Comparison of the antigenicity of African swine fever virus p35 protein as diagnostic antigen / 生物工程学报

In order to screen African swine fever virus (ASFV) diagnostic antigen with the best enzyme linked immunosorbent assay (ELISA) reactivity. By establishing the ELISA method, the diagnostic antigen of ASFV p30 protein expressed by baculovirus-insect cell expression system as reference, we explored the antigenic properties and diagnostic potential of ASFV p35 protein expressed by prokaryotic expression system as a diagnostic antigen. The results of Western blotting and immunofluorescence show that the molecular weight of the recombinant p35 protein and p30 protein obtained was 40 kDa and 30 kDa, respectively, and these two proteins had good immuno-reactivity with ASFV positive serum. Recombinant p30 and p35 proteins were used as diagnostic antigens to establish ELISA, and the sensitivity and repeatability of these methods were tested. The results show that although the detection sensitivity of the p30-ELISA established in this study was higher than that of the p35-ELISA, the sensitivity of p35-ELISA was 95.8%, and variations in intra- and inter-assay repeatability of the two methods were less than 10%. The coincidence rate between the p35-ELISA and the imported kit was 97.2%. Results show that p35-ELISA was sensitive and stable, and could detect specific antibodies against ASFV.

Comparson of the immunogenicity of genotypeⅠJapanese encephalitis virus subunit vaccine candidate antigens / 生物工程学报

To screen the best genotypeⅠJapanese encephalitis virus subunit vaccine candidate antigens, the prMEIII gene, the polytope gene and the prMEIII-polytope fusion gene of the GenotypeⅠJapanese encephalitis virus GS strain were cloned into prokaryotic expression vector pET-30a. The recombinant proteins were obtained after the induction and purification. The prepared recombinant proteins were immunized to mice, and the immunogenicity of the subunit vaccine candidate antigens was evaluated through monitoring the humoral immune response by ELISA, detecting the neutralizing antibody titer by plaque reduction neutralization test, and testing the cell-mediated immune response by lymphocyte proliferation assay and cytokine profiling. The recombinant proteins with the molecular weights of 35 (prMEIII), 28 (polytope antigen) and 57 kDa (prMEIII-polytope) induced strong humoral and cellular immune responses in mice. Compared with prMEIII-polytope and polytope proteins, the prMEIII protein induced a significant expression of IL-2 and IFN-γ (P0.05). The study suggests that the prMEIII protein can be used for the development of the Japanese encephalitis virus subunit vaccine.

Antipyretic effects of Paracetamol Tablets, Compound Paracetamol and Amantadine Hydrochloride Tablets, Compound Dextromethorphan Hydrobromide Tablets, and Chaiqin Qingning capsules on rat model with LPS and dry yeast induced fever / 药物评价研究

Objective To study the antipyretic effect of Paracetamol Tablets,Compound Paracetamol and Amantadine Hydrochloride Tablets,Compound Dextromethorphan Hydrobromide Tablets,and Chaiqin Qingning Capsules on the fever model induced by LPS and dry yeast in rats.Methods Fever was induced by ip injecting LPS (100 μg/kg) or sc injecting dry yeast (20％) in rats.We observed the changes of temperature of the rats after administration of Paracetamol Tablets,Compound Paracetamol and Amantadine Hydrochloride Tablets,Compound Dextromethorphan Hydrobromide Tablets (the acetaminophen contents were 205.67,102.83,and 51.42 mg/kg)and Chaiqin Qingning Capsules (1110.60,555.30,and 277.65 mg/kg).Maximum temperature rise height (△T) and temperature response index (TRI) were calculated,and the curve of average rise in temperature was drawn.Results Each dose group of Paracetamol Tablets,Compound Paracetamol and Amantadine Hydrochloride Tablets,Compound Dextromethorphan Hydrobromide Tablets,and Chaiqin Qingning Capsules had obvious antipyretic effect on the fever model induced by LPS and dry yeast in rats,and there was a certain dose-effect relationship.Conclusion Paracetamol Tablets,Compound Paracetamol and Amantadine Hydrochloride Tablets,Compound Dextromethorphan Hydrobromide Tablets,and Chaiqin Qingning Capsules has certain antipyretic effect on LPS and dry yeast fever model in rats,and on the whole,the Western medicine acts rapid but continue for a short time,while the traditional Chinese medicine acts slow but continues for a long time.

