A pair of specific primers was designed based on the reported Bm86 gene of Boophilus microplus,the Bm86 gene was cloned by PCR using the plasmid pMD18-T-Bm86 as templates,and subcloned into the prokaryotic plasmid pGEX-4T-1.The recombined plasmid was transformed into E.coli BL21(DE3) and followed by expression of the protein induced by different concentration of IPTG for different time.SDS-PAGE showed that the recombinant plasmid pGEX-4T-1/Bm86 expressed a fusion protein Bm86-GST(Mr 94 000) after being induced with IPTG.High level expre-ssion of Bm86-GST was found at 1 mmol/L IPTG condition after incubation for 8 h at 37 ℃,and the expression level of the recom-binant Bm86-GST reached up to 29% of total E.coli proteins.Western-blotting analysis showed that the recombinant Bm86-GST was recognized by the rabbit anti-B.microplus positive serum.

Objective:To observe the molecular mechanism of Hemoporfin-mediated photodynamic therapy on vascular endothelial cells.Methods:Human umbilical vein endothelial cells (HUVEC) were cultured in vitro and divided into four groups: PDT group(Hemoporfin concentration: 5 μg/ml, light fluences: 4 J/cm 2), drug group (only Hemporfin: 5 μg/ml), light group(only irradiation by 4J/cm 2 light), and blank control (no drug, no light). The cell viability and proliferation were detected by cck-8 cytotoxicity test and Brdu testafter different treatments as mentioned above. Expression levels of VEGF-A/MAPK/ERK pathway related molecules in the cells before and after photodynamic treatment were detected by real-time quantitative PCR, Western blot and immunofluorescence staining. Results:Compared with the black control group, the cell viability[(0.45±0.08)vs(1.02±0.11), t=12.02, P<0.05] and cell proliferation level [(0.42±0.02)vs(1.00±0.01), t=31.20, P<0.05]were significantly decreased in PDT group.The mRNA expression levels, including Ras[(0.62±0.02)vs(1.05±0.03), t=10.35, P<0.05], c-Raf [(0.72±0.04)vs(1.00±0.05), t=7.35, P<0.05], Mek[(0.73±0.12)vs(1.15±0.04), t=7.74, P<0.05], Erk [(0.56±0.11)vs(1.02±0.03), t=5.56, P<0.05], VEGF-Α [(0.34±0.04)vs(1.02±0.07), t=7.59, P<0.05], and VEGFR2[(0.54±0.05)vs(1.00±0.03), t=5.34, P<0.05] were significantly decreased. The proteinphosphorylation level of c-Raf[(0.44±0.02)vs(1.02±0.05), t=46.7, P<0.05], Mek[(0.72±0.05)vs(1.05±0.04), t=5.35, P<0.05], Erk[(0.62±0.15)vs(1.03±0.03), t=8.58, P<0.05] and the proteinexpression level of VEGF-A[(0.64±0.03)vs(1.03±0.04), t=21.65, P<0.05] were significantly down-regulated in PDT group compared with the black control group. Compared with the blank control group, there were no significant differences expression between the drug group and the light group at cell activity, molecular proliferation level and molecular expressions. Conclusions:HMME-PDT inhibits the activity and proliferation of vascular endothelial cells by inhibiting the expression of the VEGF-A/MAPK/ERK pathway to achieve the purpose of inhibiting vascular hyperplasia and repair.

