山羊痘病毒感染大鼠雪旺氏细胞研究

为探索山羊痘病毒(GPV)感染神经细胞的病理过程,采用GPV实验感染体外培养的大鼠雪旺氏细胞(RSCs),通过细胞病变(CPE)及间接免疫荧光法观察,收集不同时间细胞培养上清液,应用ELISA检测神经生长因子(NGF)、脑源性神经营养因子(BDNF)和神经营养因子3(NT-3)的分泌情况.结果表明:GPV感染72 h后,RSCs出现明显细胞病变,主要表现为细胞圆缩、聚集、脱落、极性排列不明显等现象;试验组RSCs发绿色荧光,而未感染组无荧光现象;GPV感染后,RSCs的NGF及BDNF分泌量先增加后减少,而NT-3分泌量则一直下降.说明,GPV可感染RSCs并致其细胞因子分泌量发生改变,提示外周神经细胞病变可能在痘病毒致病过程中发挥着重要作用.     

小鹅瘟病毒的分离鉴定与临床病变观察

从患病雏鹅的病料样本中分离鉴定出一株小鹅瘟野毒。采用鹅胚扩增病毒。以琼脂扩散试验从感染鹅胚尿囊液中检出小鹅瘟病毒抗原,用感染鹅胚尿囊液人工接种8日龄健康雏鹅,雏鹅表现出典型的小鹅瘟临床症状和剖检特征。     

Study on the Oxidase/Dehydrogenase Enzymes and Isozyme from Tissues and Organs of Goslings Infected by Goose Parvovirus

The enzyme was efficient active gene product of catalyzing metabolic pathway.Stress environment and genetic basis led the differences of isozyme structure or activity, which specific changes of the kinds and activities of enzyme were showed in different tissues and developmental stages.Here, lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), peroxydase (POD) and catalase (CAT) were respectively extracted from brain, heart, liver, lung and spleen tissues and organs of goslings infected by goose parvovirus (GPV) and control group.The results of PAGE and biochemical analysis showed that there respectively were 3 and 1 slow-zone CAT isozyme bands deletion in liver and spleen of goslings infected by GPV.There respectively were 1 new enzyme band of POD isozyme in the brain, heart and spleen of goslings infected by GPV, but 1 enzyme band of POD isozyme deletion in the lung of goslings infected by GPV.It only showed 1 to 2 slow-zone LDH and ADH isozyme bands in organ and tissue of goslings infected by GPV, and deleted 1 slow-zone ADH isozyme band in the lung of goslings infected by GPV.The activity of ADH from goslings infected by GPV were reduced 13.61% to 61.08%, but increased 1.2 times in the heart of goslings infected by GPV.The activity of POD, CAT and LDH from goslings infected by GPV were higher 1.2 to 6, 1.5 to 3.5, 2 to 2.5 times than that of control group, respectively.The activity of CAT reduced 40.29% and 94.68% in the brain and lung of goslings infected by GPV.The activity of LDH reduced 75.93% and 91.81% in liver and lung of goslings infected by GPV.Those results indicated that the interaction of GPV stress and host oxidase/dehydrogenase enzyme gene expression resulted in the biochemical characteristics variation of activity and isozyme patterns of oxidase/dehydrogenase enzyme, and directly or indirectly affected metabolic approach and physiological pathological function on goslings infected by GPV.Therefore, sensitive oxidase/dehydrogenase was the effective marker of pathological mechanism on investigation of virus genotype interaction with genetic susceptibility of the host.     

