摘要
以ｐＵＣ１９质粒为载体，采用鸟枪法克隆减蛋综合征病毒（ＥＤＳＶ）基因组的ＨｉｎｄⅢ酶切片段，用Ｄｉｇｏｘｉｇｅｎｉｎ－ｄＵＴＰ标记的ＥＤＳＶＨｉｎｄⅢ片段探针打点杂交，证实１３个克隆插入了ＥＤＳＶＤＮＡ的ＨｉｎｄⅢ片段。酶切分析结果表明，克隆到了Ａ、Ｂ、Ｆ、Ｇ、Ｈ、Ｉ、Ｊ７个不同大小的片段，选取含有上述插入片段的、具有代表性的７个相应质粒ｐＵＨＡ、ｐＵＨＢ、ｐＵＨＦ、ｐＵＨＧ、ｐＵＨＨ、ｐＵＨＩ、ｐＵＨＪ，在分别以ＢａｍＨＩ、ＢｇｌⅡ、ＣｌａⅠ、ＥｃｏＲＩ、ＥｃｏＲＶ、ＰｓｔⅠ、ＳｍａⅠ、ＸｂａⅠ、ＸｈｏⅠ单酶切的基础上，经适当组合的双酶切，得出各片段存在的单一酶切位点为Ａ：ＢｇｌⅡ、ＥｃｏＲＶ、ＳｍａⅠ、ＸｈｏⅠ位点；Ｂ：ＢｇｌⅡ、ＳｍａⅠ位点；Ｆ：ＢｇｌⅡ、ＥｃｏＲＶ位点；Ｇ：ＰｓｔⅠ位点；Ｈ：０；Ｉ：ＰｓｔⅠ位点；Ｊ：ＥｃｏＲＶ、ＸｈｏⅠ位点。为进一步筛选病毒复制非必需区，构建腺病毒表达载体，以及研究禽类腺病毒的分子生物学特性打下了基础。
DNA fragments of egg drop syndrome virus (EDSV) genome digested with Hind Ⅲ were inserted into the Hind Ⅲ site of pUC19 by shotgun method. Thirteen recombinant plasmids were identified by dot blot hybridization with EDSV DNA Hind Ⅲ fragments probe labelled with DigoxigenindITP. Seven fragments of EDSV DNA in sizes of 6.91(A), 5.24(B), 3.16(F), 2.02(G), 1.65(H), 1.41(I) and 1.41(J) had been successfully cloned. These recombinant plasmids in different size were called pUHA, pUHB, pUHF, pUHG, pUHH, pUHI, pUHJ and digested respectively with Bam HI, Bgl Ⅱ, Cla Ⅰ, Eco RI, Eco RV, Pst Ⅰ, Sma Ⅰ, Xba Ⅰ, Xho Ⅰ and codigested with two suitable restriction enzymes. Single sites of Bgl Ⅱ, Eco RV, Sma Ⅰ and Xho Ⅰ respectively have been located in fragment A; single sites of Bgl Ⅱ, Sma Ⅰ in fragment B; single sites of Bgl Ⅱ, Eco RV in fragment F; single site of Pst Ⅰ in fragment G; no site in fragment H; single site of Pst Ⅰ in fragment Ⅰ; single sites of Eco RV, Xho Ⅰ in fragment J. The results will facilitate the shotgun screening of the nonessentialregion(NER) for virus replication as well as the molecular virological study of EDSV and its engineering as an expression vector.
出处
《中国兽医学报》
CAS
CSCD
北大核心
1998年第1期7-10,共4页
Chinese Journal of Veterinary Science
基金
"九五"农业部畜牧业重点科研计划项目
国家自然科学基金
关键词
减蛋综合征病毒
克隆
限制酶图谱分析
egg drop syndrome virus
clone
restriction endonuclease analysis
