摘要
从鸡传染性喉气管炎病毒王岗株感染的鸡胚绒毛尿囊膜细胞中提取病毒,并抽提其核酸,经限制性核酸内切酶Pst Ⅰ完全消化后,在0.7%琼脂糖凝胶中电泳分离,然后用DE—81滤纸回收2～6kb区域的片段与质粒pBluescrip^+SK重组.有3个重组质粒(pLT—1,pLT—2和pLT—4)所含外源DNA片段的大小,分别与4.3kb,4.8kb和1.6kb的鸡传染性喉气管炎病毒DNA Pst Ⅰ片段一致,经过Southern杂交试验证明,这3个片段都是病毒DNA的特异性片段.用光生物素标记重组质粒pLT—1和pLT—2后混合作为探针,再与鸡喉气管炎病毒及其他8种鸡传染性病原进行杂交,证明此探针只与喉气管炎病毒反应,而与其他8种病原无任何交叉反应.
Infectious laryngotracheitis virus (ILTV) was purified from chorioallautoic membrane infected with Wanggang strain of ILTV. ILTV DNA was then extracted and digested completely with Pst I followed by electrophoresis on 0.7% agarose gel. Fragments between 2 and 6 kb were recovered using DE-81 paper and ligated into the correspondent site of plasmid pBluescript SK. Three recombinants (pLT-l,pLT-2 and pLT-4) were found to contain foreign DNA fragment similar in size to 4. 3, 4. 8 and 1. 6 kb fragments of Pst I cleaved ILTV DNA respectively. Through Southern hybridization, the three fragments in plasmids were indicated as ILTV DNA fragments . Recombinants pLT-1 and pLT-2 were mixed and then labelled with photobiotin. In cross hybridizations, the labelled probe only showed positive reaction too ILTV DNA, and no cross reactions to other eight different avian infectious pathogens were found.
关键词
鸡
喉气管炎
病毒
核酸探针
infectious laryngotracheitis virus
DNA probe
photobiotin