鹅细小病毒VP3基因在昆虫杆状病毒表达系统中的表达

为真核表达鹅细小病毒（GPV）融合蛋白VP3基因,本研究通过PCR扩增GPV FY89株VP3基因,并将其克隆至杆状病毒转移载体pFastBac/HBM-TOPO中,经双酶切、PCR及测序鉴定正确后,转化感受态细胞DH10Bac,提取重组杆状病毒穿梭质粒rBacmid-VP3,将经PCR鉴定正确的rBacmid-VP3转染Sf9昆虫细胞,连续传代后,细胞病变明显。SDS-PAGE分析结果表明成功表达了分子质量约为61 ku的VP3蛋白;Western blotting分析结果表明重组VP3蛋白具有良好的抗原性。     

小鹅瘟病毒H株VP3基因序列分析

试验利用PCR技术从自行分离的GPV基因组DNA中扩增出病毒衣壳蛋白VP3完整基因片段,并与pMD18-T载体连接,转化大肠杆菌DH 5α感受态细胞,提取重组质粒经PCR和酶切鉴定后,对插入片段进行序列测定及分析。结果表明,VP3基因全长1605 bp,编码534个氨基酸;分子生物学分析表明所分离到的病毒为强毒株。     

鹅细小病毒NS1基因的克隆及原核表达

本研究根据GenBank发表的GPVB株全基因核苷酸序列，设计了1对用以扩增GPV主要非结构蛋白NS1基因的引物。通过PCR技术，扩增出NS1完整基因片段1．9kb。将PCR产物克隆到pMD18-T载体后进行序列测定及分析，结果表明：GPV H1株NS1基因全长1884bp，编码627个氨基酸，与国外已发表的B株核苷酸序列同源性为98．15％。同时将该目的基因正向插入到GST融合蛋白原核表达载体PGEX-6P-1的BamH1多克隆位点，构建了GPV NS1的原核表达载体，成功的表达了约97KD的NS1融合蛋白。     

麦蚜循回传毒过程的超微结构研究

对禾谷缢管蚜和麦长管蚜传播小麦矮病毒ＧＰＶ株系的超微结构观察发现：禾谷管蚜取食８ｈ时,病毒进入蚜虫后肠肠腔中,１２－２４ｈ后进入后肠细胞质,ＧＰＶ与肠内壁顶端细胞膜接触,接触部位细胞膜逐渐下陷,形成小穴,向内凹陷发育为管状泡囊,而后进入血淋巴液,当病毒被附唾液腺吸收后蚜虫将病毒排吐进入唾液管中。     

DuCV和GPV与DEV三重PCR检测方法的建立及应用

根据GenBank中的序列,合成针对鸭圆环病毒(DuCV)REP基因、鹅细小病毒(GPV)NS1基因、鸭肠炎病毒(DEV)UL6基因的3对特异性引物,以病毒的克隆质粒作为模板,进行退火温度、引物终浓度的优化,建立一种能同时鉴别DuCV、GPV、DEV的三重PCR检测方法,并检验该方法的特异性、敏感性和重复性.结果显示:...     

番鸭细小病毒和鹅细小病毒广东株VP1基因的克隆与序列分析

参考GenBank中的MDPV FM株和GPV B株全基因序列,设计并合成一对引物,分别对MDPV GD株和GPV GD株的结构蛋白基因(VP1)进行PCR扩增,并克隆到pMD18-T载体,筛选到重组质粒并测序.通过对MDPV GD株和GPVGD株的VP1基因的核苷酸序列分析,这两种病毒VP1基因大小均为2 199 bp,核苷酸序列同源性为87%,而位于VP1基因上的VP2至VP3基因起始密码子之间的核苷酸序列的差异较大,同源性仅为64%.另外对MDPV GD株和GPV GD株与各地方毒株的VP1和VP3基因核苷酸序列的同源性比较,显示它们之间差异较小.     

鹅细小病毒VP1-VP3非重叠序列地高辛探针的制备和应用

根据GPV H1株核苷酸序列，设计了扩增VP1-VP3基因非重叠序列的1对引物，对其结构蛋白VP1与VP3非重叠核苷酸序列进行PCR扩增，将PCR产物纯化、回收后制备出GPV VP1-VP3基因DIG标记核酸探针，其标记效率达到0．1pg／μl。特异性检测结果表明，该探针能与GPV不同毒株核酸发生特异性杂交，而与对照的DPV、GPMV等病毒的核酸杂交反应均为阴性；敏感性检测结果表明该探针对GPV的最低检出量为0．032ng。上述试验结果表明该探针可以用于GPV感染临床病料的检测。