表达ibdvvp2和鸡c3d基因重组火鸡疱疹病病毒的构建(附件)【字数：9654】
1
目 录
引言 1
1.材料与方法 2
1．1材料 2
1.1.1 主要试剂 2
1.1.2 SPF鸡胚和病毒 3
1.1.3 主要仪器设备 3
1.2重组火鸡疱疹病毒转移载体的构建 3
1.2.1 重组火鸡疱疹病毒UL45和UL46同源臂的克隆 3
1.2.2 筛选基因表达盒的构建 5
1.2.3 VP2C3d串联基因表达盒的构建 6
1.2.4火鸡疱疹病毒重组病毒转移载体pUL45/46gptVP2C3d的构建 8
1.3重组火鸡疱疹病毒的获得 10
1.3.1原代鸡胚成纤维细胞(CEF)的制备 10
1.3.2 HVT DNA的提取 10
1.3.3重组火鸡疱疹病毒（rHVTVP2C3d）的包装 10
1.3.4重组火鸡疱疹病毒（rHVTVP2C3d）的筛选 10
1.3.5 夹心ELISA检测重组VP2C3d串联蛋白的表达 10
1.3.6 间接免疫荧光试验（IFA）检测重组VP2C3d串联蛋白的表达 11
1.3.7重组病毒rHVTVP2C3d的遗传稳定性的检测 11
1.3.8重组病毒与野生病毒在CEF上的生长曲线比较 11
2.结果与分析 12
2.1重组火鸡疱疹病毒转移载的构建 12
2.2重组火鸡疱疹病毒病毒的获得 13
2.3夹心ELISA检测 13
2.4间接免疫荧光试验检测 14
2.5重组病毒rHVTVP2C3d株的遗传稳定性 14
2.6重组病毒与野生病毒在CEF上的生长曲线比较 14
3.讨论 15
致谢： 16
参考文献： 16
附录 18
表达IBDV VP2和鸡C3d基因重组火鸡疱疹病毒的构建
动物医学专业学生 张炯
指导教师 严若峰
摘要：传染性法氏囊病（Infectious Bursal Disease, IBD） *好棒文|www.hbsrm.com +Q: ￥351916072$
是由传染性法氏囊病病毒引起的雏鸡急性、高度接触性疾病。本研究利用火鸡疱疹病毒（Herpesvirus of turkey, HVT）FC 126疫苗株基因组UL45/46同源序列、鼠巨细胞病毒（Mouce Cytomegalovirus, MCMV）启动子、C3d和大肠杆菌黄嘌吟鸟嘌吟磷酸转移酶基因序列（gpt）基因，构建含有VP2基因的转移载体质粒pUL45/46gptVP2C3d。再将该HVT转移载体与HVT DNA以磷酸钙沉淀法共转染鸡胚成纤维细胞（CEF），4d后出现典型病毒蚀斑，利用霉酚酸（MPA）阻断其核酸代谢途径，经过6轮筛选获得重组病毒rHVT VP2C3d。间接免疫荧光试验（IFA）检测，重组病毒具有特异性荧光；夹心ELISA检测，重组病毒呈阳性反应。PCR鉴定了30代重组病毒，其能稳定表达目的基因。结果表明，IBD重组火鸡疱疹病毒构建成功，为研制预防传染性法氏囊病和马立克氏病（MD）的二联基因工程疫苗打下基础。
Construction of recombinant Turkey Herpesvirus expressing the VP2 protein of infectious bursal disease virus strain displaying C3d
Student majoring in Veterinary Medicine Jiong Zhang
Tutor Ruofeng Yan
Abstract: Infectious bursal disease (IBD), which usually occurs in 3~6 – week old chicken, is an acute and highly contagious disease caused by infectious bursal disease virus (IBDV). In our research, the transfer vector pUL45/46gptVP2C3d which contained UL45/46 region of turkey(HVT)genome, mouce cytomegalovirus promoter, C3d, selection marker Eco gpt (Xanthine guanine phosphoribosyl transferase (gpt),and IBDV VP2 gene was constructed. And then, transfer vector and total DNA of HVT infected cells were cotransfected into chicken embryo fibroblasts (CEF). A typical virus plaque was observed after 4 days. After six rounds of selection in medium containing mycophenolic acid, xanthine and hypoxanthine, recombinant virus rHVTVP2C3d was obtained and identified. Result showed that recombinant virus had specific fluorescence by indirect immunofluorescence assay(IFA), and IBDV antibody was tested by sandwich ELISA, and the recombinant virus was positive. Recombinant IBD turkey herpesvirus is proved successful.
