Antiviral Effect of Glycyrrhizin on Porcine Respiratory Coronavirus (PRCV)

利用ST细胞研究了甘草酸对猪呼吸道冠状病毒(PRCV)体外复制过程中的抑制作用。通过RT-PCR和Western blot检测发现甘草酸能有效抑制PRCV的体外复制而对ST细胞不引起明显细胞毒作用。抑制效果与甘草酸的剂量成正比关系。PRCV病毒核衣壳蛋白的表达无论在mRNA水平还是在蛋白质水平均随着甘草酸添加量的增加而降低,提示甘草酸是一种有效的抗PRCV药物。The inhibitory effect of glycyrrhizin (GL) on porcine respiratory coronavirus (PRCV) replication was investigated in vitro in ST cells using RT-PCR and Western blot techniques.GL effectively suppressed PRCV replication without causing apparent cell cytotoxicity in ST cells.The inhibitory effect of GL on PRCV was in a dose-dependent manner.The expression levels of both mRNA and protein of the PRCV nucleocapsid protein in ST cells were impeded by the increasing addition of GL,suggesting it is a potential effective antiviral component against PRCV in vitro.This project was supported by a grant from the Bureauof Science and Technology of Fujian Province (2003N083);; agrant from the Bureau of Science and Technology of Xiamen City(3502Z20031054

Study on the cloning,expression and function of chicken interleukin-2

以RT-PCR法从鸡脾脏细胞中扩增出鸡白细胞介素2编码基因,通过双酶切、连接步骤克隆进原核表达载体pQE30及pGEX-4T-3,构建了鸡白细胞介素2基因的重组原核表达质粒pQE30-chIL-2及pGEX-chIL-2.重组质粒转化大肠杆菌JM109后经诱导表达,分别得到两种表达产物.以SDS-PAGE和Westernblot检测确认表达产物为带6组氨酸标签及GST蛋白的融合鸡白细胞介素2,分子量分别为16 kDa及39.5 kDa.表达蛋白与抗鸡白细胞介素2单抗均有良好的免疫结合性,进一步证实为鸡白细胞介素2.表达产物经过纯化复性后,具有明显促进鸡脾淋巴细胞增殖的作用.The gene encoding chicken interleukin-2(chIL-2) was amplified from chicken spleen cells by RT-PCR.Following double digestion by restriction endonucleases and ligation,chIL-2 was cloned into prokaryotic expression vectors of pQE30 and pGEX-4T-3 to construct recombinants of pQE30-chIL-2 and pGEX-chIL-2,which were consequently transformed into E.coli JM109.Two kinds of recombinant protein were obtained with IPTG induction.The molecular weights of the expressed proteins were 16?kDa and 39.5?kDa on SDS-PAGE and they were regarded as 6His and GST labeled protein(respectively) as checked by Western blot.Both proteins revealed specific reaction to anti-chIL-2 mAb,further confirming that recombinant proteins are chicken interleukin-2.After purification and(renaturation),the 6His-chIL-2 showed biological activity in stimulating chicken lymphocyte proliferation.福建省科技攻关计划重点项目(2003N083);; 厦门市科技攻关计划重点项目(3502Z20031054);; 集美大学科研启动基金(F03003

Characterization and application of nucleocapsid protein of porcine reproductive and respiratory syndrome virus expressed in E.coli

从已构建的PRRSV ORF7重组质粒pUCm-T-ORF7中用PCR扩增ORF7基因亚克隆至表达载体pGEX-4T-3,构建重组表达质粒pGEX-4T-3-ORF7并转化大肠杆菌。经SDS-PAGE及Western blotting鉴定,成功表达了谷胱苷肽转移酶(GST)融合的核衣壳蛋白(N),重组N蛋白表达量约为菌体总蛋白的35%,主要以可溶的形式存在,且能形成同源二聚体。重组N蛋白经谷胱苷肽凝胶(glutathione sepharose 4B)亲和层析后得到高度纯化,并将该蛋白作为抗原建立了间接ELISA检测方法。利用该方法对某猪场76份猪血清进行检测并将结果与IDEXX公司ELISA试剂盒检测结果作比较,2种方法的总符合率达93.4%,检测结果之间差异不显著(P>0.05)。结果表明大肠杆菌表达的重组GST融合N蛋白具有良好的抗原性,因而有望利用该重组蛋白开发为试剂盒应用于临床PRRSV抗体的检测。Porcine reproductive and respiratory syndrome virus(PRRSV) FJ-1 was a newly identified virus isolate in Fujian province.The ORF7 gene of FJ-1 was amplified by RT-PCR and cloned into vector pUCm-T,then subcloned into expression vector of pGEX-4T-3.The recombinant GST-tagged nucleocapsid protein(rN) was expressed in E.coli and the molecular weight was approximately 38 000 as identified by SDS-PAGE and Western blotting.Expression level of the rN protein was approximately 35% of the total bacterial protein and mostly soluble.The rN protein was purified to homogeneity using GST affinity chromatography.Analysis of the rN protein under nonreducing conditions revealed that similar to native protein,the rN protein also possesses homo-dimerization property.An indirect enzyme-linked immunosorbent assay(ELISA) for detecting PRRSV antibody was developed using the purified rN protein as antigen.76 serum samples were detected by the method and the result was compared with that using IDEXX PRRS HerdChek ELISA kit.An identity of 93.4% was revealed between the two ELISA kits and no significant difference(P>0.05) was detected.The data indicate the rN protein has the potential usefulness for detection of the PRRSV antibodies.福建省科技攻关计划重点资助项目(2003N083);; 厦门市科技攻关计划重点资助项目(3502Z20031054

我国传染病微生态的预防发展趋势

我国传染病微生态的预防发展趋势范明远，向近敏，苏文金，郝维善，何明清，王世荣传染病的预防战略，传统观念仍局限于疫苗和药物两大主题。从微生态学（细胞或分子生态学）角度来考虑传染病的预防战略，是当今国际上生物医学的发展趋势。微生态学是边缘学科之一，根据其..

