting the TGF-β signaling pathway, thus improving the efficiency of somatic cell reprogramming in sheep.

Iron is involved in the virulence and pathogenic effects of certain intracellular parasites.In the pathogenic process of Brucella,the uptaking and metabolism of host iron are closely related to intracellular parasitism and immunity escape of Brucella.In this paper,we elucidated the iron transport system,iron response regulators and nutrient immunity of iron based on the latest report and data about Brucella.

Background: There is an urgent need to find reliable and rapid bovine tuberculosis (bTB) diagnostics in response to the rising prevalence of bTB worldwide. Toll-like receptor 2 (TLR2) recognizes components of bTB and initiates antigen-presenting cells to mediate humoral immunity. Evaluating the affinity of antigens with TLR2 can form the basis of a new method for the diagnosis of bTB based on humoral immunity. Objectives: To develop a reliable and rapid strategy to improve diagnostic tools for bTB. Methods: In this study, we expressed and purified the sixteen bTB-specific recombinant proteins in Escherichia coli. The two antigenic proteins, MPT70 and MPT83, which were most valuable for serological diagnosis of bTB were screened. Molecular docking technology was used to analyze the affinity of MPT70, MPT83, dominant epitope peptide of MPT70 (M1), and dominant epitope peptide MPT83 (M2) with TLR2, combined with the detection results of enzyme-linked immunosorbent assay to evaluate the molecular docking effect. Results: The results showed that interaction surface Cα-atom root mean square deviation of proteins (M1, M2, MPT70, MPT83)-TLR2 protein are less than 2.5 A, showing a high affinity.It is verified by clinical serum samples that MPT70, MPT83, MPT70-MPT83 showed good diagnostic potential for the detection of anti-bTB IgG and M1, M2 can replace the whole protein as the detection antigen. Conclusions Molecular docking to evaluate the affinity of bTB protein and TLR2 combined with ELISA provides new insights for the diagnosis of bTB.

Hydatid worms, hosted by humans and animals, impose serious human health risk and cause significant livestock production loss. To better understand the disease infection status in Xinjiang, China, we investigated the disease epidemics in 4 livestock animals, i.e., cattle, sheep (both sheep and goat), camels, and horses, slaughtered at the abattoirs in Urumqi, Yining, Tacheng, and Altay areas. The results showed that the animals were infected at different rates, in the order of sheep (9.8%), cattle (8.4%), camels (6.8%), and horses (4.3%). The infection rates were found to be different between the abattoirs in various regions even for the same animals. For sheep, the rates increased significantly as the animals grew older. It was 1.9% before 1 year of age and increased to 8.2% in the age of 1-2 years, and further increased to 12.3% when the animals were 3-4 years old, and reached 17.2% when they were 5-6 year old. Sheep older than 6 years had an infection rate of 19.5%. This study demonstrates that the 4 livestock animals in the pastoral areas in Xinjiang were infected by the parasites to various extend. This study is the first systematic investigation of the hydatid worms in various livestock animals in Xinjiang, China, which provides epidemiological information about the infection of hydatid worms in livestock, and is valuable in developing strategies for prevention and control of the hydatid disease.
Age Factors ; Animals ; China/epidemiology ; Echinococcosis/epidemiology/*veterinary ; *Livestock ; Prevalence

A fluorescent microsphere-based immunochromatographic strip test (FICT) was developed for the rapid, sensitive, and quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) antibodies at the pen-side. The assay was based on the formation of a sandwich immune-complex (anti-pig IgG-PRRSV antibodies-NSP7/N), which was validated by a comparison with IDEXX-ELISA using 3325 clinical specimens. The diagnostic specificity, sensitivity, and accuracy of FICT were 97.28, 93.41, and 94.95%, respectively. FICT showed a good correlation with the virus neutralization assay. Overall, a promising pen-side diagnostic tool was developed for the rapid and quantitative detection of PRRSV antibodies within 15 min.

Objective To prepare monoclonal antibodies (mAb) against the type Ⅳ secretion system protein VirB5 of Brucella melitensis and to provide a basis for pathgenic diagnosis and research of brucellosis.Methods Four SPF female BALB/c mice were subcutaneously immunized with purified VirB5 protein at a dose of 60 μg/mice,and immunization was strengthened every 2 weeks at a dose of 30 μg/mice,three times in total.Two weeks later,the orbital venous blood of mice was taken to determine the antibody titer,and then intraperitoneally injected for the fourth time to strengthen immunization.Three days later,mouse spleen cells were fused with mouse myeloma SP2/O cells in a ratio of 5:1.After 3 times of cell screening and monoclonal cloning,the hybridoma cell lines with stable secretion of VirB5 antibody were established;one BALB/c mouse was intraperitoneally injected with hybridoma cells,and ascites were collected and antibody was purified when the mouse abdomen was significantly enlarged.The immunological characteristics of mAbs were identified by indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting.Results A total of 6 monoclonal cell lines (2-2,2-12,2-19,2-25,2-31 and 2-40) capable of secreting VirB5 antibody were established.Among them,the cell line 2-19 can stably secrete an antibody that specifically recognized the VirB5 protein,and the VirB5 antibody secreted by the cell line was identified as an IgG1 subtype,a kappa light chain,a mAb affinity constant of 1.6 × 108.The titer of ascites antibody of mouse intraperitoneally injected with hybridoma cell 2-19 was 1:51 200.Conclusion The high-affinity mAb of type Ⅳ secretion system protein VirB5 is successfully prepared,and the antibody can rapidly bind specifically to pathogens,providing an alternative material for establishment of brucellosis pathogen diagnostic method.