A corn straw-based diet increases release of inflammatory cytokines in peripheral blood mononuclear cells of dairy cows / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Recent studies have shown that diet can affect the body's immunity. Roughage of dairy cows consists of a variety of plant materials which make different contributions to health. This study investigated the effect of different roughages on the immunity of dairy cows. Serum, peripheral blood mononuclear cells (PBMCs), and milk samples were collected from 20 multiparous mid-lactation cows fed mixed forage (MF)- or corn straw (CS)-based diets. Expression profile analysis was used to detect the differentially expressed genes (DEGs) from PBMCs. The results showed that milk protein in the MF group increased to 3.22 g/100 ml, while that of the CS group milk was 2.96 g/100 ml; by RNA sequencing, it was found that 1615 genes were differentially expressed between the CS group and the MF group among the 24 027 analyzed probes. Gene ontology (GO) and pathway analysis of DEGs suggested that these genes (especially genes coding cytokines, chemokine and its receptors) are involved in the immune response. Results were confirmed at the protein level via detecting the levels of interleukin-2 (IL-2), IL-6, IL-10, IL-12, leptin (LEP), interferon-γ (IFN-γ), transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) in peripheral blood by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay analysis. Our data supported the conclusions that the protein content in milk of the MF group was higher than that of the CS group, the CS-based diets induced more release of cytokines than the MF-based diets in dairy cows' PBMCs, and milk protein content may be affected by cytokines.

Comparison of plaque-forming characteristics and morphogenetic changes of Newcastle disease virus isolated from chickens and geese on host cells / 病毒学报

The plaque-forming characteristics of Newcastle disease viruses of chickens and geese source were compared on various cells. The result showed that there were obvious differences of plaque formation between F48E9 and NA-1 on Vero cells, chicken embryo fibroblast cells (CEF) and goose embryo fibroblast cells (GEF). The plaque-forming ability of NA-1 was higher than F48E9 on GEF, but lower than F48E9 on CEF. On Vero cells, the plaque-forming ability of NA-1 was slightly stronger than F48E9. It demonstrated that the plaque-forming characteristics were consistent with host tropism of virus. The morphogenesis of F48E9 and NA-1 on Vero cells was observed with transmission electron microscope. There were different replication processes between F48E9 and NA-1 on cells at different stages. NA-1 had stronger adaptability to host than F48E9 according to budding processes and envelope integrity.

Comparison of effectiveness of whole viral, N and N199 proteins by ELISA for the rapid diagnosis of severe acute respiratory syndrome coronavirus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although severe acute respiratory syndrome (SARS) has been controlled, the subsequently emerging sporadic cases in 2004 emphasize the necessity of developing a rapid diagnostic method, which would be of great help in clinical diagnosis and also wild host screening. This study aims to establish an effective and rapid serological tool for the diagnosis of SARS-CoV by comparison among whole viral, N and N199 proteins by ELISA.</p><p><b>METHODS</b>SARS-CoV N and N199 (a truncated nucleocapsid gene) genes were cloned, expressed, identified by Western blotting, and applied in screening of human and swine samples. Sera of SARS convalescent-phase patients, normal human sera, sera of patients with other respiratory diseases, and swine sera were screened by ELISA, with whole SARS-CoV F69, N and N199 proteins as antigens.</p><p><b>RESULTS</b>The sensitivity and specificity of N and N199 proteins in human sera diagnosis were approximate (P = 0.743), which was higher than whole viral protein but the difference was not significant (P = 0.234). The N199 protein proved to be more specific in swine sera screening than whole viral and N protein (P < 0.001).</p><p><b>CONCLUSION</b>N199 protein is feasible in both clinical diagnosis and SARS-CoV reservoir screening.</p>

Salmonella choleraesuis C500 delivering DNA immunization against classical swine fever virus / 生物工程学报