Morphology of the intervertebral disc in high fat combined with streptozotocin-induced type Ⅱ diabetic rats / 中国组织工程研究

BACKGROUND:Rat models of diabetes mellitus type 2 (T2DM) are usually induced by the combination of high-fat diet and low-dose streptozotocin, but their effects on the intervertebral disc have not yet been reported. Endplate sclerosis is an important factor contributing to intervertebral disc degeneration. OBJECTIVE:To evaluate whether the rat T2DM model induced by high-fat diet combined with low-dose streptozotocin is suitable for the study of T2DM related intervertebral disc degeneration. METHODS:Thirty-two 3-month-old female Sprague-Dawley rats were divided into four groups (n=8 per group), including sham, bilateral variectomy, DM, and bilateral variectomy plus DM groups, followed by subjected to bilateral ovariectomy and/or high-fat diet combined with low-dose streptozotocin, respectively. The blood glucose level, body mass and glucose tolerance were determined. The bone mineral density of the lumbar spine was measured after 8-week streptozotocin treatment. The L5-6 segments were removed and cut through midst sagittal plane after decalcification, and then underwent Von Gieson staining and histological degeneration scoring, and the disc height and endplate thickness were measured. RESULTS AND CONCLUSION:Fasting blood glucose and random blood glucose levels in the DM and bilateral variectomy plus DM groups were significantly higher than those in the other two groups, and the insulin sensitivity in the DM and bilateral variectomy plus DM groups were significantly lower than that in the other groups (P<0.05). L4-6 vertebral bone mineral density in the bilateral variectomy group was significantly lower than that in the sham group (P<0.05);L5-6 vertebral bone mineral density in the DM and bilateral variectomy plus DM groups was significantly lower than that in the sham group (P<0.05). L5-6 vertebral histological scores in the DM and bilateral variectomy plus DM groups were significantly higher than those in the other groups (P<0.05). Similar with the bilateral variectomy group, there were chondrometaplasia and mucoid degeneration of nucleus pulposus cells in the bilateral variectomy plus DM group, and the histological scores were significantly higher than those in the sham and DM groups (P<0.05). Compared with the sham group, the intervertebral disc height in the bilateral variectomy and bilateral variectomy plus DM groups was significantly decreased (P<0.05). While, there was no significant difference in the endplate thickness among groups. These results indicate the combination of high-fat diet and low-dose streptozotocin-induced rat T2DM models possess diabetic characteristics, but the rat intervertebral disc tissues show no significant differences from the normal ones;therefore, this model may be unsuitable for the study on T2DM-related intervertebral disc degeneration.

Analysis a family outbreak of epidemic hemorrhagic fever in Yingkou City of Liaoning Province / 中华地方病学杂志