Purification and Properties of Soluble Hydrogenase from Photosynthetic Bacterium Chromatium vinosum

联系人: 龙敏南, 男, 1965 年1 月生, 博士, 副教授 T el : 0592-2186392 E-mail : longmn@ jin gxian. xmu. edu. cn光合细菌 Chromatium vinosum可溶性氢酶经 4次柱层析 (Whatman DE- 52 ,TSK- DEAE,Ultragel Ac A- 44 ,Mono Q)分离提纯后被纯化 630倍 ,得率为 33.5% .可溶性氢酶催化放氢比活性为 7.5μmol· min-1· mg-1.SDS- PAGE结果显示 ,可溶性氢酶由 52 k D和 2 1 .5k D两个亚基组成 .大亚基 N端氨基酸序列为 :SRTITIEPVTRXEGHAR;小亚基 N端氨基酸序列为 :STQPKIT-VATXLDG.氧化态可溶性氢酶在 54K时产生了典型的 Ni( )电子顺磁共振 (EPR)信号 (gxyz=2 .37、2 .1 6、2 .0 1 6和 gxyz=2 .30、2 .2 3、2 .0 1 6) .研究结果表明 ,C.vinosum可溶性氢酶是一种新的催化放氢的 Ni Fe-氢酶 .教育部重点科技项目!资助 (No.990 70

Studies on Detection of Porcine reproductive and respiratory syndrome virus in Tissue Sample by Reverse Transcription Polymerase Chain Reaction

根据猪繁殖和呼吸综合征病毒(PRRSV)已发表的核苷酸序列 ,在其核衣壳蛋白基因 (ORF7)保守区设计了一对跨幅约 380bp的特异性引物N1/N2 ,对已知PRRSVJK10 0 1毒株和PRRS临床病料进行RT PCR扩增 ,均扩增出约 380bp的特异片段。用此方法检测 5份疑似PRRS病例肺组织病料 ,结果均为阳性 ,对 2个阳性样品扩增产物进行克隆、测序 ,全长均为 372bp ,与PRRSVVR 2 332毒株ORF7同源性为 95%,证实为PRRSV的核衣壳蛋白基因。结果表明 ,建立的RT PCR检测PRRS临床组织病料特异、快速 ,可用于PRRS临床病料检测According to the published sequence of PRRSV genome, a pair of specific primers (N1/N2) was designed based on the gene encoding the N protein of PRRSV. The region between the primers was about 380 bp. A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of PRRSV in clinical lung tissue samples. Five clinical lung samples were detected by RT-PCR and all were identified as PRRSV positive, two of them were further cloned and sequenced. The result showed that both genes are 372 bp in length and shared very high homology to the ORF7 gene of PRRSV VR-2332 strain.In conclusion, the RT-PCR is specific and rapid and can be applied to diagnose PRRS clinically.福建省科技攻关计划重点项目 (2 0 0 3N0 83) ;; 厦门市科技攻关计划重点项目 (35 0 2Z2 0 0 310 5 4

Characteristics of well tests in the carbonate reservoirs of the eastern Tazhong-1 Gas Field,Tarim Basin

塔里木盆地塔中Ⅰ号气田东部试验区储层高温高压,流体性质和流动机理复杂;试井曲线具有多样性、复杂性及多解性的特点,试井分析面临巨大的挑战。为此,根据研究区地质特点及有关资料录取情况,采用现代试井分析方法与生产动态分析相结合的非均质气藏动态描述综合技术,将储层划分为视均质、双重孔隙、复合模型以及裂缝、裂缝孔洞模型等5种类型进行试井分析。结果表明:该区储层非均质性较强,平面连通性差;试井特征与稳产能力有着紧密的联系,对不同类型的井,应制订相应的开发技术对策

Study on Liquid-Phase Hybridization in PCR-ELISA for the Detection of Genetically Modified Organisms

确定杂交液成分,选择探针浓度,将5’端标记生物素的PCR扩增产物与5’端标记地高辛的探针呈液相混合,在PCR管中按优化后的条件(92℃,5 min;55℃,5 min)进行液相杂交.杂交产物通过链霉亲和素被固定在微孔表面,同时在含高浓度二甲基亚砜(dimethyl sulfoxide,DMSO)的杂交液中将非特异结合的探针解离,然后对被固定的特异杂交产物进行ELISA检测.PCR-ELISA液相杂交检测方法操作简便,特异性高,避免了繁琐的杂交程序和对人体有害的溴化乙锭,适用于转基因产品及其加工食品的快速检测.Optimizing ingredient of hybridization solution and concentration of probes was conducted. 5'-biotinylated PCR product was automatically hybridized to a digoxigenin-labeled probe presented in the PCR reaction. The hybridization is performed as part of the PCR program. Streptavidin bound to the wall of tube were used to capture the biotin- digoxigenin- labeled fragments, meanwhile the non-specifically hybridized probes were removed in the solution of high concentration dimethyl sulfoxide (DMSO) . The biotin-digoxigenin hybrids were served in ELISA detection. Liquid-phase hybridization in PCR-enzyme linked immunosorbent assay (PCR-ELISA) method enabled a fast, specific and accu-rate detection of GMOs and thus appeared to be a useful tool for routine analysis of raw and processed food products. Laborious blotting procedures and hazardous ethidium bromide in gel staining could be avoided.福建省财政厅资助项目(1401-621601);厦门市科技计划资助项目(3502Z2001109)