猪和牛轮状病毒抗原和抗体的检测

应用ELISA直接双抗体夹心法检查轮状病毒抗原,24份仔猪和29份犊牛的腹泻粪样,分别有12和16份阳性。用病毒RNA电泳分析检查阳性粪样,各出现两种病毒RNA电泳型,用中和试验检查17份成年牛和16份成年猪血清,分别有16和15份病毒抗体阳性。将其与ELISA间接法和结合法进行了比较。     

成人腹泻轮状病毒的提纯及其高价免疫血清的制备

本文采用交叉免疫电泳技术,首先提纯了成人腹泻轮状病毒(Adult DiarrhoeaRotavirus简称:ADRV),并经电镜确认,用作免疫抗原,首次制备了高价兔与豚鼠抗ADRV血清和小鼠抗ADRV腹水,其特异性经交叉免疫电泳和对流免疫电泳鉴定,其效价经酶联免疫吸附试验(ELISA)检测均在1:2,000以上。     

小儿副轮状病毒的检出

用聚丙烯酰胺凝胶电泳法,从婴幼儿急性腹泻粪便中检出轮状病毒RNA电泳阳性293份,发现一株副轮状病毒(青-27株),此株病毒经电泳观察,呈典型的轮状病毒形态,但易破碎。ELISA检测表明不具有一般轮状病毒的特异性群抗原,病毒RNA基因组由11个片段组成,但电泳图型特殊,吴4:3:2:2排列模式,本文证实,此一小儿副轮状病毒与国外报道的,散发罕见的小儿副轮状病毒RNA电泳图型相同,提示此病毒的重要意义。     

Calcium influences the stability and conformation of rotavirus SAH glycoprotein VP7 expressed in Dictyostelium discoideum

We have previously reported expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of Ca²⁺ since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca²⁺ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca²⁺ reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca²⁺ chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca²⁺ and that conformational changes in VP7 which occur following depletion of Ca²⁺ reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7.     

A组轮状病毒SA11VP6基因的克隆和表达

从ＳＡ１１ＶＰ６基因全序列克隆开始,设计一对两端带有酶切位点的引物,逆转录ＰＣＲ扩增出ＶＰ６全基因ＣＤＮＡ。经酶切后插入ＰＵＣ１９,构建了ＶＰ６全基因克隆ＰＲＡ６。再经酶切后插入痘苗病毒载休质凿ＰＪＳＡ１１７５中。利用Ｌｉｐｏｆｅｃｔｉｎ导入ＴＫ１４３细胞,利用ＴＫ基因和Ｌａｃ基因作为重组病毒的筛选标记。表达产物用单克隆抗体ＥＬＩＳＡ法检测,发现细胞培养上清和细胞裂解液都是阳性。Ｗｅｓｔｅｒｎ ｂ     

轮状病毒新敏感细胞的筛选

轮状病毒（RV）的细胞培养问题的尚未完全解决,其原因之一可能与敏感细胞较少有关。为此,我们进行了RV新敏感细胞的筛选,发现恒河猴胚肾传代细胞Frhk-4感染不同血清型的人和动物RV标准株Wa、DS－1、YO、ST－3、SA－11和UK,37℃旋转或静止培养均能产生明显CPE。培养物经ELISA测定含RV抗原,用PAGE检测有特征性RV核酸图型,电镜超萍切片检查感染细胞可见RV颗粒,证实Rrhk-4是RV的敏感细胞,这在国内外尚未见报道。同时与MA104－CV－1细胞的比较研究证明,三种细胞对Wa和DS－1的敏感性及增殖滴度,以CV－1最高,Frhk-4次之,MA－104最低。