Objective To investigate and analyze the epidemiological characteristics of epidemic hemorrhagic fever (EHF),and explore the causes and possible routes of transmission in Yingkou City for proposing prevention and control measures.Methods A case restropective investigation was done to all outbreak cases of EHF in Yingkou City,relevant epidemiological investigation,clinical examination and laboratory test were done.Search of case and new case were carried out in the population.Density of rats (night clip) and rate of rat infection (Hanta virus antigenemia) in the region of the epidemic area of EHF were investigated.Blood samples were collected,antibody of EHF was detected with enzyme linked immunosorbent assay (ELISA).The rat lung was detected virus antigen using immunofluorescence.EHF diagnosis was based on Diagnostic Criteria for Epidemic Hemorrhagic Fever (WS 278-2008).Results The results of the case study showed that a family of three was infected with the same disease in one month.It was a family cluster of EHF.Of the three cases,one case was Seoul virus (SEO) positive and showed infection for rattus type.Totally 54 human serum samples from the case search in key groups were tested.Two cases were seropositive with IgG,through epidemiological investigation,all people were healthy.On physical examination they had no symptoms such as viral infection with fever,fatigue,symptoms of three red and pains of EHF.Two cases were determined latent infection.The patient's living and health conditions were very poor.The floor of indoor was soil,all of the things were placed in chaos in this family,there were many signs of rats and rat holes in the yard,and there were several rat holes around the wellhead.Rat density surveillance showed that cloth clip 108 times,effective clamp 105 times,4 rats were captured,and the density of rats was 3.81％.The density of rats exceeded the national standard of no more than 1％ of the residential area.All the rats were Rattus norvegicus.Detection of lung antigen was negative in rats by immunofluorescence.Conclusions This event is a family cluster of EHF caused by Rattus norvegicus.Rodent prevention and control,improving family conditions and environmental management are essential measures for blocking of the disease transmission and infection controlling.

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

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.

The effects of autologous blood transfusion and acute normovolemic hemodilution inflammatory response in patients with spinal surgery / 国际检验医学杂志

Objective To investigate the effects of autologous blood transfusion and acute normovolemic hemodilution inflamma‐tory response in patients with spinal surgery .Methods 60 patients with spinal surgery ,were randomly divided into the control group ,autologous blood transfusion group ,acute normovolemic hemodilution group ,20 cases in each group .The control group in in‐traoperative bleeding time and blood input variants of inventory .Autologous blood transfusion group was used in combination with intraoperative autologous blood recovery unit ,lose banked blood hemoglobin was low .Acute normovolemic hemodilution group via peripheral vein input must first crystal liquid or gel liquid ,then through internal jugular vein slowly pulled out the body′s blood into the special bags containing anticoagulant ,swing machine through blood kept shaking ,input when appropriate .Preoperative(T1) , After surgery 2 h(T2) ,6 h(T3) ,12 h(T4) and 24 h(T5) blood at each time point 5 mL ,detect the WBC ,IL‐6 ,TNF‐α.Records with and without postoperative complications .Results WBC in autologous blood transfusion group and acute normovolemic he‐modilution group were higher than that of preoperative ,difference was statistically significant(P<0 .05);In T2 -T5 ,serum IL‐6 , TNF‐a and the WBC concentration compared with the basis of their respective value(T1) increased significantly ,but were signifi‐cantly lower than control group ,the difference was statistically significant(P< 0 .05) .Postoperative follow‐up of the autologous blood transfusion group and acute normovolemic hemodilution group and no complications .Conclusion The autologous blood trans‐fusion group and acute normovolemic hemodilution group could effectively reduce the intraoperative and postoperative systemic in‐flammatory response ,which obviously save blood resources .

Simvastatin effects on the expressions of specific osteogenic genes in bone marrow stromal stem cells / 中国组织工程研究

