摘要
将犬瘟热病毒 ( canine distemper virus,CDV ) H基因表达盒克隆入转移质粒 p VAXΔE3,构建含 CDV H基因表达盒的转移质粒 p VAXΔ E3L PH,用 Nru 和 Sal 分别酶切转移质粒 p VAXΔ E3L PH和犬 2型腺病毒全基因组质粒p Poly2 - CAV- 2 ,将 CDV H基因表达盒定向克隆入 p Poly2 - CAV- 2质粒中 ,构建 E3区部分缺失的包含 CDV H基因表达盒的犬 2型腺病毒重组质粒 p CAV- 2 / CDVL PH。 Asc 和 Cla 对 p CAV- 2 / CDVL PH进行双酶切 ,释放 CAV- 2 /CDVL PH重组基因组 ,利用脂质体将 CAV- 2 / CDVL PH重组基因组与去除 Sal 和 Nru 片段的 CAV- 2基因组两端片段共转染 DK细胞 ,盲传和重复转染 3次 ,4 d后出现典型 CAV- 2病变 ,获得重组病毒 CAV- 2 / CDVL PH。用 CDV H基因特异性引物经 RT- PCR扩增重组病毒 DK细胞培养物 ,能够扩增出相应的核酸片段 ,表明 CAV- 2 / CDVL PH在DK细胞繁殖的过程中能够转录 CDVL P H的 m RNA,重组病毒免疫犬 ,可以诱导犬产生特异的抗 CAV- 2 HI抗体和抗
The CDV H expression cassette was released from pVAXLPH plasmid by being digested with MluⅠ/SpHⅠ, blunted and ligated into SspⅠ site of pVAXΔE plasmid. The recombinant plasmid was named PVAXΔE3LPH. The orientation of the expression cassettes in PVAXΔE3LPH is consistent with the transcript orientation of adenovirus E3 region. PVAXΔE3LPH and pPoly2-CAV-2 were digested with NruⅠ/SalⅠ, respectively. The purified NruⅠ/SalⅠ DNA fragment containing the CDV H expression cassettes was cloned into pPoly2-CAV-2 plasmid to generate recombinant plasmid pPoly2-CAV-2/CDVLPH. The cloned recombinant genome was released from pPoly2-CAV-2/CDVLPH by ClaⅠ/AscⅠ digestion, and was cotransfected into DK cells with CAV-2 DNA without the NruⅠ/SalⅠ segment by Lipofectamine. After three transfections into DK cells with the mixed DNA, the recombinant virus, named CAV-2/CDVLPH, was obtained, producing typical cytopathic effects in DK cells. The expression of CDV H gene by CAV-2/CDVLPH in infected DK cells was confirmed by RT-PCR. The specific immune response against CDV was induced by CAV-2/CDVLPH in dogs. It indicated that the H protein was encoded by CAV-2/CDVLPH in dogs.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2004年第5期425-428,共4页
Chinese Journal of Veterinary Science
基金
"十五"军队医药卫生重点项目 (Z2 0 0 1 0 95 )
