摘要
根据已发表的序列，分别在鸡贫血病毒（ＣＡＶ）环形基因组ＤＮＡ（全长２．３ｋｂ）的ＥｃｏＲＩ位点和ＢａｍＨＩ位点的两侧选择适当序列合成两对引物，用ＰＣＲ技术，从斑点杂交检测到病毒核酸的ＣＡＶ感染的ＭＤＣＣＲＰ１细胞基因组ＤＮＡ中，分别扩增出包含ＥｃｏＲＩ和ＢａｍＨＩ分割开的病毒基因组两部分（１．５ｋｂ和０．８ｋｂ）约１．５ｋｂ和约１．２５ｋｂ的两个片段。再将其中相应序列拼接克隆进ｐＵＣ１８载体，获得包含ＣＡＶ全基因组序列ＤＮＡ片段的克隆质粒ｐＣＡＶ２．４。酶切分析表明，该质粒具有预期的ＢａｍＨＩ位点、ＰｓｔＩ位点、ＨｉｎｄⅢ位点，而预期的ＥｃｏＲＩ位点消失。重组质粒插入ＤＮＡ片段的两端序列分析表明，质粒ｐＣＡＶ２．４是包含ＣＡＶ全基因组序列的重组质粒，插入ＤＮＡ片段序列中的ＥｃｏＲＩ位点序列发生了一个碱基突变。
By two pairs of primers complementory to the published sequences at the both sides of EcoRI site and BamHI site on CAV circular genome, two DNA fragments containing two parts (1.5 kb and 0.8 kb) of CAV genome divided by EcoRI and BamHI sites were amplified by PCR from the circular CAV genome (2.3 kb) detected by dot blot in genomic DNA of CAV infected RP1 cells. Through re ligation of the selected sequences from the amplified fragments, the whole CAV genomic DNA was cloned into pUC18, and the recombinant plasmid was designated as pCAV2.4. Restriction endonucleases analysis of pCAV2.4 showed that there were sites of BamHI、PstI、HindⅢ,but no site of EcoRI. Two ends of cloned DNA sequence analysis showed that the recombinant plasmid pCAV2.4 containes the whole genomic DNA sequence of CAV, and that the site sequence of EcoRI in the cloned DNA was changed by one base substitution.
出处
《中国病毒学》
CSCD
1999年第1期80-86,共7页
Virologica Sinica
