摘要
采用９～１０日龄非免疫鸭胚增殖的减蛋综合征病毒（ＥＤＳＶ）ＡＡ－２毒株，经差速离心法浓缩纯化后，提取病毒基因组ＤＮＡ。采用碱变性法除去病毒基因组共价结合的末端蛋白（ＴＰ）。用限制性内切酶ＨｉｎｄⅢ水解纯化的ＥＤＳＶ基因组ＤＮＡ。经低熔点琼脂糖凝胶电泳后，回收Ｃ、Ｄ、Ｅ片段。克隆到ｐＵＣ１９载体的ＨｉｎｄⅢ和ＳｍａⅠ双酶切位点及ＨｉｎｄⅢ位点上，经蓝白斑筛选和单、双酶切鉴定，获得了ｐＵＨＣ、ｐＵＨＤ、ｐＵＨＥ重组质粒，其中ｐＵＨＣ含有末端片段。将ＥＤＳＶＳａｌⅠ水解产生并回收的大片段与ｐＵＨＣ在９５℃水浴中变性，６５℃复性后，用钙离子介导法，共转染５０％～７０％的单层鸭胚成纤维细胞，转染后３６ｈ开始产生细胞病变（ＣＰＥ）。４８ｈ后将病变细胞反复冻融，经尿囊腔接种９～１０日龄鸭胚，回收的尿囊液能凝集鸡红细胞，这种血凝性能被ＥＤＳＶ高免血清抑制，电镜下观察到腺病毒样颗粒。
Egg
drop syndrome virus (EDSV)AA2 strain was purified from the duck embryo allantonic fluids
using differential centrifugation. The typical adenovirus virons were observed under electron
microscope.The viral DNA that was prepared and purified showed a clear band about 33 kb in
agarose gel electrophoresis. The terminal peptides(TP) were removed with alkaline
treatment.Then the EDSV DNA was digested by Hind .The C,D,E fragments were retook,and
cloned into pUC19 between Hind and Sma sites.The mixture of recombinant plasmid and the
leftend fragment of viral DNA digested by Sal ,were denatured at 95 for 10 min and annealed at
65 for 30 min, then cotransfected into duck embryo fibroblast of 50%70% confluent cells in 35
mm well by Ca2+ reagent .The typical cytopathic effects(CPE) were observed at 36 h later,in
contrast, Cla fragment or recombinant plasmid did not make the DEF pathic .The medium was
passaged in 9or 10dayold embryonated eggs by allantonic cavity inoculation and the allantonic
fluid havested was tested by HA with HI and adenoviruslike virons were also found using
electron microscope.This research established a method for the aviadenovirus vector
constructing at the right region of EDSV genomic DNA.
出处
《中国兽医学报》
CAS
CSCD
北大核心
1999年第3期226-229,共4页
Chinese Journal of Veterinary Science
基金
国家自然科学基金
农业部重点资助
关键词
减蛋综合征病毒
末端蛋白
末端片段
重组
DNA
egg drop syndrome virus
terminal protein
terminal fragment
recombinant DNA