Objective:To observe the molecular mechanism of Hemoporfin-mediated photodynamic therapy on vascular endothelial cells.Methods:Human umbilical vein endothelial cells (HUVEC) were cultured in vitro and divided into four groups: PDT group(Hemoporfin concentration: 5 μg/ml, light fluences: 4 J/cm 2), drug group (only Hemporfin: 5 μg/ml), light group(only irradiation by 4J/cm 2 light), and blank control (no drug, no light). The cell viability and proliferation were detected by cck-8 cytotoxicity test and Brdu testafter different treatments as mentioned above. Expression levels of VEGF-A/MAPK/ERK pathway related molecules in the cells before and after photodynamic treatment were detected by real-time quantitative PCR, Western blot and immunofluorescence staining. Results:Compared with the black control group, the cell viability[(0.45±0.08)vs(1.02±0.11), t=12.02, P<0.05] and cell proliferation level [(0.42±0.02)vs(1.00±0.01), t=31.20, P<0.05]were significantly decreased in PDT group.The mRNA expression levels, including Ras[(0.62±0.02)vs(1.05±0.03), t=10.35, P<0.05], c-Raf [(0.72±0.04)vs(1.00±0.05), t=7.35, P<0.05], Mek[(0.73±0.12)vs(1.15±0.04), t=7.74, P<0.05], Erk [(0.56±0.11)vs(1.02±0.03), t=5.56, P<0.05], VEGF-Α [(0.34±0.04)vs(1.02±0.07), t=7.59, P<0.05], and VEGFR2[(0.54±0.05)vs(1.00±0.03), t=5.34, P<0.05] were significantly decreased. The proteinphosphorylation level of c-Raf[(0.44±0.02)vs(1.02±0.05), t=46.7, P<0.05], Mek[(0.72±0.05)vs(1.05±0.04), t=5.35, P<0.05], Erk[(0.62±0.15)vs(1.03±0.03), t=8.58, P<0.05] and the proteinexpression level of VEGF-A[(0.64±0.03)vs(1.03±0.04), t=21.65, P<0.05] were significantly down-regulated in PDT group compared with the black control group. Compared with the blank control group, there were no significant differences expression between the drug group and the light group at cell activity, molecular proliferation level and molecular expressions. Conclusions:HMME-PDT inhibits the activity and proliferation of vascular endothelial cells by inhibiting the expression of the VEGF-A/MAPK/ERK pathway to achieve the purpose of inhibiting vascular hyperplasia and repair.

In order to determine the effect of various hosts on feeding performance of Rhipicephalus (Boophilus) microplus, we used 3 mammalian species as hosts, cattle (Qinchuan), sheep (T an), and rabbits (Japanese white rabbit) for infest-ing ticks. Five hundreds of R. microplus larvae were exposed to each animal (3 animals/host species). Tick recoveries were 11.0%, 0.47%, and 5.5% from cattle, sheep, and rabbits, respectively. The averages of tick feeding periods were not significantly different on cattle, sheep, and rabbits, 28.8, 25.3, and 26.7 days, respectively. The average weights of individual engorged female from cattle, sheep, and rabbits were 312.5, 219.1, and 130.2 mg, respectively and those of egg mass weights each to 85.0, 96.6, and 17.8 mg. The highest egg hatching rate was in the ticks from cattle (96.0%), fol-lowed by those from rabbits (83.0%) and sheep (19.2%). These data suggest that rabbits could be as an alternative host to cultivate R. microplus for evaluating vaccines and chemical and biological medicines against the tick in the laboratory, although the biological parameters of ticks were less than those from cattle.
Animals ; Cattle* ; Female ; Humans ; Larva ; Ovum ; Rabbits* ; Rhipicephalus* ; Sheep* ; Ticks ; Vaccines ; Weights and Measures

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.
Animals ; B-Lymphocytes/parasitology ; Cattle ; Cell Line ; Cells/*parasitology ; Cells, Cultured ; Gene Expression Profiling ; Host-Parasite Interactions/*genetics ; Real-Time Polymerase Chain Reaction/*veterinary ; Reproducibility of Results ; Signal Transduction/*genetics ; Theileria annulata/physiology ; Theileriasis/*physiopathology

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.
Animals ; Base Sequence ; China ; DNA, Ribosomal/chemistry/genetics ; Molecular Sequence Data ; Phylogeny ; RNA, Ribosomal, 28S/genetics ; Ruminants ; Sequence Alignment ; Sequence Analysis, DNA/veterinary ; Theileria/*classification/genetics/isolation & purification ; Theileriasis/*parasitology