摘要
根据伪狂犬病病毒 (PRV) Ka株序列 ,合成 1对包含 g K基因完整编码区的引物 ,以 PRV Ea株 Bam H 5′片段为模板 ,扩增出 0 .9kb特异性带。将纯化的扩增产物克隆到 p Bluescript SK+ 中 ,构建了重组质粒 p SKg K,用双脱氧末端终止法进行序列测定 ,并同国外 Ka株进行比较。结果表明 ,Ea株 g K基因全长 939bp,编码 313个氨基酸 ,与 Ka株比较 ,Ea株 g K基因有 2 2处点突变 ,但无插入突变和缺失 ,同源性达 97.78%。将 g K基因克隆到原核表达载体p GEX- 2 T中 ,构建了 g K全基因原核表达载体 p2 Tg K939。再用 Bam H 、Xho 酶切 p2 Tg K939,回收 5 88bp g K小片段 ,克隆到原核表达载体 p GEX- KG中 ,构建了 g K部分基因原核表达载体 p KGgk5 88。经 IPTG诱导 ,g K全基因不表达 ,而 g K部分基因表达约 4 0 0 0 0融合蛋白。用 PRV高免血清经 Western blotting证明 ,g K有免疫原性。提取 g K包涵体并制备高免血清 ,EL ISA检测抗体效价为 1∶ 6 4
A pair of primers was synthesized according to the sequence of PRV Ka strain which contains whole gK gene coding domains.About 0.9 kb fragment was amplified by polymerase chain reaction(PCR) using BamHⅠ 5′ fragment of PRV Ea strain as template,and cloned into transfer vector pBluescript ⅡSK +,resulting in a recombinant plasmid pSKgK.The sequence was obtained by dideoxy chain-termination method and compared with PRV Ka strain.The whole gK gene was cloned into porkaryote expression vector pGEX-2T,resulting in p2TgK939.The partial gK gene was obtained by cutting p2TgK939 with BamHⅠ,XhoⅠ,and cloned into pGEX-KG,resulting in pKGgK588.After induction by IPTG,the whole gK gene didn′t express while the partial gK gene expressed about 40 000 fusion protein at a high level.The Western blotting showed the immunogenicity of expressed gK with PRV high-immune serum.The inclusion bodies were extracted for preparation of the gK high-immune serum.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2003年第2期138-141,共4页
Chinese Journal of Veterinary Science
