【作者】 苏春霞；

【导师】 张彦明； 陈溥言；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2004， 硕士

【摘要】 新城疫是由新城疫病毒引起的鸡和火鸡的一种急性高度接触性传染病。20 世纪40 年代就开始使用新城疫疫苗，通常使用的疫苗为活病毒苗。但活疫苗会导致鸡群产生轻微呼吸道症状，从而继发细菌感染。这些疫苗产生的免疫反应持续时间短，必须进行多次免疫接种；且雏鸡具有较高的母源抗体，会干扰疫苗的免疫效果。因此，构建重组病毒来表达与新城疫病毒免疫有关的抗原基因，试图解决以上问题。 马立克氏病是由马立克氏病病毒引起的鸡的一种高度接触性传染性肿瘤病。马立克氏病病毒疫苗株被认为是最有潜力的病毒载体之一，通过构建表达外源基因抗原的多价活疫苗来诱导免疫达到预防禽病的目的。 考虑多价重组疫苗的诸多优点，本试验利用马立克氏病病毒的 gB 启动子控制新城疫病毒 F 基因的表达来构建重组马立克氏病病毒，使其能有效地预防新城疫和由马立克氏病病毒超强毒株引起的马立克氏病，且既可以避免病毒对外源启动子的排斥作用，又可以避免母源抗体的影响来提高疫苗对商品鸡的免疫保护作用。 根据 GenBank 己发表的新城疫病毒 F48E9 株 F 基因序列，设计了一对引物，以含有新城疫病毒 F48E9 株 F 基因的质粒 pz1/z2 为模板，将扩增的 F 基因(1 678bp)片段克隆到真核表达载体 pIRES 中，构建成表达 F 基因的载体 pIRESF。再根据 GenBank己发表的载体 pIRES 基因序列，设计了一对引物，以载体 pIRESF 为模板将扩增的包含 F 基因及其上游的内含子和下游的 polyA 的 2 900bpDNA 片段，再克隆入包含马立克氏病病毒的 gB 启动子的 pBluescriptⅡSK 载体中，并使其克隆到 gB 启动子 580bp的下游，最后将包含gB 启动子和F基因表达盒3 500bp克隆到马立克氏病病毒CVI988非必需区 US10 基因中，并命名为 pUS10F。 在试验中，我们选择 CVI988 疫苗株作为病毒载体来构建重组病毒，原因是致弱的 MDVI 型疫苗如 CVI988，它们的免疫效果明显优于 HVT，同时疫苗株 MDVI 型与 vvMDV的抗原有较高的同源性，理论上可以比其他血清型更能诱导具有针对性的免疫应答反应，特别是针对 vvMDV 和 vv+MDV。为进一步提高重组病毒的表达水平，将成功构建的转移质粒载体转染 293T 细胞后，对转染条件进行了优化，通过利用转染后 293T细胞的基因组作为模板进行 PCR，结果说明真核细胞中已有带 F 基因的细胞存在，质粒载体已经进入细胞 DNA。间接免疫荧光检测结果表明，转染质粒载体后的 293T 细胞可特异性地表达 NDV 的 F 基因，所表达基因的抗原性较好，能够与抗 NDV 的标准血清反应。从而说明转移质粒载体中的基因表达盒可以在细胞中有效地表达，为进一步开发新型的抗 ND 和 MD 疫苗的研究打下基础。更多还原

【Abstract】 Newcastle disease is an acute infectious disease caused by Newcastle disease virus in chicken andturkey. Newcastle disease’ vaccination with live virus has been used since 1940s，which lead torespiratory complications and cause bacterium infection. These vaccines were used many times toproduce immunity protection in chicken and their immunes were disturbed by mother’ antibody inchicken .So we construct recombinant virus which express NDV antigen gene to deal with theseproblem. Marek’s Disease is a highly contiguity infectivity tumour disease caused by Marek’s Disease Virusin chicken. The MDV vaccine viruses are considered one of the most potent vectors for polyvalent livevaccines expressing foreign antigens related to vaccine-induced immunity against poultry diseases.There are great homologue and similarity between MDV 1 type and vvMDV，so MDV1 can preventMD efficiently. Considering polyvalent recombinant vaccine with much advantage to prevent poultry diseasesefficiently，we try to construct recombinant MDV expressing NDV F gene controlled by MDVglycoprotein B promoter .we hope this recombinant virus can avoid virus rejecting to foreign promoterand avoid disturb of mother’ antibody to prevent NDV and vvMDV efficiently， One pair of primers were synthesized according fusion protein gene of NDVF48E9 strainnucleotide sequence published on GenBank The NDV F48E9 strain Fusion Protein gene was amplifiedand cloned into the multi clone site of eukaryotic expression vector pIRES to form pIRESF. One pair ofthe primers were synthesized according vector pIRES gene nucleotide sequence published onGenBank .This pair of primers were used to amplify 2900bp DNA fragment containing F gene with IVSand polyA and was cloned into multi clone site of vector pBluescprint II+SK with gB promoter of MDV.At last ,the DNVF gene expression cassette was inserted into us10 gene to give rise to the transferringvector pUS10F. We choose CVI988 strain as virus carrier to construct recombinant virus, because the immunityeffect of CVI988 strain is better than HVT, at the same time, there are great homologue and similaritybetween antigen of MDVI and vvMDV .In theory,CVI988 strain has better pertinence immunityresponse reaction ,especially for vvMDV and vv+MDV. In order to improve expression level ofrecombinant virus ,the transferring vector was transfected 293T cells and the specific fluorescent andresult of PCR in the transfected 293T cells was observed. The result is the basis of obtainingrecombinant MDVCVI988 expressing NDV F gene.更多还原