BACKGROUND:Previous studies have demonstrated that simvastatin that can promote osteogenic differentiation of bone marrow stromal stem cel s in vitro, is likely to be a new osteogenic drug. While it is stil unknown whether there is time-dependent stimulation of simvastatin on the expressions of bone morphogenetic protein 2 and col agen type I. OBJECTIVE:To investigate the expressions of bone morphogenetic protein 2 and col agen type I in rat bone marrow stromal stem cel s in vitro stimulated by simvastatin at different time points. METHODS:Passage 1 bone marrow stromal cel s were divided into control and simvastatin group, fol owed by cultured in osteogenic differetiation medium with or uithout 10-7 mol/L simvastatin. After 7-day intervention, expression of alkaline phosphatase was detected in passage 3 cel s. Passage 4 cel s were divided and cultured as described above, and afterwards, RNA and proteins were extracted at 12 and 36 hours to detect the expressions of bone morphogenetic protein 2 and col agen type I using real-time PCR and western blot assay. RESULTS AND CONCLUSION:Both two groups could express alkaline phosphatase, while the rate of positive cel s significantly increased in the simvastatin group compared with the control group (P<0.05);at 12 and 36 hours after intervention, mRNA expressions of bone morphogenetic protein 2 and col agen type I in the simvastatin group were significantly higher than those in the control group (P<0.05). Besides, western blot assay showed:at both 12 and 36 hours, simvastatin significantly enhanced the expression of bone morphometric protein 2, while the expression of col agen type I significantly increased at 12 hours (P<0.05), but not at 36 hours. In conclusion, simvastatin can promote the expressions of bone morphometric protein 2 and col agen type I in rat bone marrow stromal cel s, with more favorable outcomes after 12-hour treatment.

Characterization of follistatin-related protein from the hard tick Haemaphysalis longicornis / 生物工程学报

We designed the primers based on the sequence of the follistatin-related protein from Haemaphysalis longicornis Okayama strain accessed in GenBank. We cloned a gene encoding follistatin-related protein by RT-PCR, and the length cDNA is 814 bp, encoding a deduced protein of 289 amino acids. The alignment with the sequence of follistatin-related protein from the H. longicornis Okayama strain showed that the percent of nucleotide sequence and amino acid sequence is 97.8% and 99%, respectively. The expected size of GST-fused recombinant protein was 57 kD. We purified the recombinant protein through MagneGST protein purification system. Western blotting revealed that stronger reaction happened with the antiserum against eggs, but not clear with antisera against other developmental stages.

A case-control study of intravenous drip and local artery infusion in treatment of severe acute pancreatitis / 医学研究生学报

Objectives:To explore a new way in treatment of severe acute pancreatitis(SAP). Methods:From 1995 to 2001,23 patients with SAP proved by clinic and CT were treated, and compared the local artery infusion with intravenous drip on effect,mortality and time of hospitalization. Results:The mortality and time of hospitalization in 12 artery infusion and 11 intravenous drip were (14.4?3.1),(29.3?6.1) days of hospitalization and 8.33%,27.27%(mortality),respectively. Conclusions:The mortality and the time of hospitalization can be reduced by local artery infusion of medicine.

Preparation of ~(99)Tc~m-MAG_3-K237 and its pharmacokinetics and distribution in health animals / 第三军医大学学报

95%) and perfect stability,which could be quickly cleared from blood and excreted though liver and kidney.

Construction of cDNA Expression Library of Unfed Female Haemaphysalis longicornis and Immuno-Screening / 中国寄生虫学与寄生虫病杂志

Objective To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes.Methods Total RNA was isolated from unfed female ticks,mRNA was purified and a library of oligo(dT)-primed cDNA with added directional EcoR Ⅰ/Hind Ⅲ linkers was constructed from the purified mRNA.The constructed cDNA was ligated to the EcoRⅠ/HindⅢ arms of the ?SCREEN vector.Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8.Recombinant plasmids that were subcloned to E.coli BM25.8 were isolated and transformed into E.coli JM109.Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing.Rusults The recombinant phage DNA was packaged by using phage-marker packaging extracts,resulting in a primary cDNA library with a size of 1.8?106 pfu.Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4?109 pfu/ml.Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library.Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H.longicornis tropomyosin mRNA,Rhipicephalus annulatus unknown larval protein mRNA,chromosome 2R of Drosophila melanogaster,mitochondrial DNA of H.flava,clones HqL09 unkown mRNA and Hq05 mRNA of H.qinghaiensis,and myosin alkali light chain protein mRNA.Conclusion The cDNA expression library from unfed female H.longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.