Preparation and immunogenicity evaluation of mRNA vaccine against porcine epidemic diarrhea / 生物工程学报

Porcine epidemic diarrhea (PED) is a highly contagious disease that causes high mortality in suckling piglets. Although several licensed inactivated and live attenuated vaccines were widely used, the infection rate remains high due to unsatisfactory protective efficacy. In this study, mRNA vaccine candidates against PED were prepared, and their immunogenicity was evaluated in mice and pregnant sows. The mRNA PED vaccine based on heterodimer of viral receptor binding region (RBD) showed good immunogenicity. It elicited robust humoral and cellular immune responses in mice, and the neutralizing antibody titer reached 1:300 after a single vaccination. Furthermore, it induced neutralizing antibody level similar to that of the inactivated vaccine in pregnant sows. This study developed a new design of PED vaccine based on the mRNA-RBD strategy and demonstrated the potential for clinical application.

Nucleomodulin BspJ as an effector promotes the colonization of Brucella abortus in the host

Background@#Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified. @*Objectives@#To verify the functions of nucleomodulin BspJ in Brucella's intracellular infection cycles. @*Methods@#Constructed Brucella abortus BspJ gene deletion strain (B. abortus ΔBspJ) and complement strain (B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro. @*Results@#BspJ gene deletion reduced the survival and intracellular proliferation of Brucellaat the replicating Brucella-containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1β, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella. @*Conclusions@#BspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella's intracellular survival.

A combined application of molecular docking technology and indirect ELISA for the serodiagnosis of bovine tuberculosis

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.

miR-200c-141 Enhances Sheep Kidney Cell Reprogramming into Pluripotent Cells by Targeting ZEB1

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.

A rapid and quantitative fluorescent microsphere immunochromatographic strip test for detection of antibodies to porcine reproductive and respiratory syndrome virus

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.

Interaction between Brucella melitensis 16M and small ubiquitin-related modifier 1 and E2 conjugating enzyme 9 in mouse RAW264.7 macrophages

Brucella is an intracellular pathogen that invades a host and settles in its immune cells; however, the mechanism of its intracellular survival is unclear. Modification of small ubiquitin-related modifier (SUMO) occurs in many cellular activities. E2 conjugating enzyme 9 (Ubc9) is the only reported ubiquitin-conjugating enzyme that links the SUMO molecule with a target protein. Brucella's intracellular survival mechanism has not been studied with respect to SUMO-related proteins and Ubc9. Therefore, to investigate the relationship between Brucella melitensis 16M and SUMO, we constructed plasmids and cells lines suitable for overexpression and knockdown of SUMO1 and Ubc9 genes. Brucella 16M activated SUMO1/Ubc9 expression in a time-dependent manner, and Brucella 16M intracellular survival was inhibited by SUMO1/Ubc9 overexpression and promoted by SUMO1/Ubc9 depletion. In macrophages, Brucella 16M-dependent apoptosis and immune factors were induced by SUMO1/Ubc9 overexpression and restricted by SUMO1/Ubc9 depletion. We noted no effect on the expressions of SUMO1 and Ubc9 in B. melitensis 16M lipopolysaccharide-prestimulated mouse RAW264.7 macrophages. Additionally, intracellular survival of the 16M△VirB2 mutant was lower than that of Brucella 16M (p < 0.05). VirB2 can affect expression levels of Ubc9, thereby increasing intracellular survival of Brucella in macrophages at the late stage of infection. Collectively, our results demonstrate that B. melitensis 16M may use the VirB IV secretion system of Brucella to interact with SUMO-related proteins during infection of host cells, which interferes with SUMO function and promotes pathogen survival in host cells.

Production and identification of monoclonal antibodies against Brucella melitensis VirB5 protein / 中华地方病学杂志

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.

Iron uptake systems of Brucella and anti-infectious immunity of the host / 中华地方病学杂志

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.

Prokaryotic expression, reactogenicity, and function of Brucella secretary protein BspE / 中华地方病学杂志

Objective To investigate the prokaryotic expression and immunoreactivity of BspE,a type Ⅳ secretion protein of Brucella,and the effect of recombinant protein BspE on cytokines.Methods According to the BspE gene of Brucella M5-90 published in GenBank,the gene fragments were synthesized by a company and then ligated into PUC57 vector for sequencing.The sequenced gene was cloned into a prokaryotic expression vector pET-28α and transformed.Induced expression was performed in E.coli DE3 competent cells.The obtained target protein was purified by a Ni-NTA affinity column,and its reactogenicity was analyzed by Western blotting.Mouse RAW264.7 cells were treated with 25 g/L BspE recombinant protein for 12,24,48 h,and the control group was treated with the same amount of BSA instead of BspE,and enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin (IL)-1β level.Results The recombinant expresed plasmid of pET-28α-BspE was successfully obtained.The results of Western blotting showed a single band with a relative molecular mass of about 30.1 × 103,and the recombinant protein BspE had good reactogenicity,and IL-1β levels (ng/L)were significantly elevated by the recombinant protein BspE (12 h:43.27 ± 2.13 vs 30.24 ± 1.66,24 h:57.78 ± 3.44 vs 41.22 ± 1.22,48 h:72.52 ± 3.04 vs 46.77 ± 2.75,t =8.38,7.86,10.89,P ＜ 0.05).Conclusions BspE recombinant protein has better immunoreactivity and can increase the expression level of IL-1β in mouse macrophages.This study provides a scientific basis for the role of effector proteins in the pathogenesis of Brucella.

