摘要
从马传染性贫血病毒（ＥＩＡＶ）弱毒株感染的驴胎皮肤（ＦＤＤ）细胞中提取前病毒ＤＮＡ，以其为模板，通过ＰＣＲ扩增出ＥＩＡＶ弱毒株的ＬＴＲ，并将其克隆到载体质粒ｐＵＣ１９中。经酶切分析，ＰＣＲ及Ｓｏｕｔｈｅｒｎ杂交筛选、鉴定，获得含有ＥＩＡＶ弱毒株ＬＴＲ片段的重组质粒ｐＬＴＲ。对该质粒的序列分析发现，在中国ＥＩＡＶ弱毒株ＬＴＲ的Ｕ３区含有４个与细胞转录因子结合的位点，其中有３个ＰＵ．１位点和１个ＡＰ－１位点，Ｕ３区还存在１个ＴＡＴＡＴＡＡ启动子序列；Ｒ区长为８１ｂｐ，含有１个帽位点ＧＧ和１个Ｐｏｌｙ（Ａ）信号序列ＡＡＴＡＡＡ；在帽位点下游是１个病毒Ｔａｔ蛋白结合位点，即ＴＡＲ位点；Ｕ５区长为３９ｂｐ。中国ＥＩＡＶ弱毒株ＬＴＲ序列与国外发表的其他毒株序列相比，在ＬＴＲ的一些调控部位有变异发生，中国ＥＩＡＶ弱毒株与美国ＥＩＡＶ原型株（Ｗｙｏｍｉｎｇ株）ＬＴＲ区核苷酸序列有１８．７２％的差异。
Provirus DNA was extracted from fetal donkey dermal (FDD) cells which were infected with the attenuated equine infectious anemia virus (EIAV). A pair of primers were designed and synthesized according to the published nucleotide sequence of EIAV Wyoming strain for amplification of long terminal repeat (LTR). A 0.5 kb DNA fragment containing most of LTR sequence was obtained by polymerase chain reaction (PCR) amplification using DNA from infected FDD cells as template. The PCR product was cloned into the plasmid vector pUC19, and a recombinant plasmid (designated as pLTR) with the LTR sequence was identified by restriction enzyme analysis, PCR and Southern hybridization. The nucleotide sequence of the attenuated EIAV LTR was determined. There are four transcriptional control elements (three PU.1 elements and one AP1 element) in the LTR U3 region. A promoter sequence with “TATA” box is present in U3 region. A cap site (GG) and a poly (A) signal (AATAAA) are present in R region (81 bp). There is a transactivating responsive element (TAR) downstream of the cap site. U5 region is 39 bp in length. By comparison with LTR sequences of EIAV prototype strain (Wyoming strain), it was found there was nucleotide divergence of 18.72% between the attenuated EIAV and Wyoming strain in the LTR, and a hypervariable region within the U3 region. The differences were also seen in the hypervariable region of U3 between the attenuated EIAV and other EIAV strains.
出处
《中国兽医学报》
CAS
CSCD
北大核心
1998年第1期1-6,共6页
Chinese Journal of Veterinary Science
基金
国家自然科学基金
关键词
马传染性贫血
克隆
病毒
弱毒株
序列分析
equine infectious anemia virus
attenuated strain
long terminal repeat
nucleotide sequence analysis