目 录
引言 1
1.材料与方法 2
1．1材料 2
1.1.1 主要试剂 2
1.1.2 SPF鸡胚和病毒 3
1.1.3 主要仪器设备 3
1.2重组火鸡疱疹病毒转移载体的构建 3
1.2.1 重组火鸡疱疹病毒UL45和UL46同源臂的克隆 3
1.2.2 筛选基因表达盒的构建 5
1.2.3 VP2C3d串联基因表达盒的构建 6
1.2.4火鸡疱疹病毒重组病毒转移载体pUL45/46gptVP2C3d的构建 8
1.3重组火鸡疱疹病毒的获得 10
1.3.1原代鸡胚成纤维细胞(CEF)的制备 10
1.3.2 HVT DNA的提取 10
1.3.3重组火鸡疱疹病毒（rHVTVP2C3d）的包装 10
1.3.4重组火鸡疱疹病毒（rHVTVP2C3d）的筛选 10
1.3.5 夹心ELISA检测重组VP2C3d串联蛋白的表达 10
1.3.6 间接免疫荧光试验（IFA）检测重组VP2C3d串联蛋白的表达 11
1.3.7重组病毒rHVTVP2C3d的遗传稳定性的检测 11
1.3.8重组病毒与野生病毒在CEF上的生长曲线比较 11
2.结果与分析 12
2.1重组火鸡疱疹病毒转移载的构建 12
2.2重组火鸡疱疹病毒病毒的获得 13
2.3夹心ELISA检测 13
2.4间接免疫荧光试验检测 14
2.5重组病毒rHVTVP2C3d株的遗传稳定性 14
2.6重组病毒与野生病毒在CEF上的生长曲线比较 14
3.讨论 15
致谢： 16
参考文献： 16
附录 18
表达IBDV VP2和鸡C3d基因重组火鸡疱疹病毒的构建
动物医学专业学生 张炯
指导教师 严若峰
摘要：传染性法氏囊病（Infectious Bursal Disease, IBD） *好棒文|www.hbsrm.com +Q: ￥351916072$
是由传染性法氏囊病病毒引起的雏鸡急性、高度接触性疾病。本研究利用火鸡疱疹病毒（Herpesvirus of turkey, HVT）FC 126疫苗株基因组UL45/46同源序列、鼠巨细胞病毒（Mouce Cytomegalovirus, MCMV）启动子、C3d和大肠杆菌黄嘌吟鸟嘌吟磷酸转移酶基因序列（gpt）基因，构建含有VP2基因的转移载体质粒pUL45/46gptVP2C3d。再将该HVT转移载体与HVT DNA以磷酸钙沉淀法共转染鸡胚成纤维细胞（CEF），4d后出现典型病毒蚀斑，利用霉酚酸（MPA）阻断其核酸代谢途径，经过6轮筛选获得重组病毒rHVT VP2C3d。间接免疫荧光试验（IFA）检测，重组病毒具有特异性荧光；夹心ELISA检测，重组病毒呈阳性反应。PCR鉴定了30代重组病毒，其能稳定表达目的基因。结果表明，IBD重组火鸡疱疹病毒构建成功，为研制预防传染性法氏囊病和马立克氏病（MD）的二联基因工程疫苗打下基础。
Construction of recombinant Turkey Herpesvirus expressing the VP2 protein of infectious bursal disease virus strain displaying C3d
Student majoring in Veterinary Medicine Jiong Zhang
Tutor Ruofeng Yan
Abstract: Infectious bursal disease (IBD), which usually occurs in 3~6 – week old chicken, is an acute and highly contagious disease caused by infectious bursal disease virus (IBDV). In our research, the transfer vector pUL45/46gptVP2C3d which contained UL45/46 region of turkey(HVT)genome, mouce cytomegalovirus promoter, C3d, selection marker Eco gpt (Xanthine guanine phosphoribosyl transferase (gpt),and IBDV VP2 gene was constructed. And then, transfer vector and total DNA of HVT infected cells were cotransfected into chicken embryo fibroblasts (CEF). A typical virus plaque was observed after 4 days. After six rounds of selection in medium containing mycophenolic acid, xanthine and hypoxanthine, recombinant virus rHVTVP2C3d was obtained and identified. Result showed that recombinant virus had specific fluorescence by indirect immunofluorescence assay(IFA), and IBDV antibody was tested by sandwich ELISA, and the recombinant virus was positive. Recombinant IBD turkey herpesvirus is proved successful.
版权保护: 本文由 hbsrm.com编辑，转载请保留链接: www.hbsrm.com/yxlw/dwyx/86.html