Genetic Diversity of Echinococcus granulosus Genotype G1 in Xinjiang, Northwest of China

Cystic echinococcosis (CE) caused by E. granulosus is a serious helminthic zoonosis in humans, livestock and wildlife. Xinjiang is one of high endemic province for CE in China. A total of 55 sheep and cattle livers containing echinococcal cysts were collected from slaughterhouses in Changji and Yining City, northern region of Xinjiang. PCR was employed for cloning 2 gene fragments, 12S rRNA and CO1 for analysis of phylogenetic diversity of E. granulosus. The results showed that all the samples collected were identified as G1 genotype of E. granulosus. Interestingly, YL5 and CJ75 strains were the older branches compared to those strains from France, Argentina, Australia. CO1 gene fragment showed 20 new genotype haploids and 5 new genotype haplogroups (H1-H5) by the analysis of Network 5.0 software, and the YLY17 strain was identified as the most ancestral haplotype. The major haplotypes, such as CJ75 and YL5 strains, showed identical to the isolates from Middle East. The international and domestic trade of livestock might contribute to the dispersal of different haplotypes for E. granulosus evolution.

Rapid detection of isoniazid resistance in clinical Mycobacterium tuberculosis isolates by high-resolution melting curve analysis / 中国人兽共患病学报

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.

Isolation of Borrelia burgdorferi in ixodes from four counties, in North Xinjiang / 中华流行病学杂志

<p><b>OBJECTIVE</b>To identify ticks and determine the Borrelia (B.) burgdorferi genotype from four counties of northern Xinjiang.</p><p><b>METHODS</b>Sheep ticks were collected from 6 surveillance sites in four counties including Shihezi, Shawan,Yining and Chabuchaer. All ticks were initially screened out based on morphological methods and 16S rRNA sequence analysis. B. burgdorferi was detected and cultivated with BSK-H medium. Combined with nested PCR, silver nitrate staining was employed to detect B. burgdorferi. Genotype of isolated B. burgdorferi was determined by Sequencing and phylogenic analysis based on 11 conference sequences.</p><p><b>RESULTS</b>Hyalomma asiaticum asiaticum, Haemaphysalis punctata, Dermacentor marginatus and Rhipicephalus turanicus were identified from more than 900 ticks. Out of 24 tubes from 102 representative tick specimens, 16 tube were positive for B. burgdorfer. Sequencing of 5S-23S rRNA intergenic spacer showed 98.6%-99.5% identities to B. burgdorferi Sensu Stricto(B31). Results from the analysis of OspC genotype showed consistent with that of 5S-23S rRNA intergenic spacer.</p><p><b>CONCLUSIONS</b>16 strains of B. burgdorferi Sensu Stricto were isolated in four counties, from northern Xinjiang. Additionally, B. burgdorferi Sensu Stricto was isolated from Rhipicephalus turanicus first time in China.</p>

Function analysis of outer membrane protein VirB4 in Brucella infecting embryo trophoblastic cells / 中国人兽共患病学报

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 .

Screening of receptor that interacted with outer membrane protein 25 during macrophage infection by Brucella abortus / 中华地方病学杂志

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.

A rapid, sensitive and reliable diagnostic test for scrub typhus in China.

Purpose: To evaluate the performances for detection of IgM and IgG antibodies to Orientia. tsutsugamushi (Ot) using a gold conjugate-based rapid diagnostic test (RDT). Materials and Methods: The RDT employing mixture recombinant 56-kDa proteins of O. tsutsugamushi and the mIFA assay was performed on 33 patients from Fujian and Yunnan province respectively and 94 positive sera (36 from Hainan province and 58 from Jiangsu province) from convalescent stages of the patients with scrub typhus respectively and 82 negative sera from healthy farmers from Anhui province and Beijing City respectively in 2009. A comparison of the RDT and mIFA assay was performed by using the c2 test and the P level of ≤0.05 was considered to be significant. Results: Among these 94 positive sera from convalescent stages of the illness and 82 sera from control farmers, the specificity of RDT was 100% for both IgM and IgG tests. In 33 cases with scrub typhus, 5 cases were positively detected earlier by RDT than by mIFA for the IgM test, and 2 cases were positive for the IgG test. The sensitivities of RDT were 93.9% and 90.9% for IgM and IgG, respectively. Considering IgM and IgG together, the sensitivity was 100%. The geometric mean titre (GMT) of IFA and the RDT assay in diluted sera from confirmed cases were 1:37 versus 1:113 respectively (P<0.001) for IgM test and 1:99 versus 1:279 respectively (P<0.016) for IgG. Conclusions: The RDT was more sensitive than the traditional IFA for the early diagnosis of scrub typhus and was particularly suitable for use in rural areas.