The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung (FBL) cells.Bovine Herpesvirus 1(BHV-1),which is a viral pathogen of cattle,can infect FBL cells and induce cytopathic effects.Real-time PCR assays showed that BHV- 1 's infection could repress the basal or inducible transcription of bISG15 in FBL cells.It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis.Our previous work showed that bIRF-3 was the key factor in the stimulation of bISG 15 in FBL cells,so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed.The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3.Taken together,our work suggested that BHV-I had some molecular mechanism to resist the cellular bISG15'santiviral functions.

Objective To develop prototype foamy virus (PFV) detection method by loop-mediated isothermal amplification. Methods Three pairs of primers targeting core region of PFV integrase were designed in this study and Bst DNA polymerase was used to amplify target sequence at 63℃. The system was established with all the conditions optimized. Results The method was established with the plasmid containing target sequence as the template. This method could specifically detect PFV infectious clone, no crossreaction was observed with human immunodeficieney virus infectious clone, bovine immunodefieiency virus infectious clone and bovine foamy virus infectious clone as templates. The detection capability of this system was 50 copy, one order more sensitive than PCR. The amplification could be finished in 15 min and human genomic DNA did not adversely affect the amplification efficiency. Conclusion The PFV detection method by loop-mediated isothermal amplification was established and it had potential usefulness in PFV detection.

ISG15, the first ubiquitin-like molecule identified two decades ago, is encoded by interferon stimulated gene 15 ( ISG 15), where its robust expression can be induced by viral infections or interferon treatments. ISG 15 conjugate to other proteins as the ubiquitin and was found to be involved in innate immune response. However, the functions of ISG15 modification remained unclear. We cloned bovine ISG15(bisg15) into a prokaryotic expression vector pET28a( + ) with a His-tag to generate a soluble form of bISG15 fusion protein, and purified with Ni-NTA Sepharose chromatography. The purified protein was concentrated and used to immune Balb/c mice to raise the antiserum, which could specifically recognize bISG15 expressed in eukaryotic cells by Western blot analysis. The concentrated bISG15 protein and its antiserum were then used to establish an in vitro bISG15 modification system. Our studies have demonstrated that cellular proteins could be conjugated to bISG15 with this system.

Objective:To analyze the relationship of lipoprotein associated phospholipase A 2 (Lp-PLA2) and severity of coronary atherosclerosis, and evaluate the effects of rosuvastatin at different doses on the concentration of plasma Lp -PLA2.Methods: Totally 152 cases of patients with suspected coronary heart disease were treated with coronary angiography .According to the results of angiogra-phy, the patients were divided into the coronary heart disease group ( n=117 ) and the normal control group ( without coronary heart disease,n=35).Gensini integral scale was performed and referring to the number of diseased coronary arteries , the degree of coronary atherosclerosis was evaluated .The concentration of serum Lp-PLA2 was detected and the relationship of Lp-PLA2 and the severity of coronary plaque was evaluated .Meanwhile , the patients with coronary heart disease were divided into 2 groups and orally treated with rosuvastatin respectively at the routine dose (10 mg· d-1 ) and the loading dose (20 mg· d-1 ).The changes of the plasma concentra-tion of Lp-PLA2 before the treatment, in the 2nd, 4th,8th and 12th week after the medication were measured and the effect of atorvastatin at different doses on the plasma concentration of Lp-PLA2 was summarized .Results: The plasma Lp-PLA level in the control group was (22.22 ±1.75) μmol· ml-1, while that in the coronary heart disease group was (29.03 ±3.99) μmol· ml-1(P<0.05).The differences in Lp-PLA2 levels between the groups with different Gensini scores of coronary heart disease were statistically significant ( P<0.05).The higher scores were, the higher Lp-PLA2 levels were.The results of multivariate analysis showed that the severity of cor-onary atherosclerosis was significant and positive correlated with Lp-PLA2 level (OR=1.613,P<0.05).In the 2nd, 4th, 8th and 12th week after the medication , Lp-PLA2 levels in the loading dose group were significantly lower than those in the routine dose group ( P<0.05).In the 2nd, 4th, 8th and 12th week after the medication, the degree scores of coronary artery stenosis in the loading dose group were reduced.The decreasing range was significantly greater than that in the routine dose group (P<0.05).The incidence of adverse cardiovascular events in the routine dose group (27.12%) was significantly higher than that in the loading dose group (6.90%) ( P<0.05).The incidence of adverse drug reactions in the routine dose group was 11.86%, while that in the loading dose group was 18.97%(P>0.05).Conclusion:Lp-PLA2 is correlated with the severity of coronary plaque .High dose of rosuvastatin can reduce plasma Lp-PLA2 concentration in the patients .