Classical Swine Fever Virus (CSFV) E2 protein eukaryotic expression plasmid pVAXE2 was constructed. The plasmid pVAXE2 was transformed into Salmonella choleraesuis C500 (S. C500) attenuated vaccine strain by electroporation to generate Salmonella choleraesuis engineering strain S. C500/pVAXE2. The characterization of S. C500/pVAXE2 in morphology, growth, biochemistry and serology indicated that it retained the same properties as its original strain S. C500 with exception of kanamycin resistance originated from the plasmid pVAXE2. The plasmid stable in the bacteria after 15 passages. Kunming mice and rabbits were vaccinated three times at two weeks interval with S. C500/pVAXE2 in oral and intramuscular routes at the dosage of 1 x 10(8) CFU for mice and 2 x 10(9) CFU for rabbits each time. The specific antibody response against CSFV and Salmonella choleraesuis was detected by ELISA. Two weeks after the third boost the immunized rabbits were challenged with 20 ID50 of hog cholera lapinized virus (HCLV), followed by a virulent strain of Salmonella choleraesuis two week later than HCLV challenge. The results showed that all immunized mice and rabbits produced significant antibodies against CSFV and Salmonella choleraesuis, and the immunized rabbits demonstrated the effective protection against the challenge of HCLV and virulent Salmonella choleraesuis. These results indicated the potential of developing multiplex swine DNA vaccine by using this bacteria as the vector.

Construction of recombinant fowlpox virus coexpressing gp120 of Chinese HIV-1 strain and IL-18 and its immunogenicity in mice / 生物工程学报

To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.

Effects of oxLDL and simvastatin on PKC activity and level of cytosolic free Ca 2+ in cultured human umbilical vein endothelial cells / 第二军医大学学报

Objective: To investigate the effects of oxLDL and HMG-CoA reductase inhibitor simvastatin on PKC activity, and level of cytosol ic free Ca 2+ in cultured human umbilical vein endothelial cells. Methods: Th e activity of PKC was determined by its ability to transfer phosphate from 32P-ATP to lysine-rich histone and level of cytosolic free calcium［Ca2+ ］i was measured by flow cytometric analysis loading with the Ca2+ dye F luo-3/Am. Results: oxLDL increased PKC total activity in a dose-de pendent manner and peaked after 12 min, then decreased slowly and maintained for at least 30 min, while oxLDL induced biphasic ［Ca2+］i responses includ ing the rapid initial transient phase and the sustained phase. Removal of extrac ellular Ca2+ did not inhibit the rapid transient phase, but abolished the sustained phase. When simvastatin was added, the activity of PKC wasmarkedly dec reased with no impairment to the initial peak response, but significantly reduce d the sustained phase. Conclusion: oxLDL can induced dynamic changes of signal transduction of PKC and level of cytosolic free Ca2+ in HUVEC, these 2 events are closely linked. The change of rapid initial transient phase i s the result of mobilization of Ca2+ from intracellular pool and the chang e of sustained phase is from the influx of extracellular Ca2+. The inhibit ion of PKC activity induced by simvastatin may contribute to the changes of ［Ca 2+］i.

Effects of oxLDL and simvastatin on PKC activity and level of cytosolic free Ca 2+ in cultured human umbilical vein endothelial cells / 第二军医大学学报

Objective: To investigate the effects of oxLDL and HMG-CoA reductase inhibitor simvastatin on PKC activity, and level of cytosol ic free Ca 2+ in cultured human umbilical vein endothelial cells. Methods: Th e activity of PKC was determined by its ability to transfer phosphate from 32P-ATP to lysine-rich histone and level of cytosolic free calcium［Ca2+ ］i was measured by flow cytometric analysis loading with the Ca2+ dye F luo-3/Am. Results: oxLDL increased PKC total activity in a dose-de pendent manner and peaked after 12 min, then decreased slowly and maintained for at least 30 min, while oxLDL induced biphasic ［Ca2+］i responses includ ing the rapid initial transient phase and the sustained phase. Removal of extrac ellular Ca2+ did not inhibit the rapid transient phase, but abolished the sustained phase. When simvastatin was added, the activity of PKC wasmarkedly dec reased with no impairment to the initial peak response, but significantly reduce d the sustained phase. Conclusion: oxLDL can induced dynamic changes of signal transduction of PKC and level of cytosolic free Ca2+ in HUVEC, these 2 events are closely linked. The change of rapid initial transient phase i s the result of mobilization of Ca2+ from intracellular pool and the chang e of sustained phase is from the influx of extracellular Ca2+. The inhibit ion of PKC activity induced by simvastatin may contribute to the changes of ［Ca 2+］i.