【作者】 孙公军；

【导师】 李劲松； 李银； 陈溥言；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 自行设计并合成引物，用PCR方法扩增出了分子量分别为519bp和2101bp的鸡γ干扰素基因（ChIFN-γ基因）和马立克的gB基因。然后将chIFN—γ基因克隆于穿梭质粒pEGFP—C₁中，构建的质粒命名为pC₁-chIFN—γ，经过双酶切鉴定证明已将ChIFN—γ基因插入到了pEGFP—C₁的多克隆位点中。gB基因的PCR产物克隆到pMD18-T载体中，构建的质粒命名为pT-gB，构建的质粒也用双酶切的方法进行鉴定。随后，我们从pT-gB中分离出gB基因，并将之连接到自行构建的载体pEGFP—C₁中，构建的质粒命名为pC₁-IFN-gB，经过双酶切鉴定证明将gB基因也插入到了pC₁-chIFN-γ中。 从自行设计构建的基因表达盒载体pC₁-IFN-gB上，用Apal Ⅰ和Nae Ⅰ酶切并分离出分子量为4958bp的基因表达盒，然后应用平端连接反应将表达盒插入到pTK₂B载体的Nhe Ⅰ的单酶切位点位置，构建出含有chIFN-γ、gB和EGFP基因表达盒的转移质粒载体，并命名为pTKG-IFN-gB；从自行设计构建的基因表达盒载体pC₁-chIFN—γ上，分离出分子量为3480bp的基因表达盒，并平端插入到pTK₂B载体的Nhe Ⅰ酶切位点位置，构建出含有chIFN-γ和EGFP基因表达盒的转移质粒载体，并命名为pTKG-IFN-γ。 将自行设计构建的pTKG-IFN-gB用Pvu Ⅱ酶切，电泳回收后用脂质体方法进行转染，并用有限稀释的方法筛选重组病毒，转染的病毒在荧光显微镜上进行观察。经过笔者的摸索，发现适合本试验的最佳转染条件是：细胞的接毒量为0.5ml，脂质体的用量为25ul，转染时间为细胞接毒吸附2小时后培养8小时。初步筛选到了较为理想的重组病毒。 该重组火鸡疱疹病毒的获得，使得进一步研究它的生物学特性和预测它的应用前景变得切实可行。此外，本实验也为进一步研究ChIFN—γ对病毒活载体复制的影响提供了物质基础，并且可为阐释chIFN—γ做为基因工程活载体疫苗免疫佐剂的可能性提供实验依据。更多还原

【Abstract】 The objective of this paper is to construct recombinant Herpesviruse of Turkey. On designing construction strategy, the research focuses on the following.Based on the former studies, two pairs of primers were designed and synethesized to obtain chicken 1FN- Y (ChIFN- Y ) and gB genes, before these two genes were amplified by PCR, with pT- ChlFN- Y and pURMDV-EgB as template. The gene fragement of ChIFN- Y and gB were then respectively cloned into pEGFP-Cl and pMD18-T, the accordingly recombiant plasmids were designed as pC1-IFN-rand pT-gB. Both of these clones were identified by Bgl II and Hind III, then the gene fragement of gB was inserted into pC1 -IFN-yto obtain the recombinant plasmid pC1-IFN-gB.From both the constructed plsmid pTK2B containing non-essential fragement and the plasmid pCl-IFN-Y containing the eukargotic gene expression casstle, an isolated gene expression casstle frgement of about 495 8bp was inserted into the Nhe I site of HVT TK gene by blunting ligation and recombinant plsmid designed as pTK-IFN-gB was generated. In the same way , another recombinant plasmid named pTK-IFN- Y was generated ,too.The constructed plasmid pTK-IFN-gB was transfected into secondly CEFs several times by using the methods of lipsome transfection and limiting dilution, then a recombinat virus was obtined by the following condition: adding 25ul liposomal in transfection, inoculating 0.5ml diluted vaccine viruses and inoculationg cells 8 hours after inoculating .In conclution, the construction of the recombinant HVT had paved a path for investigating further their biological characteristics and for prediction of the prospect of their potential application. In addition, this experiment provide a basis for further study of the effect of ChlFN- Y on the replication of recombinated vaccine virus and of possibility of ChlFN- Y as adjuvant of recombinat vaccine vector.更多还原