The literature study and survey revealed existed problems and factors such as the difficulties to maintain quality of traditional Chinese medicine (TCM), sustainable development, homogenization and indeterminacy of clinical orientation diagnosis, lack of favorable evidence-based medicine and pharmacoeconomics data to support, lack of ev-idence of safety evaluation, which restrict the development of TCM enterprises. Based on problems and factors men-tioned above, recommendations were made on the healthy development of TCM enterprises, which include guarantee of the quality of TCM herbs and the sustainable development of its resource; conduct systematic evidence-based pharmacoeconomic research, identify the clinical orientation; carry out safety assessment systematically, improve the specification; improve clinical effectiveness-oriented quality standards.

Objective To construct the competitiveness evaluation index system of listed TCM pharmaceutical companies and provide efficient technology and methods for the evaluation in related field.Methods Index base was founded by the means of the literature research method at first. Then 20 experts were asked to score all these indexes according to the importance of each index. Dimensionality and index base of competitiveness evaluation index system of listed TCM pharmaceutical companies were screened. With two rounds of questionnaires, the evaluation index system was constructed finally.Results The positive coefficients of two rounds of expert consultation were 95% and 100%;the cooperative coefficients were 0.659 and 0.639;the authoritative coefficient was 0.713 2. Evaluation system consisted of 5 first grade indexes and 26 second grade indexes.Conclusion The positive coefficients and the authoritative coefficients are both high enough through Delphi method. Opinions of all the experts in the two round of expert consultation tend to be uniform, which reveals that the evaluation index system of listed TCM pharmaceutical companies is relatively scientific.

Objective To study the chemical constituents from the exocarp of Juglans mandshurica.Methods The compounds were isolated by Diaion HP-20,Sephadex LH-20,MCI CHP-20,and silica gel column chromatography.Their structures were identified by physicochemical properties and spectroscopic analysis.Results Twelve compounds were elucidated as pinostrobin (Ⅰ),quercetin (Ⅱ),quercetin-3-O-?-D-glucoside (Ⅲ),chrysophanol (Ⅳ),gallic acid (Ⅴ),4-hydroxyl-cinnamic acid methyl ester (Ⅵ),vanillin (Ⅶ),caffeic acid (Ⅷ),4-hydroxylcinnamic acid (Ⅸ),?-sitosterol (Ⅹ),daucosterol (Ⅺ),and 5-hydroxyl-1,4-naphthoquinone (ⅩⅡ).Conclusion The compounds Ⅰ,Ⅳ,and Ⅵ—Ⅸ are isolated from J.mandshurica for the first time.

Virus infection or interferon can stimulate robust expression of the protein ISG15 that is encoded by interferon stimulated gene 15, which was the first unbiquitin-like molecule identified two decades ago. While ubiquitin and its many important functions have been well established, the functions of ISG15 and its post-translational conjugation are still largely unknown . Recently, some specific enzymes have been identified to be involved in the ISG15 modification system, suggests that ISG15 and its modification system play important roles in the innate immune response and regulation of interferon signaling. The history of ISG15 discovery and its biochemical characterization were briefly introduced. Then such topics as the ISG15 gene expression and the ISG15 modification will be focued on, and finally summarize new findings which have implications for ISG15 and its modification system in immunology and interferon signal transduction were summarized.

In order to improve the accuracy for quantitating the bovine foamy virus(BFV)in vitro,we developed a baby hamster kidney cell(BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter(LTR,from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone,designated BFVL,was selected from ten neomycin-resistant clones.BFVL showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV infection.Although the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection(MOI)of BFV and the activated ratio of luciferase expression in BFVL. Moreover,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay. BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.