摘要
以伪狂犬病病毒 Ea株为模板 ,通过 PCR扩增含 g E基因主要抗原表位区的 80 1 bp的片段 ,扩增产物酶切后克隆于 p Bluescript SK+ 中 ,双脱氧末端终止法测定序列并同国外 Rice株进行同源比较。将该片段插入原核表达载体 p ET-1 7b的 T7噬菌体启动子下游 ,构建的原核表达质粒 p ETE80 4在大肠杆菌 BL2 1( DE3 )中获得了高效表达。 SDS-PAGE结果显示 ,表达的蛋白 Mr为 380 0 0 ,并形成包涵体。 Western印迹分析表明 ,该蛋白能与 Ea株野毒高免血清发生特异性反应 ,但与 g E缺失的 Bartha株高免血清不反应。上述结果说明 ,该原核表达产物不仅具有生物学活性 。
A0 .8kb DNA fragmentencoding the majorepitope domain of glycoprotein g E of pseu- dorabies virus Ea strain was amplified by PCR technique and cloned into p Bluescript SK+ .The se- quence of the fragmentwas obtained by Sanger′s sequencing technique.Then,the fragmentwas insert- ed into downstream of the T7promoter of an expression vector,p ET-1 7b,to yield the recombinant plasmid p ETE80 4.After induction by IPTG,a high expression of fusion protein was obtained,SDS- PAGE analysis and Western blotting showed that the fusion protein was measured of3 80 0 0 specific to antisera against PRV Ea strain than against Bartha strain in Western blotting.This indicated that the expression products could be useful for the differentiation of natural infection from vaccinated herds.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2001年第5期459-462,共4页
Chinese Journal of Veterinary Science
基金
国家"九五"科技攻关计划生物技术项目 ( 96 -C0 1-0 4-0 3)
关键词
伪狂犬病病毒
GE基因
抗原表位
克隆
表达
pseudorabies virus
glycoprotein g E
major epitope
cloning
expression
