【作者】 项朝荣； 李文刚； 吴凤笋； 唐桂芬；

【Author】 Xiang Chao-rong~1 Li Wen-gang~2 Wu Feng-sun~2 Tang Gui-feng~2 (1.Livestock farming bureau Jiyuan,Jiyuan Henan;2.Zhengzhou college of Animal Husbandry Engineering,Zhengzhou Henan)

【机构】 济源市畜牧局； 郑州牧业工程高等专科学校；

【摘要】 根据Genebank公布的质粒pGFPuv序列、质粒pUSK序列和PRV基因gG启动子,设计一对含限制性内切酶PstⅠ和NotⅠ酶切位点的引物,以含绿色荧光蛋白基因的质粒pGFPuv为模板,扩增了完整的GFPuv基因,然后用PstⅠ和NotⅠ双酶切后的扩增片段与经PstⅠ和NotⅠ双酶切处理的pUSK质粒连接,转化大肠杆菌DH5a,构建了转移重组质粒pUSK-pGFPuv。经酶切鉴定与PCR方法证明重组质粒大小正确,且插入了绿色荧光蛋白,并证明插入方向正确。进一步测序鉴定,测序结果表明插入的片段与预期相符。利用脂质体介导的方法,将质粒pUSK-pGFPuv和PRV基因组共转染VERO细胞进行同源重组并观察到荧光,这为筛选和进一步鉴定重组病毒打下了基础,也为构建伪狂犬gG基因缺失疫苗打下基础。更多还原

【Abstract】 According to Genebank the announcement Plasmid pGFPuv sequence,the plasmid pUSK sequence and PRV the gene gG promotor,designed a pair of primers which contain restriction enzyme PstI and the NotI sites,Containing green fluorescent protein gene pGFPuv plasmid as a template,amplified a complete GFPuv gene,Amplified fragment length assay,Then PstI and NotI-digested and the amplified fragment by PstI and NotI-digested the cloned pUSK vector and transformed into E.coli DH5a, constructing a recombinant plasmid pUSK-pGFPuv transfer.Was identified by PCR and the recombinant plasmid correct size,and insert a green fluorescent protein,methods and proved to insert the right direction.Further sequencing,sequencing results showed the footage inserted in line with expectations.The use of lipofectamine method,The plasmid pUSK-pGFPuv and PRV genome were transfected VERO homologous recombination and observed that the cells fluorescence,for further screening and identification of recombinant virus laid the foundations Construction for pseudorabies vaccine gG gene deletion lay the groundwork.更多还原