含增强型绿色荧光蛋白（EGFP）的伪狂犬病病毒Fa株转移载体质粒的构建-预防兽医学专业论文.docx

中文摘要本实验应用酶切、连接、插入的方法，用增强型绿色荧光蛋白(EGFP)作为筛选 中文摘要 本实验应用酶切、连接、插入的方法，用增强型绿色荧光蛋白(EGFP)作为筛选 标记，构建了含EGFP的伪狂犬病病毒(PRV)Fa株转移载体质粒。在PRV Fa株BamHI一7 重组质粒的基础上，用限制性内切酶．％co I酶切除去gE和llk基因及部分gI和部分 28k基因后回收8．6Kb的目的片段，经T；DNA连接酶自身连接之后进行转化，经筛选 鉴定，得到阳性转化子，命名为pPB7一l：用妇』I消化pPB7一l DSA，除去gG及部分 gD基因后回收约6．ORb的目的片段，经TIDNA连接酶自身连接之后进行转化，得到的 阳性转化子命名为pPI；用EcoRI和BamHI消化pPI DXA，除去EeoRl、BamHl、 HindII等酶切位点，回收约5．7Kb的电泳目的片段，以K1eIlOW大片段酶对其未端进 行补平，之后进行平端连接及转化，筛选得到的阳性重组子命名为pPI一1；然后用 ^rp门I消化pPI-1 DNA、回收目的片段(约5．7Kb)、补平、平端连接及转化，筛选白色 菌落，得到的阳性重组子命名为pPI一2。再将绿色荧光蛋白载体pEGFP-C1上古EGFP 及其多克隆位点的完整的基因表达盒插入pPI一2中gI抗原基因起始密码子ATG F游 的Stu I位点，在选择培养基中筛选表达EGFP的转化子，构建了以gI为同源序列的 含EGFP报告基因的转移载体质粒，其上含有DR I、胁枷1、Ba／r蚪I和an I四个 单一的多克隆位点，命名为pPI一2．EGFP。大小为7．7Kb经酶切、核酸分子杂交技术 鉴定．结果表明该转移载体质粒的构建是成功的。 关键词伪狂犬病毒转移载体重组EGFP Constrction Constrction of Shuttle Vector Plasmid Expressing EGFP of Pseudorabies Virus—Fa Strain Liu Yanli(Major in Preventive Veterinary Sicience) Directed by Professor Guo Wanzhu Abstract By means of restriction enzyme digestion、ligation and insertion the shuttle vector plasmid pPI-2．EGFP of Pseudorabies virus-Fa strain(PRV Fa)was obtained based on plasmid pPB7．PRV Fa strain BamHl·7 was d!gested with Nco[．deleted gE、l 1 k and part of gI and 28k genes，was ligated with T4DNA Ligation．After translated and filtrated，we got PPB7·l：after digested with Sail． PPB7-1 deleted gG and part of gD．we got pPI：after digested with EcoRl and BamHI．deleted EcoRl、BamHI、Hindlll enzyme sites．pPI turned tO pPl-1；after digested with KpnI，pPI·2 was got；afterward EGFP and its integrallty gene expression case which lie in the plasmid pEGFP—C 1 were inserted into Stul site which lies in gl gene startup backward site of pPI·2，recombinants were filtrated in the culture medium，pPI-2．EGFP was got which contains four multi·cloning sites．Identified using the mothods of enzyme digestion and Dot-bolt．the result indicated the construction of pPI一2．EGFP was successful Key words：Pseudorabies virus：Suttle vector；EGFP；Recombinant 附件一论文独创性声明 附件一 论文独创性声明 本人郑重